Document 0qrgbNVovO3BGOw1L7yGLKRQR

EPA United States Environmental Protection Agency Research and Development FINAL DRAFT ECA0-CIN-405 March, 1985 EPA-60Q/X-84-194-1 P B 8 6 - 1 17983 DRINKING WATER CRITERIA DOCUMENT FOR 2 , 3 , 7 , 8 - T E T R A C H L O R O D I B E N Z O - jd- D I O X I N Prepared for OFFICE OF DRINKING WATER Prepared by Environmental Criteria and Assessment Office Cincinnati OH 45268 DRAFT: DO NOT CITE OR QUOTE NOTICE This document 1s a preliminary draft.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 1s being circulated for comments on Its technical accuracy and policy Implications. TECHNICAL REPORT DATA . /fM tf m d Instructions on the rtvtru before compteUni) i 2 ---------------------------- " b . PUCieiEMT*SACESSON MO. ` ________________ . PB8h 1179 83 7S /u t it lc Drinking Water Criteria Document for REPORT DATE 3/85 ,_______________________ per fo r m in g o r g a n iz a tio n co d e 2,3,7,8-TCDD h S rsi PERFORMING ORGANIZATION REPORT NO. Environmental Protection Agency Pe r fo r m in g o r g a n iz a t io n n a m e a n d a d d r e s s 10. P R O G R A M E L E M E N T NO. Ti. o n Y r a c t /g r a n t n o . 12. SPO NSORING A G E N C Y N A M E A N D A D D R E S S U.S. Environmental Protection Agency Office of Drinking Water (WH-550) 401 M St., S.W., Washington, D.C. 20460 IS. SU PPLEM EN TA R Y NO TES 13. T Y P E O F R E P O R T A N D PE R IO O C O V E R E D Scientific r e v i e w __________ 1. S PO N S O R IN G A G E N C Y CODE 16. A BS TR A C T The Office of Drinking Water (COW), U.S. Environmental Protection Agency has prepared a Drinking Water Criteria Document on 2,3,7,8J-TCDD. This Criteria Document is an extensive review of the following topics: - Physical and chemical properties of 2,3,7,8-TCDD - Toxicokinetics and human exposure to 2,3,7,8-TCDD - Health Effects of 2,3,7,8-TCDD in humans and animals - Mechanisms of toxicity of 2,3,7,8-TCDD - Quantification of toxicological effects of 2,3,7,8-TCDD ) . t n a. D E S C R IP T O R S 2,3.7,8-TCDD properties . . 2 , 3,7,8-TCDD exposure 2,3,7,8-TCDD toxicity Drinking Water KEY m o r d s a n d d o c u m e n t a n a ly s is b.ID EN TIFIERS/OPEN EN D ED TERM S c. c o s a t i F k ld /C to u p IS. D IS T R IB U T IO N S T A T E M E N T OPEN IB. SEC U RITY CLASS f i l i t i R tp o rt1 Non-sensitive 20. S E C U R IT Y C LA S S ( T h ii p a g r i Non-sensitive EPA Ft! 2120-1 (*. 4-77) pwcviout b d i t i o n < osiottu 21. NO. OF PA G ES 22. PR IC E DISCLAIMER Mention of trade names or commercial products does not constitute endorsement or recommendation for use. AVAILABILITY NOTICE . This draft document 1s for Internal review and should not be released. For Information contact Dr. Jerry F. Stara, Director, Environmental Criteria and Assessment Office, Cincinnati, OH (513/684-7531). 11 DOCUMENT DEVELOPMENT Helen H. Ball, M.S., Project Officer Environmental Criteria and Assessment Office, Cincinnati U.S. Environmental Protection Agency Authors D1pak K. Basu, Ph.D. Syracuse Research Corporation Syracuse, New York ... Patrick R. Durkin, Ph.D. Syracuse Research Corporation Syracuse, New York Denzll L. T u l H s , Ph.D. Syracuse Research Corporation Syracuse, -New York 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 Scientific Reviewers Larry D. Anderson, Ph.D. Office of Drinking Water U.S. Environmental Protection Agency Washington, DC . Document Preparation Technical Support Services Staff: C. Cooper, P. Daunt, C. Fessier, K. Mann, B. Zwayer, Environmental Criteria and Assessment Office, Cincinnati Special Note: Since this document was developed from the comprehensive Information found 1n Ambient Water Quality Cr1ter1' for 2,3,7,8-Tetrachlorod1benzo->-d1ox1n (EPA 440/5-84-00. and Health Assessment Document for PolvchlorInated D1benzo-dloxlns (EPA 600/8-84-014 A ), portlr s of this document were extracted from these two documents. 111 TABLE OF CONTENTS Page I. SUMMARY . . . . "................................................. 1-1 II. PHYSICAL AND CHEMICAL PROPERTIES................................ H-l CHEMICAL STRUCTURE AND SYNONYMS............................ PHYSICAL PROPERTIES......................................... STABILITY................................................. SUMMARY................................................... H-l H-l H -4 11-7 III. TOXICOKINETICS. . . '....................................... H 1" 1 ABSORPTION . . ............................................ H I - 1 Absorption From the Gastrointestinal Tract.......... III-l , D I S T R I B U T I O N .............................................. M E T A B O L I S M ............................................... EXCRETION.................................... SUMMARY........ .......................................... i IV. HUMAN EXPOSURE......... ...................................... HI-5 IH-H III-15 111-20 IV-1 EXPOSURE ESTIMATION ............................................. IV-1 Drinking Water ............................................. D i e t ........................................................ A 1 r ....................................... IV~1 Iv "2 S U M M A R Y .......................................................... REFERENCES........................................................ V. HEALTH EFFECTS IN ANIMALS ..................................... EXPERIMENTAL ANIMALS .................................... Acute Toxicity.................................... .. Subchronic Toxicity ................................ Chronic Toxicity.................................... TARGET ORGAN TOXICITY.................................... IV" 5 IV" 6 V-l V-l V-l V-46 V-53 V-58 Hepatic Effects . . . . . .......................... Immunological Effects .............................. Other Organ S y s t e m s ................................ OTHER EFFECTS............................................. Carcinogenicity .................................... Mutagenicity........................................ Teratogenicity and Reproductive Toxicity........... V-58 V-62 V-64 V-66 V-66 V-94 V-108 SUMMARY................................................... V-l 20 1v TABLE OF CONTENTS (cont.) VI. HEALTH EFFECTS IN HUMANS.......................... > .......... Page vi-1 CLINICAL CASE STUDIES................................... EPIDEMIOLOGICAL STUDIES.................................. HIGH RISK SUBPOPULATIONS................................ . SUMMARY................................................... VI-1 VI-5 VI-61 VI-61 VII. MECHANISM OF T O X I C I T Y ........................................... VII-1 RECEPTOR-MEDIATED TOXICITY . . VII-T 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 VI1-4 M ETABOLISM.......................................... VITAMIN A DEPLETION..................................... LIPID PEROXIDATION ................................... ENDOCRINE IMBALANCE...................................... SUMMARY................................................... VII-8 VII-10 VII-10 VII-11 vi I--13 VIII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS ............... . . . VIII-1 NONCARCINOGENIC EFFECTS.................................... VIII-5 1-Day HA.............................................VII1--6 10-Day HA ........................................... VIII-9 Long-Term Exposure......................... .. VIII-9 QUANTIFICATION OF NONCARCINOGENIC EFFECTS............... VII1-13 Calculation of 1-Day H A ....................... .. . VIII-13 Calculation of 10-Day HA............................ VIII-14 Quantification of Lifetime AADI ........... . . . . VIII-15 CARCINOGENIC EFFECTS .......................................VIII-16 QUANTIFICATION OF CARCINOGENIC RISK..................... VII1-19 SPECIAL CONSIDERATIONS . ................... VIII-21 Synergistic Effects ................................ VIII-21 High Risk Subpopulatlons. .......................... VIII-23 S U M M A R Y ...................................................... VIII-23 IX. REFERENCES. ..................................................... Ix_-j v LIST OF TABLES Wo. Title Page _ II-l Solubility of 2,3,7,8-TCDD.................................. II-2 I1-2 Phys^Jal Parameters of 2,3,7,8-TCDD ....................... II-3 III-l Percentage of 2,3,7,8-TCDD In the Liver of Rats 24 Hours After Oral Administration of 0.5 ml of Various Formulations Containing TCDD................................ 111-3 II1--2 Tissue Distribution of 2,3,'%8-TCDD ...................... . III-6 II1-3 Elimination of 2,3,7,8-TCDD............. . . T . . . . III-17 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-ll V-3 Estimated Single Oral LD50 - 30 Values forPCDDs............ V-12 V-4 Immunological Effects of 2,3,7,8-TCDD In 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 1n Male Sprague-Dawley R a t s ......................................... V-74 V-9 Benign and Malignant Tumors In 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 2,3,7,8-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 tvphlmurlum .................................. V-96 vl LIST OF TABLES (cont.) No. Title Page V-15 Studies on the Potential Teratogenic Effects of 2,3,7,3-TCDD Contaminated 2,4,5-T .......................... V-109 V-16 Studies on the Potential Teratogenic and Reproductive Effects of 2,3,7,8-TCDD ..................................... V-112 VI-1 Distribution of Tumor Types 1n Two Case-Contro7, . Studies of Soft-Tissue Sarcoma. , . ........................ VI-23 VI-2 Exposure Frequencies 1n 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 S t u d i e s ........................... VI-26 VI-4 Distribution of Histological Types of Soft-Tissue Sarcomas......................................... VI-31 VI-5 Midland County Soft and Connective Tissue Cancer Deaths 1960-1981 ............................................. VI-40 VI-6 Other Occupations (Mlnus/Forestry/Agrlculture)........ VI-47 VI-7 Other Occupations (M1nus/Forestry/Agr1culture/ Woodworkers)............................................. VI-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 In 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-TCDD................. .. VIII-7 VII 1-2 Effects of 4-13 Weeks Exposure to 2,3,7,8-TCDD..........VIII-10 VII1-3 Effects of Long-Term Exposure to 2,3,7,8-TCDD .......... VIII-12 VIII-4 Carcinogenicity Bioassays of 2,3,7,8-TCDD by Oral and Dermal Exposure ................................... . . . VIII-17 VII1-5 Summary of Human Potency Estimates for 2,3,7,8-TCDD. . . . V I I 1-20 VIII-6 Responses and Parameters of the Koclba Feeding Study. . . . VII1-22 VIII-7 Health Advisories for 2,3,7,8-TCDD....................... VIII-24 vii ADI AHH bw BCF 8romoPeCDD DCDD DHBA DMSO DNA EC/GC ED50 FEL GC/MS 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 8romopentachlorodlbenzo--d1ox1n 01chlorodlbenzo--dloxln Dlmethylbenzanthracene Dimethylsulfoxide Deoxyribonucleic acid Electron capture/gas chromatography Median effective dose Frank-effect level Gas chromatography/mass spectrometry Sas chromatography/spec!f 1c Ion monltorlng/mass spe trometry Gastrointestinal High performance liquid chromatography High resolution gas chromatography High resolution mass spectrometry Hexachloro derivatives of dlbenzo-i>-dloxlns Intraperitoneal Concentration lethal to 50% of recipients Dose lethal to 50% of recipients Lowest-observed-adverse-effect level Low resolution mass spectrometry 3-Methylcholanthrene MFO NICI NOAEL NOEL OCDD .PCDOs KP PeCODs PPb ppm ppt RBC RNA SA s.c. TCODs TrICDD 2,4,5-T THA UV WCOT ' Mixed function oxidase Negative Ion chemical Ionization No-observed-adverse-effect level No-observed-effect level Octachlorlnated dlbenzo-jj-dloxlns All polychlorinated d1benzo-{>-d1ox1ns Pentachlorophenol Pentachloro derivatives of d1benzo-j>-d1ox1ns Parts per billion Parts per million Parts per trillion Red blood cel Is Ribonucleic acid Satellite association Subcutaneous Tetrachloro derivatives of d1benzo--d1ox1ns Tr1chlorod1benzo-j)-d1ox1n 2,4,5-Trlchlorophenoxy acetic acid Time-weighted average Ultraviolet Wall-coated open tubular lx . SUMMARY 2.3.7.8- Tetrachlorod1benzo-{)-dlox1n (2,3,7,8-TCDD) 1s one of the most toxic and environmentally stable tricyclic aromatic compounds belonging to chlorinated d1benzo-j)-d1ox1ns. It 1s a contaminant formed 1n the production of 2,4,5-trlchlorophenol. It Is also a contaminant of a few chlorinated phenoxy acids (especially the herbicide 2,4,5-trlchlorophenoxy acetic acid and s11vex) and hexachlorophene. 2,3,7,8-TCDD Is considered relatively stable toward heat, acids and alkalies. It begins to decompose at 500C and virtually complete decomposition occurs within 21 seconds at a temperature of 800C. It Is very slightly soluble In water (0.2 yg/i). In aquatic media, the compound 1s expected to persist for a long time since 1t 1s 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 I1p1d solubility, 2,3,7,8- TCDD Ts rapidly distributed to tissues with a high I1p1d content. The Uver also represents a major site of accumulation 1n many species. Metabolism occurs slowly, and the polar metabolites are excreted 1n the urine and feces. Biliary excretion of 2,3,7,8-TCDD metabolites also occurs. Unmetaboll zed 2,3,7,8-TCDD 1s also excreted In the feces and 1n the milk of lactatlng animals. There are great differences 1n species sensitivity to 2,3,7,8-TCDD, with LDgjjS ranging from 0.6 jig/kg bw In 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, 2,3,7,8-TCDD produces acute liver injury which is not observed in either monkeys, hamsters or guinea pigs. Suppression of the immune system has been observed in mice, rats and guinea pigs. Clinical case studies and epidemiological studies have Implicated 2,3,7,8-TCDD 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 1n the dermal symptomology elicited. Chloracne Is the major dermal finding in humans. In monkeys, common dermal effects Include loss of hair, toenails and fingernails. 2,3,7,8-TCDD has been demonstrated to be teratogenic In rats, mice and rabbits, and fetlcldal -1n .monkeys. The major toxic signs and terata observed were cleft palate 1n 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 In vitro 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 In 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 1s probably carcinogenic for humans. Because 2,3,7,8-TCDD 1s usually found 1n association with other materials (e.g., chlorophenols, phenoxyacetlc acids, combustion products, etc.), 1t 1s not presently possible to evaluate the carcinogenicity of 2,3,7,8-TCDD by Itself 1n humans. A few possible mechanisms of toxicity have been proposed for 2,3,7,8TCDD. These Include receptor mediated toxicity, metaboHsm/dlsposItlon, vitamin A depletion, Increased 11 p1d 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 1n 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 1n dogs exposed to 2.3.7.8- TCDD. The major metabolite 1s 1 ,3,7,8-tetrachloro-2-hydroxyd1benzoji-d1o x 1n. A 1-day HA of 3.5xl0"a yg/a for a 70 kg adult and l.OxlO-3 yg/a for a 10 kg child were calculated from a single-dose oral LOAEL 1n guinea pigs, the species most sensitive to the toxicity of this compound. Ten-day HAs were calculated by dividing the 1-day HAs by 10-4. The resulting HAs '. were 3.5xl0" 5 yg/a for a 70 kg adult and l.OxlO-4 yg/a for a 10 kg child. A lifetime AADI of 3.5xl0-5 yg/a was estimated from a LOAEL derived 1n a 3-generat1on reproductive study 1n rats (Hurray et al., 1979) along with the rationale developed by the EPA. 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 10/21/85 female rats, the concentrations of 2,3,7,8- TCDD 1n drinking water that would result 1n Increased lifetime cancer risks of 10 4 , 10 5 , and 10-6 were estimated to be 2 .2x10"*, 2 .2x10 * and 2 .2x10 7 vg/8. respectively. 01290 1-4 10/21/85 II. PHYSICAL AND CHEMICAL PROPERTIES Chemical Structure and Synonyms . 2,3,7,8-Tetrachlorod1benzo-i>-d1ox1n (2,3,7,8-TCOD) CAS Registry Number: 1746-01-6 Chem. Abst. Name: 2,3,7,8-tetrachlorod1benzo[b,e](l,4)-d1ox1n RTECS Number: HP35000 Synonyms: D1ox1n; TCDBD; TCDD; 2.3,7,8-tetrachlorod1benzod1ox1n, 2,3,7,8tetrachlorod1benzo-l,4-d1ox1n (IARC, 1977). Physical Properties 2,3,7,8-TCDO has a molecular formula of C^H^Cl^O^ and a molecu lar weight of 321.9. In the pure form, 1t exists as colorless needles with a melting point of 303-305C (Crummett and Stehl, 1973). In a chloroform solution, the maximum absorption of 2,3,7,8-TCDO 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-TCOD 1s a highly lipophilic substance. . Values for other physical properties of 2,3,7,8-TCDO 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-DIchlorobenzene Chloroform Acetone n-Octanol Methanol 2 x 10" 44 570 1400 370 no SO 10 Source: Adapted from Crummet and Stehl, 1973 . 01300 11-2 / 08/10/84 TABLE II-2 Physical Parameters of 2,3,7,8-TCDO Parameter Vapor pressure (mm of Hg) Octanol/water partition coefficient Sorption partition coefficient (Koc) Value 1.7 X 10" ... 1.4 X 10 6.9 X 10 1.9 X 107 9.9 X 10= 3.3 X 10 Reference NRCC, 1981 NRCC, 1981 Mabey et al., 1981 U.S. EPA, 1984a NRCC, 1981 Mabey et al., 1981 01300 11--3 09/18/84 Stability 2,3,7,8-TCDD 1s considered relatively stable toward heat, acids and alkalies (Albro, 1979). It begins to decompose at a temperature of 500C with virtually complete degradation at 800C within 21 seconds (Stehl et al.:, 1973). Gamma radiation degrades 2,3,7,8-TCDD (FanelH et a!., 1978). 1 The four transformation processes (photoreaction, biotransformation, hydrolysis and radical oxidation) that control the' fate of a chemical 1ri aquatic media do not appreciably transform 2,3,7,8-TCDD (Hatsumura et al., 1983). In organic solvents, 2,3,7,8-TCDD undergoes reductive photodechlorlnatlon at wavelengths <320 nm (Crosby et al., 1971; Libert1 et al., 1978). In aqueous solution, hydroxylatlve dechlorination has not been seen. Pllmmer 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 1n water 1n the presence of a surfactant, underwent substan tial photodegradation under sunlamp or sunlight Irradiation (Pllmmer et al., 1973; Crosby et al., 1971). The surfactant, l-hexyldecylpyrid1n1um chlo ride, sensitized the photodecomposition of 2,3,7,8-TCDD 1n aqueous solution (Botre et al., 1978). In order to explain the longer half-Hfe 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 photo sensitization of 2,3,7,8-TCDD was speculated to have caused some photodecom position of this compound. From the available Information, 1t 1s difficult 01300 II-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. 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 1n sediment-containing Wisconsin lake waters was 550-590 days. In lake water alone, -70X 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-Hfe 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 b1odegradabH1ty of 2.3.7.8- TCDD. 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 In the literature or on structure-activity analogy values given by Mabey et al. (1981). Assuming the blotransformatlpn rate constant of 1x10" ma cell'* hr-* (Mabey et al., 1981) and the concentration of microorganisms capable of degrading 2,3,7,8-TCDD as 5x10s cell mi" 1 (Burns et al., 1981), the half-Hfe of biodegradation 1s estimated to be >1 year. 01300 II-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 solution during microbial studies, little quantitative Information regarding the volatilization of 2,3,7,8-TCDD from aquatic media 1s 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 Llss and Slater (1974), a vapor pressure value of -10" torr (0.1 m Pa) and a solubility value of `6.2x10_1 mole/i, 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-life 1s directly proportional to water depth and Inversely proportional to mass transfer coefficient (Thibodeaux, 1979). The limitations of the Llss-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 1n 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^ in NRCC (1981), volatilization has been estimated to account for 100% of the fraction lost and biodegradation has been calculated to be 0%. The volatilization halr-llfe 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 the adsorbed state (Ward and Matsumura, 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. (19^9) 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.4C1.40_2 and a molecu- lar weight of 321.9. In pure form, 1t exists as colorless needles with a melting point of 303-305C (Crummett 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 (Crummett and Stehl, 1973). The compound has a low vapor pressure (1.7x10"* mm 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. ERA, 1980; Hu-etter and Philippi, 1982). The eslmated biodegradation half-life of 2,3,7,8-TCDD 1s >1 year (Habey et al., 1981; Burns et al., 1981). Hydrolysis and radical oxidation do not appear to be significant processes for 2,3,7,8-TCDD In aquatic media (NRCC, 1981). 01300 11--7 04/05/84 Little quantitative Information regarding the volatilization of 2,3,7,8TCOD from aquatic media 1s available. Theoretical modeling of 2,3,7,8-TCDD (EXAMS model) provides a volatilization half-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 Is likely to remain sorbed to sediments and biota 1n 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.f 1982; Gas1ew1cz 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-TCDD 1n various mammalian species. Absorption The dermal and gastrointestinal absorption of 2,3,7,8-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]2,3,7,8TCDD/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 acetonercorn 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., 1979). The gastrointestinal absorption of 2,3,7,8-TCDD was also examined 1n 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 I I I - l 09/18/84 [1,6--3H]--2,3,7,8-TCDD 1n olive oil {650 pg/kg) and reported that 7414 of the dose was absorbed. When 2,3,7,8-TCDD was administered to rats 1n the diet at 7 or 20 ppb (0.5 or 1.4 pg/kg/day) for 42 days, 50-60% of the consumed dose was absorbed {Fries and Marrow, 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 1n 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-TCDD was administered 1n 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 1n 50% ethanol (for doses of 12-280 ng, equivalent to 0.06-1.4 pg/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 1n Table III-l. Admin istration of 2,3,7,8-TCDD 1n an aqueous suspension of soil resulted 1n a decrease 1n the hepatic levels of 2,3,7,8-TCDD as compared with hepatic levels resulting from administration of 2,3,7,8-TCDD 1n 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 II1-2 08/10/84 TABLE III-l Percentage of 2,3,7,8-TCDD 1n the Liver of Rats 24 Hours After Oral Administration of 0.5 ml of Various Formulations Containing TCOO* Formula M o n TCOO Dose (ng) No. of Animals 50% Ethanol 14.7 Aqueous suspension of soil (37%, w/w) that had been 1n contact with TCOO for: 10-15 hours - 8 days 12.7, 22.9 21.2, 22.7 Aqueous suspension of activated carbon (25%, w/w) 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 III-3 08/10/84 in an aqueous suspension of activated carbon, absorption was almost totally eliminated (<0.07% of the dose in hepatic tissues). Philippi et al. (1981) and Hutter and Philippi (1982) have shown that radiolabeled 2,3,7,8-TCDO becomes progressively more resistant with time to extraction from soil. Similarly, the feeding of fly ash, which contains PCODs, to rats in the diet for 1? : days resulted 1-n considerably lower hepatic levels of PCODs than did the feeding of an extract of the fly ash at comparable dietary concentrations of PCODs (van den 8erg et al., 1983). The PCDOs were tentatively identified as 2,3,7,8-TCDO, 1,2,3,7,8-PeCDO, 1,2,3,6,7,8-HxCDD and ,1,2,3,7,8,9-HxCDD and the difference in hepatic levels noted between fly ash-treated and extract-treated rats was greater for the more highly chlorinated isomers than It was for 2,3,7,8-TCDO. These results indicate the importance of'"the formulation or vehicle containing the toxin(s) on the relative bicavailability of 2,3,7,8-TCDO, PeCOD and HxCDDs following oral exposure. Information on the absorption of 2,3,7,8-TCDO through the skin 1:; found only in a study by Polger and Schlatter (1980). The authors administered 26 ng 2,3,7,8-TCDO In 50 yi 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 1n 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 1s equiv alent to absorption from 50% ethanol. As compared with dermal application 01310 111--4 08/10/84 1n methanol, dermal application of 2,3,7,8-TCDD to rats 1n vaseline or poly ethylene glycol reduced the percentage of the dose 1n hepatic tissue to 1.4 and S.3%, respectively, but had no observable effect on the dose of 2,3,7,8TCDD required to Induce skin lesions {-1 pg/ear) 1n the rabbit ear assay. Application of 2,3,7,8-TCDD 1n a so1l/water paste decreased hepatic 2,3,7,8TCDD to -2% of the administered dose and Increased the amount required to produce skin lesions to 2-3 pg 1n rats and rabbits, respectively. Appli cation 1n an activated carbon/water paste essentially eliminated absorption, as measured by percent of dose 1n the Uver, and Increased the amount of 2,3,7,8-TCDD required to produce skin lesions to -160 pg. These results suggest that the dermal absorption and acnegenlc potency of 2,3,7,8-TCDD are dependent on the formulation (vehicle or adsorbent) containing the toxin. Distribution .. . The tissue distribution of 2,3,7,8-TCDD 1n a number of species 1s sunmarlzed 1n Table III-2. From these data 1t 1s apparent that 2,3,7,8-TCDD dis tributes preferentially to the Uver and adipose tissue of most species that have been examined. Piper et al. (1973) used a single oral dose of [14C]2,3,7,8-TCDD to study """distribution and excretion in male Sprague- 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 Uver and adipose tissue to contain the highest percent of the dose per gram of tissue, with 3.18 and 2.60%, respectively. 01310 111--5 08/10/84 01310 TADLE III-2 Tissue Distribution of 2,3,7,8-TCDD II1-6 Species 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 1.p. oral ; 1.p. 1.p. I.p. or oral oral 1.p. Tissues with the Highest Concentration of 2,3,7,8-TCDD liver , Uver > fat Uver > fat liver > fat liver > fat Uver > fat liver > fat > kidney > lung liver > fat > kidney > lung > spleen fat > skin > liver > adrenal = thymus IJver > fat fat > liver > adrenals > thymus > skin fat > liver > 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 09/18/84 Rose et al. (1976) also examined the distribution of [14C]2,3,7,8-TCDD 1n the rat. Twenty-two days after a single oral dose of 1.0 pg/kg, Uv e r 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 activity was again localized mainly 1n the U v e r and adipose tissue, but the U v e r had five times as much radioactivity as did the fat. With the single oral dose, no radioactivity was detected In either the urine or expired air, Indicating that most 1f not all of the elimination of 2,3,7,8-TCDD and/or Its metabolites was through the feces. With repeated oral doses, the 14C activity was also excreted prjmarlly through the feces, but significant amounts were found In the urine, especially of the female rats. Male rats given 1.0 pg/kg/day of 2,3,7,8-TCDD for 7 weeks excreted an average of 3 .1% of the cumulative dose 1n the urine while the female rats excreted an average of 12.5% 1n the urine (Rose et al., 1976). Fries and Marrow (1975) have also reported evidence of sex differences 1n tissue distribution In rats. During 42 days of administration of_ 2,3,7,8TCDD, -85% of the total body residue of male rats.was located 1n the Uver, whllje 70% of the total body residue of female rats was located 1n this organ. Studies performed by Van Miller et al. (1976) on rhesus monkeys and rats using single 1.p; doses of trltlated 2,3,7,8-TCDD (400 pg/kg bw) showed that while rats had over 40% of the 2,3,7,8-TCDO 1n the liver 7 days after dosing, the monkeys had only about 10% 1n 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 1.p. administration (Manara et al., 1982). The Uv e r was also found to be the major site of accumulation of 2;3,7,8TCDD 1n the hamster, with 20% of the dose localized 1n the U v e r (5.3% of 01310 III--7 08/10/84 dose/g liver) at 3 days following a sublethal dose of 650 pg 3H-2,3,7,8TCDD/kg {Olson et al., 1980a). In all three species, 1-22 days after single-dose oral or i.p. administration, levels of 2,3,7,8-TCDD in adipose tissue were generally slightly lower than levels in the liver, and were con siderably higher than concentrations 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 dally dietary 2,3,7,8-TCDl) intake of 0.1 pg/kg/day for 2 years had an average 2,3,7,8-TCDD content of 8100 ppt in fat and 24,000 ppt in the liver. Rats given 0.01 pg/kg/day had an average of 1700 ppt of 2,3,7,8-TCDD in the fat and 5100 ppt in the liver. For both of these daily dosages the liver:body fat ratio of 2,3,7,8-TCDD was 3:1. At the lowest dose* level of 0.001 ' pg/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 administration of a single oral dose of 1 pg/kg of 2,3,7,8-TCDD to an adult rhesus macaque monkey, tissue levels of the compound were 100 ppt 1n adipose tissue and 15 ppt 1n liver. These results Indicate that prolonged retention of 2,3,7,8TCDD may occur 1n this species. The tissue distribution of 2,3,7,8-TCDO 1n the guinea pig appears to be similar to the monkey, with the highest concen tration of the toxin being found in adipose tissue {Gasiewicz and Neal, 01310 111-8 09/18/84 1979; Nolan et al., 1979). The interspedes difference in the tissue dis tribution of 2,3,7,8-TCDO 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 in doubt. Fjr example, the hepatotoxlclty of 2,3,7,8-TCDD 1n a given species does not appear to be related to the hepatic concentration of the toxin (Neal et al., 1982). m 2,3,7,8-TCDD has been demonstrated to be teratogenic and fetotoxlc in 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 [1*C]2,3,7,8-TCDD was Investigated by Moore et al. (1976). They found low concentrations of 2,3,7,8-TCDD 1n the fetus at gestation days 14, 18 and 21. The radioactivity..appeared to be evenly distributed throughout the fetus on days 14 and 18; however, Increased levels of radioactivity were detected 1n fetal Uv e r on day 21. Nau and Bass (1981) (more recently reported by Nau et al., 1982) Investigated the fetal uptake of 2,3,7,8-TCDD 1n NMRI mice following oral, 1.p. or s.c. administration of the compound at dose levels of 5, 12.5 or 25 pg/kg 1n DMSOrcorn oil or acetone:corn 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,B-TCDD at 5 and 12.5 pg/kg. At 25 pg/kg, higher maternal and fetal tissue levels were obtained with s.c. administration, and much higher levels were obtained with 1 .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 1n the embryo and fetus between gestational days 11 01310 111-9 09/18/84 and 18. This sharp decrease In 2,3,7,8-TCDD concentration coincides with placentatlon. 2,3,7,8-TCDU concentrations 1n the placenta were an order of magnitude greater than In the fetus Itself. The affinity of fetal liver for 2.3.7.8-TCDD was relatively low, as compared to maternal liver; however, 2.3.7.8-TCDD levels In fetal livers were 2-4 times higher than the levels 1n other fetal organs.' An attempt was made to correlate 2,3,7,8-TCDD levels In the fetuses with the observed Incidence of cleft palate, but no clear rela tionship was observed. Autoradiographic studies of tissue localization following 1.v. adminis tration of [l4C]2,3,7,8-TCDD 1n DMSO to three strains of mice Indicated that the Uver had the highest concentration and longest retention of radio activity 1n the body, followed by the nasal mucosa (Appelgren et al., 1983). In pregnant mice, the concentration of radioactivity In the fetuses was lower than In the dams, but a similar, selective labelling of the liver and the nasal mucosa 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 Uver at 1 hour after dosing, but thereafter was much lower than 1n the Uver. Labelling of the thymus, lymph nodes, bone marrow and prostate were low at all observation times (l.e., 5 minutes to 61 days after Infection). Very few data are available on the tissue distribution of 2,3,7,8-TCDD 1n humans. Facchettl et al. (1980) reported tissue concentration of 2.3.7.8- TCDD at levels of 1-2 ng/g In Uver and <0.1 ng/g In thyroid, brain, lung, kidney and blood 1n a woman who died 7 months after potential exposure to 2,3,7,8-TCDD from the Seveso accident. This pattern of 2,3,7,8-TCDD distribution, however, may not be representative for humans since the woman 01310 III-10 09/18/84 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 al. (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 321.8335 and 319.8965. Three groups were studied consisting of 20 veterans claiming health problems related to Agent Orange exposure, 3 A1r Force officers with known heavy exposure to Agent Orange during disposal operations, and 10 controlveterans with no known herbicide exposure. In the first group, 10 of the 20 had measurable levels of 2,3,7,8-TCDD (5 with 5-7 ppt, 3 with 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 between7 and 14 ppt. Levels of 2,3,7,8-TCDD 1n adipose tissue did not appear to be associated In this study with 111 health or any particular symptom. However, 1t was considered that Information on background levels of 2,3,7,8-TCDD In adipose tissue was too limited to draw any firm conclusions. Metabolism Vlnopal and Caslda (1973) found no evidence of water soluble metabolites of 2,3,7,8-TCDD following Incubation with mammalian Uver mlcrosomes or l.p. Injection into mice. In the same experiment, only unmetabolized 2,3,7,8TCDD was extractable from mouse Uver 11-20 days after treatment. Van Miller et al. (1976) claimed that the slow elimination of 2,3,7,8-TCDD they observed In both rats and monkeys after 1.p. Injections suggested that 01310 III-ll / 08/10/84 2,3,7,8-TCDD was not readily metabolized. Metabolites of 2,3,7,8-TCDD have been detected 1n the bile and urine of Syrian Golden hamsters after single oral cr l.p. doses (Olson et al., 1980a) and 1n 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 al. (1982) demonstrated biliary excretion of several metabolites of [l4C]2,3,7,8-TCDD by rats after repeated oral dosing. The metabolites were tentatively Identified as glucuronldes of hydroxylated 2,3,7,8-TCDD. The amounts of metabolites found were small, Indicating that 2,3,7,8-TCDD 1s only slowly metabolized 1n the Uver. Previous work by Piper et al. (1973) using single oral doses of 2,3,7,8-TCDD concluded that, since small amounts of radioactivity were found fn-the urine and expired air of male rats during the first 10 days, metabolic alteration or breakdown must occur. The study by Rose et al. (1976) using oral doses stated that while the l,C activity In the rat livers appeared to be present as unchanged 2,3,7,8-TCDD, a significant amount of radioactivity found In the feces appeared to come from substances other than 2,3,7,8-TCDD; the excretion of 14C In the urine also Indicated that metabolism had occurred. Polger et al. (1982a) Investigated the toxicity of 2,3,7,8-TCDD metabo lites by administering extracts of bile from 2,3,7,8-TCDD-treated dogs to male guinea pigs 1n single oral doses equivalent to 0.6, 6.0 and 60 pg/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 01310 III-12 08/10/84 guinea pigs was at least 100 times higher than was the acute toxicity of Its metabolites. More recently, Olson et al. (1983) reported that all of the radioactiv ity 1n urine and bile from l4C-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-TCDO metabolites from the rat and hamster altered the chromatographic profile of the metabolites, Indicating the presence of glucuronlde conjugates 1n bile and sulfate conjugates 1n urine (Olson and Bittner, 1983). The apparent absence of these metabolites 1n extracts of hamster and rat Uver suggest that once formed, the metabolites of 2,3,7,8TC00 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-TCDO 1s dependent upon metabolism "Of the toxin. Although urine and bile appear to be free of unmetabollzed TCDO, data from the hamster and rat Indicate that from 10 to 40% of the 2,3,7,8-TCDD-derlved radioactivity 1n feces represents unchanged 2,3,7,8-TCDD (Olson et al., 1983; Olson and Bittner, 1983). The dally presence of unchanged 2,3,7,8-TCDD 1n feces and Its absence 1n bile suggests that direct Intestinal elimination 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 In vivo rate of 2,3,7,8-TCDD metabolism 1n a given animal. Neverthe less, the metabolism of 2,3,7,8-TCDD does 1n part regulate U s elimination or relative persistence 1n a given animal. Several metabolites of 2,3,7,8-TCDD have, recently been Identified. Sawahata et al. (1982) Investigated the In vitro metabolism of 2,3,7,8-TCDD 01310 III-13 08/10/84 in isolated rat hepatocytes. The major product was deconjugated with B-glucuronidase, derivatized with diazomethane, and separated into two com pounds by high performance liquid chromatography (HPLC). These metabolites were subsequently identified as l-hydroxy-2,3,7.8-TCDD and 8-hydroxy-2,3,7tr1chlorodi benzo-jj-dioxin. Poiger et al. (1982a) identified six metabolites in the bile of dogs that were given a lethal dose of [3H]2,3,7,8-TCDD. The major metabolite was 1,3,7,8-tetrachloro-2-hydroxydibenzo--d1oxin; 3,7,8tr1chloro-3-hydroxyd1benzo-]D-d1ox1n 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 trichlorohydroxydi benzo--di oxi ns and the third was apparently a chlorinated 2-hydroxydiphenyl ether. 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 in. vivo binding of [1,6-3H]-2,3,7,8-TCDD derived radioactivity to rat hepatic macromolecules. They found maximum levels equivalent to 60 pmol 2,3,7,8-TCDD/mole of amino acids in protein, 12 pmol 2,3,7.8-TCDD/mole of nucleotide in rRNA, and 6 pmol of 2,3,7,8-TCDD/mole of nucleotide in DNA. This corresponds to one 2,3,7,8-TCDD-DNA adduct/35 cells. Poland and Glover (1979) suggest that it is unlikely that 2,3,7,8-TCDD-induced oncogenesis is through a mechanism of covalent binding, to DNA and somatic mutation. Further studies in other species,; possibly 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-TCDO. 01310 III-14 09/18/84 Isolated rat hepatocytes In suspension have been used as an In. vitro system for assessing 2,3,7,8-TCDD metabolism under various conditions (Olson et al., 1981). Data Indicate that the rate of 2,3,7,8-TCDD metabolism 1n rat hepatocytes correlates directly with drug Induced changes 1n hepatic cytochrome P-450 monooxygenase activity, suggesting that 2,3,7,8-TCDD 1s 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-TCOD metabolism In Isolated hepatocytes, demonstrating that 2,3,7,8-TCDO 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 2,3,7,8- TCDD 1n rats. In both naturally occurring age and sex-related differences 1n MFQ activity, and following administration of Inducers and Inhibitors of MFO enzyme systems, hepatic HFO activity was directly correlated with the 20-day LD5Q. ....... Olson and Bittner (1983) reported that the rate o-f 2,3,7,8-TCDD metabo lite formation In vitro was higher 1n hepatocytes from the hamster than 1n hepatocytes from the rat. Qualitative evaluation of In. vivo and In. vitro metabolites by HPLC also suggested significant 1nterspec1es variability. The authors suggested that such differences 1n metabolism may partially .explain the differences 1n toxicity among species. Excretion The following discussion assumes that elimination 1s a first order process. With the exception of the guinea pig, which may follow zero order kinetics (Gaslewlcz and Neal, 1979), elimination data yield a straight line on a semi logarithmic plot, Indicating that elimination 1s a single, first 01310 III-15 08/10/84 order process. Hlles 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 Its metabolites has been Investigated 1n a number of species. Table III-3 summarizes results on the elimination of 2,3,7,8-TCDD-der1ved 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 excreted from the bodies of all species tested, with a half-life 1n 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; Gaslewlcz et al., 1983a). The nigh levels found In the urine of Infant monkeys were probably due to the Incomplete separation of urine and feces (Van Miller et al., 1976). In all. the other species tested so far, excretion occurred mainly through the feces (80-100% of total urinary and fecal radioactivity) with only minor amounts of 2,3,7,8-TCDD metabolites found In the urine (Piper et al., 1973; Allen et al., 1975; Rose et al., 1976; Gaslewlcz and Neal, 1979). Only Piper et al. (1973) reported the excretion of metabolites 1n the expired air. During 21 days following administration of a single oral dose of [14C]2,3,7,8-TCDD to rats, 3.2% of the administered radioactivity (4.6% of the excreted radioactivity) was recovered In the expired air. 01310 III-16 09/18/84 TABLE II1-3 Elimination of 2,3,7,8-TCDD Species ----,---------- ----- Single Treatment pg/kg (route) Half-Life for Elimination (days) Relative % of TCDD-Oertved Radioactivity . Feces Urine Reference - II1-17 Guinea pig Guinea pig Rat Rat Rat Rat 2 (l.p.) . 1.45 (oral) 1.0 (oral) 50 (oral) 50 (oral) 400 (l.p.) Monkey (adult) 400 (l.p.) Monkey (Infant) 400 (l.p.) Mouse C57B1/65 DBA/2J D602Fi/J* Hamster Hamster 10 (l.p.) 10 (l.p.) 10 (l.p.) 650 (l.p.) 650 (oral) 30.2 + 5.8 22 - 43 31 6 17.4 5.6 21.3 2.9 NT NT NT 11.0 + 1.2 24.4 * VI12.6 0. 10.8 2.4 15.0 2.5 94.0 NT >99 ; 80.0 95.5 91.0 78.0 6.0 NT <1 20.0 4.5 9.0 22.0 39.0 61.0 72.0 54.0 72.0 59.0 NT 28.0 46.0 28.0 41.0 NT 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 Hiller et al., 1976 Van Miller et al., 1976 Gaslewlcz et al., 1983a,b Gaslewlcz et al., 1983a,b Gaslewlcz et al., 1903a,b Olson et al., 1980a Olson et al., 1980a 09/18/8A Offspring of C57B1/6J and DBA/2J which are heterozygous at the Ah locus NT = Not tested Rose et al. (1976) Investigated the elimination of [14C]2,3,7,8-TCDD 1n rats given repeated oral doses of 0.01, 0.1 or 1.0 yg/kg/day Monday through Friday for 7 weeks, or a single dose of 1.0 yg/kg. In the single dose study, no l4C was excreted 1n the urine or expired air; 1n the repeated-dose study, however, 3-18% of the cumulative dose was excreted 1n the urine by 7 weeks. This study Indicated that steady-state concentrations will be reached 1n the bodies of rats 1n -13 weeks. The rate constant defining the approach to steady-state concentrations was Independent of the dosage of 2,3,7,8-TCDD over the range studied. This 1s consistent with the observations of Fries and Marrow (1975) who found that the total retention 1n the bodies of rats was proportional to total Intake. When rats were maintained on a diet containing either 7 or 20 ppb 2,3,7,8-TCDD, the amount of 2,3,7,8-TCDD retained 1n 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 1n Table II1-3 suggest some 1nterspec1es differences 1n the half-Hfe for elimination (t 1/2) of 2,3,7,8-TCDD. In the hamster, the least sensitive species to the..acute toxicity of 2,3,7,8-TCDD, a mean t 1/2 of 10.8 days was observed (Olson et al., 1980a,b), and 1n the guinea pig, the most sensitive species to the acute toxicity of 2,3,7,8-TCDO, the mean ;t 1/2 was 30.2 days (Gas1ew1cz and Neal, 1979). The observed 1nterspec1es differences In the t 1/2 of2,3,7,8-TCDD may In part be related tothe relative sensitivity of a given species to the acute toxicity of 2.3.7.8- TCDD. The Intraspecies differences In the t 1/2 of 2,3,7,8-TCDD In three mouse strains may be due to the finding that the 08A/2J strain possesses -2-fold 01310 III-18 08/12/84 greater adipose tissue stores than the C57B1/6J and B6D2F^/J strains (Gaslewlcz et al., 1983b). The sequestering of the lipophilic toxin 1n adipose tissue stores of the DBA/2J mouse may contribute to the greater persistence of 2,3,7,8-TCOI) In this strain. In all of the rat studies shown 1n Table III-3, urinary and fecal elimi 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-der1ved radioactivity In rats over a 35-day period following a single 1.p. exposure at 1 yg aH-2,3,7,8-TCDD/kg. They observed 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 suggested. A preliminary study In the rhesus monkey Indi cates that.2,3,7,8-TC00 may be exceptionally persistent In adipose tissue. McNulty et al. (1982) estimated the apparent half-life of 2,3,7,8-TCDD ln~ the fat of a monkey to be ~1 year. Studies 1n the rat, guinea pig, hamster and mouse have found that all of the 2,3,7,8-TCDD derived radioactivity excreted 1n the urine and bile corre sponds to metabolites of 2,3,7,8-TCDD (Olson et al., 1983). The apparent absence of 2,3,7,8-TCDD metabolites In Uver and fat suggests that, once formed, the metabolites of 2,3,7,8-TCDD are readily excreted. Thus, urinary and biliary elimination of 2,3,7,8-TCDD Is dependent upon metabolism of the 01310 II1--19 09/18/84 toxin. Although urine and bile appear to be free of unmetabollzed 2,3,7,8- TCDD, 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). Unmetabollzed 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 1n lactatlng rats have also found that unchanged 2,3,7,8-TCL may be excreted 1n the milk' of lactatlng animals (Moore et al., 1976; lucler 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 In vivo half-1.1fe for elimination of 2,3,7,8- TCDD may not directly reflect the rate of 2,3,7,8-TCDD metabolism 1n a given animal (Neal et al., 1982). Summary The toxicokinetics of 2,3,7,8-TCDD has been Investigated 1n a number of laboratory animals; the reader 1s referred to recent reviews (Neal et al., 1982; Gas1ew1cz et al., 1983a; Olson et al., 1983) for Indepth discussions j of this subject. Because 2,3,7,8-TCDD 1s a strongly lipophilic compound (Crummett and Stehl, 1973), gavage treatment with single or repeated doses of the compound 1n oil has resulted 1n absorption of -50% of the dose administered 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). 01310 I 11-20 09/18/84 Dietary administration of comparable dose-ranges to rats resulted 1n some what reduced GI absorption (-50-60% of administered dose was absorbed) (Fries and Harrow, 1975). Using hepatic concentration of 2,3,7,8-TCDD as a endpoint, Polger and Schlatter (1980) demonstrated a linear relationship In rats between 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 (Polger and Schlatter, 1980) were able to show a decrease 1n GI absorption of 2,3,7,8-TCDD directly proportional to the length of time the compound had been In contact with soil before the suspension was made and the rats were treated. H1x1ng 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 (Polger and Schlatter, 1980). That adsorbant materials may reduce the GI absorption of 2,3,7,8TCDD 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 absorption of 2,3,7,8-TCDO has been estimated In rats to be -40% of the absorption of an equivalent dose orally administered (Polger and Schlatter, 1980). Dermal application of the compound 1n vaseline, poly ethylene glycol or soil/water paste substantially reduced dermal absorp tion. Application of the compound 1n activated carbon/water paste virtually eliminated absorption. 01310 III--21 09/25/84 Tissue distribution following oral or 1.p. administration of 2,3,7,8TCOD to rats appears to be preferentially to the liver and adipose tissue {Fries and Marrow. 1975; Rose et al., 1976'; Van Miller et al., 1976; Koclba et al., 1978a). Other tissues showed substantially lower concentrations of 2,3,7,8-TCDO. Soon after treatment the liver may have concentrations =3 (Koclba et al., 1978a) to 5 (Rose et al., 1976) times that In adipose tis sue. It was suggested that male rats accumulate 2,3,7,8-TCDD 1n the liver _ more efficiently than female rats (Fries and Marrow, 1975). Tissue distri bution 1n mice (Manara et al., 1982) and hamsters (Olson et al., 1980a) seems to be similar to that In rats. Monkeys, however, appear to accumulate 2,3,7,8-TCDD preferentially 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-TC00 (McNulty et al., 1982). Prolonged tissue retention of the compound was thus demonstrated. Tissue distribution In guinea pigs appears similar to that In monkeys (Gaslewlcr. and Neal, 1979; Nolan et al., 1979) 1n that tissue levels 1n fat exceed those 1n the liver. Evidence that 2,3,7,8-TCOD accumulates 1n the adipose tissue of exposed humans was presented by Young et al. (1983) who reported levels of 3-99 ppt 1n the adipose tissue ofarmed forces veterans claiming health problems related to Agent Orange. Distribution of 2,3,7,8-TCDD 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 1n rat fetuses on gestation days 14 and 18 01310 I 11--22 09/18/84 and appeared to be evenly distributed 1n all fetal tissues. At gestation day 21, the fetal liver showed a marked affinity for 2,3,7,8-TCOD (Moore et al., 1976). 2,3,7,8-TCOD was distributed to the fetuses of mice following oral, l.p. or s.c. administration (Nau et al., 1982). Maximum fetal concen trations occurred on gestation days 9 and 10; lower fetal concentrations were observed on gestation days 11-18, coincident with placentation. The fetal Uver had less affinity for the compound that did the maternal Uver. In an early metabolism study, Vlnopal and Caslda (1973) reported that In vivo or l vitro studies with mice showed that polar metabolites of 2,3,7,8-TCOD were not produced by this species. In rats, however, hydroxy lation and conjugation with glucuronlde and sulfate have been demonstrated (Polger and Schlatter, 1979; Olson et al., 1983; Polger et al., 1982a. Glu curonlde conjugates tended to-predominate 1n the bile (Polger and Schlatter, 1979) and sulfate conjugates were located 1n the urine (Olson et al., 1983).. Polger and Schlatter (1979) stated that U v e r metabolism of 2,3,7,8-TCOD proceeds slowly In the Uver. Neal et al. (1982) demonstrated that the rate of hepatic metabolism was enhanced by activated cytochrome P-450 mono oxygenase. It was suggested that metabolism of 2,3,7,8-TCOD 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 al. (1982) who Identified trl-- and d1chlorod1benzo-j>-d1ox1ns as metabolites 1n In vitro rat hepatocyte systems. From the bile of dogs; six major metabolites have been Identified (Polger et al., 1982a); hydroxylated conjugates of tetra-, tr1- and d1chlorod1benzo--d1ox1n predominated. 01310 II1--23 09/25/84 When the excretion data are subjected to a sem1-logar1thmlc plot, a straight line results, suggesting that elimination of 2,3,7,8-TCDD Is a first-order phenomenon, particularly for rats. Excretion In the guinea pig may be a zero-order process (Gas1ew1cz and Neal, 1979). The half-Hfe for body elimination varied considerably with ranges of -10-15 days In the hamster (Olson et al., 1980a), the species least sensitive to the toxic effects of 2,3,7,8-TCDD, to estimates of -11-24 days 1n the mouse (Gaslewlcz et al., 1983a,b), -17-31 days 1n the rat (Rose et al., 1975; Piper et al., 1973; Allen et al., 1975) and -22-30 days 1n the guinea pig (Gaslewlcz and Neal, 1979; Nolan et al., 1979). One strain of mice, 0BA/2J, had a half life for elimination (-24 days) about twice as high as other strains tested by Gaslewlcz et al. (1983a,b). These authors also noted that this strain of mice accumulated 2,3,7,8-TCDD 1n adipose tissue more strongly than other strains and that this phenomenon probably resulted 1n slowed, body elimina tion. Half-Hves for body elimination of 2,3,7,8-TCDD have not been calcu lated for the monkey, but 1t was suggested that the tendency of this species to accumulate 2,3,7,8-TCDD 1n body fat may well result In slowed body elimi nation (Van Miller et al., 1976). Recently, Olson and Bittner (1983) examined the elimination of 2,3,7,8TCDD In rats over a longer period than the studies previously summarized and determined that a blphaslc elimination occurred. They suggested a half-11fe of -7 days for the Initial rapid phase and a half-life of -75 days for the slower phase, probablv related to release from stores of body fat. McNulty et al. (1982) estimated the half-Hfe for elimination from the fat of mon keys to be -1, year. 01310 II1-24 09/18/84 The fecal route seems to be the major pathway for the elimination of 2,3,7,8-TCDD-deMved radioactivity from rats {Rose et al., 1976; Piper et al., 1973; Allen et al., 1975; Van Hiller et al., 1976), guinea pigs . {Gas1ew1cz and Neal, 1979) and mice {Gaslewlcz et al., 1983a,b). Urinary excretion played less of a role 1n these species, accounting for <l-28?4 of total excreted radioactivity while fecal excretion accounted for 72->99% of the eliminated radioactivity. Urinary excretion accounted for a more sub- stantlal proportion of body elimination 1n hamsters (41% compared to 59% by feces) {Olson et al., 1980a) and that strain of mice {DBA/2J) which prefer entially accumulated 2,3,7,8-TCDD 1n body fat {Gaslewlcz et al., 1983a,b). The failure to detect metabolites of 2,3,7,8-TCDD 1n Uver and fat {Olson et al., 1983) Indicates that elimination of the metabolites occurs rapidly and that the rate of-elimination 1s governed primarily by the rate of hepatic metabolism. 01310 II1-25 09/25/84 IV. HUMAN EXPOSURE Humans may be exposed to chemicals such as dioxin from a variety of sources, Including drinking water, food and ambient air, occupational settings and consumer products. This analysis of human exposure to dioxin 1s limited to drinking water, food and ambient air because those media are considered to be sources common to all: Individuals. Even 1n limiting the analysis to these three sources, 1t must, be recognized that Individual expo sure will vary widely based on many personal choices and on several factors over which there 1s little control. Where one lives, works and travels, what one eats, and physiologic characteristics related to age, sex and health status can all profoundly affect dally exposure and Intake. Individ uals living 1n the same neighborhood or even 1n the same household can experience vastly different exposure patterns. . . In the Exposure Estimation Section of this chapter, available Informa tion 1s presented on the range of levels of human exposure and Intake for dioxin from drinking water, food and ambient air for the 70 kg adult male. It 1s not possible to provide an estimate of the number of Individuals experiencing specific combinations of Intake from those three sources. However, the Summary Section of this chapter provides some Insight Into the relative contributions of the three sources, especially drinking water, for the range of Intake values suggested by the available data. Exposure Estimation Drinking Water. No Information was found 1n either the Federal surveys or state data on measured levels of dioxins 1n drinking water from community water supplies. 01320 IV-1 01/16/85 Diet. Limited Information was obtained on the occurrence of dioxins 1n food. Dioxins are not analyzed for 1n the Food and Drug Administration's Market Basket Studies. However, a few studies address the occurrence of dioxin [In particular, the occurrence of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (2,3,7,8-TCDD)] In specific foods. The Ambient Water Quality Criteria Document for 2,3,7,8-tetrachloro- d1benzo-p-d1ox1n (U.S. EPA, 1984) reported studies conducted by others for dioxin (2,3,7,8-TCDD) determinations In food. In a study by Jensen et al. (1983), rice grain from fields In Arkansas, Louisiana and Texas were anal yzed after the application of 2,4,5-T (containing 0.4 mg/kg 2,3,7,8-TCDD). None of the grain analyzed contained 2,3,7,8-TCDD (detection limit = 2-10 ng/kg), nor was any found In 30 samples of rice bought 1n retail stores throughout the United States.- Other studies on the contamination of fruits, vegetables or grains 1n the U.S. with 2,3,7,8-TCDD have apparently not been conducted (U.S. EPA, 1984). . Levels of 2,3,7,8-TCDD In the fat of cattle that grazed on pasture treated with 2,4,5-T ranged from 4-70 ng/kg In one study (U.S. EPA, 1980). In another Investigation by Kocher et al. (1978), levels were reported as undetected (detection limit = 1 ng/kg). The number of samples were not reported for either study. Bumb et al. (1980) analyzed charcoal-broiled steak to determine 1f 2,3,7,8-TCDD was formed due to the broiling process. TCDD was not detected (detection limit = 1-10 ng/kg). 01320 IV--2 01/16/85 2,3,7,8-TCDD has been detected 1n many species of commercial and non commercial fish 1n several rivers and lakes In the United States (U.S. EPA, 1984). Levels ranged from 1-695 ng/kg (Including all sites and types of fish studied). The maximum dally Intake of 2,3,7,8-TCDO was estimated by Cordle (1983) for residents of the Great Lakes region who regularly consume fish from the Great Lakes. The Intake ranged from 0.39-8.4 ng/day. Bovine milk surveillance samples have been analyzed for 2,3,7,8-TCDD (detection limit = 1 ng/kg) by several Investigators after normal applica tion of 2,4,5-T on pasture In Oklahoma, Arkansas and Missouri, as well as from quarantined milk samples In Michigan (U.S. EPA, 1984). 2,3,7,8-TCDO was undetected 1n all samples. ; A study of 103 samples of. breast milk from mothers living In sprayed areas of the United States (U.S. EPA, 1980) reported no detection of 2,3,7,8-TCDO (detection limit * 1-4 ng/kg). About six of nine human milk samples analyzed by Langhorst and Shadoff (1980) may have contained 2,3,7,8TCDD at levels slightly higher than the detection limits. (0.2-0.7 ng/kg). However, due to lack of Information on the accuracy of the data, 1t was concluded by those authors that 2,3,7,8-TCDD was not present. The available studies on the occurrence of dioxins 1n food do not repre sent the food groups that comprise an average diet. Since no Information, was obtained on most food groups, It was Impossible to calculate dietary Intake. Consequently, the contribution of dietary Intake to total exposure of dioxins could not be assessed. 01320 IV--3 01/16/85 Air. Information on Inhalation exposure to dioxins 1s limited. Dioxins usually exhibit very low vapor pressures and, therefore, tend not to be found In ambient air (Cleverly, 1984). No data on Inhalation exposure of dioxins 1s currently available, however, some other studies as reported 1n U.S. EPA (1984) are cited below. Nash and Beall (1980) reported very low air levels of 2,3,7,8-TCDD following the application of emulsified and granular Sllvex. Levels were reported to range between 0.09x10" ng/m3 and 7.98xl0"2 ng/m3 . A1r filter samples collected after an Industrial fire were analyzed for 2.3.7.8- TCDD by Harvan et al. (1981). ` Of nine samples analyzed, one contained 0.02 ng/m3 of 2,3,7,8-TCDD, four contained <0.009 ng/m3 of 2.3.7.8- TCDD and four (as- reported 1n U.S. EPA, 1984) "probably" contained 0.005-0.012 ng/m3 of unspecified TCDD Isomer (not the 2,3,7,8-TCDD Isomer). A1r levels near two hazardous waste sites have been monitored. 2,3,7,8TCDD was not detected near Love Canal at a detection limit of 1-20 ng/kg (U.S. EPA, 1982). However, at a disposal site near Jacksonville, Arkansas, Thibodeaux (1983) reported an average concentration of 1100 ng/kg of 2.3.7.8- TCDD In two air particulate samples. _ Atmospheric emissions from municipal Incinerators and from certain fires are other sources of polychlorinated dioxins. Quantities emitted may vary due to differing Incinerator efficiencies, and the quantity measured depends on the capability to reliably characterize such emissions. Bumb et al. 01320 IV-4 01/16/85 (1980) and Baser and Rappe (1980) detected 0.4 ng/g of 2,3,7,8-TCDD In fly ash from a United States municipal Incinerator (location not given 1n U.S. EPA, 1984). . .i At an Industrial boiler 1n the U.S. where pentachlorophenol was known to have been burned (location not given), Rappe et al. (1983) reported -5 mg/kg of polychlorinated d1benzo-p-d1ox1ns (PCDDs) 1n the baghouse and bottom ash.Among the many Isomers found, only a small' amout of 2,3,7,8-TCDD could be quantified (quantification not reported 1n U.S. EPA, 1984). Soot samples from'a transformer fire 1n Binghamton, NY were analyzed and revealed that 2,3,7,8-TCDD and 1,2,3,7,8-pentachlorod1benzod1ox1n (1,2,3,7,8-penta-CDD) were the predominate Isomers of the PCDDs formed (Buser and Rappe, 1983; Rappe-et al., 1983). 2,3,7,8-TCDD was quantified at 0.6 mg/kg, 1,2,3,7,8-penta-CDD was not quantified. Analyses of wipe tests from a garage adjacent to this accident site showed the presence of PCDDs prior to the cleaning of the garage (Tlernan et al., 1982; Tlernan, 1983). Summary . .. Sufficient Information was not available to assess the extent of human exposure to dioxins or to determine the relative source contribution to total exposure by the three common media (drinking water, food and ambient air). 01320 IV--5 01/16/85 References Bumb, R.R., W.B. Grummet, S.S. Cutle, et al. 1980. Trace chemistries of fire: A source of chlorinated dioxins. Science. 210: 385. (Cited 1n U.S,. EPA, 1984) . Buser, H.R. and C. Rappe. 1980. High resolution gas chromatography of the 22 tetrachlorod1benzo-p-d1ox1n Isomers. Anal. Chem. 52: 2257-2262. (Cited 1n U.S. EPA, 1984) Buser, H.R. and C. Rappe. 1983. Isomer-speclf1c separation and analysis of 2,3,7,8-substltuted polychlorinated d1benzo-p-d1ox1ns (PCDDs) using high resolution gas chromatography and mass spectrometry. Anal. Chem. (In review). (Cited 1n U.S. EPA, .1984) . .. Cleverly, D. 1984. Personal communication between Dave Cleverly, Environ mental Scientist, Strategy and A1r Standards Division, Office of A1r Planning and Standards, U.S. EPA, Research Triangle Park, NC and Denis Borum, 3RB Associates. September 6, 1984. Cordle, F. 1983. Use of epidemiology 1n. the regulation of dioxins 1n the food supply. In: Accidental Exposure to Dioxins: Human Health Aspects, F. Coulston and F. Pocchlarl, Ed. Academic Press, NY. p.' 245-256. (Cited 1n U.S. EPA, 1984) '; . Harvan, D.J., J.R. Hass, J.L. Schroeder and B.J. Corbett. 1981. Detection of tetrachlorod1ber.zod1ox1ns 1n air filter samples. Anal. Chem. 53(12): 1755-1759. (Cited 1n U.S. EPA, 1984) 01320 IV--6 01/16/85 Jensen, O.A., M.E. Getzendaner, R.A. Hummel and J. Turley. 1983. Residue studies for (2,4,5-trlchlorophenoxy) acetic acid and 2,3,7,8-tetrachlorod1benzo-p-d1ox1n 1n grass and rice. J. Agrlc. Food Chem. 31: 118-122. (Cited 1n U.S. EPA, 1984) Kocher, C.H., N.H. Hahle, R.A. Hummel, L.A. Shadoff and M.E. Getzendaner. 1978. A search for the presence of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n 1n . beef fat. Bull. Environ. Contam: Toxicol. 19:. 229-236. (Cited 1n U.S. EPA, 1984) Langhorst, M.L. and L.A. Shadoff. 1980. Determination of part-per-tr1ll1on. concentrations of tetra-, hexa-, hepta-, and octachlorcd1benzo-p-d1ox1ns 1n human milk samples. Anal. Chem. 52: 2037-2044. (Cited 1n U.S. EPA, 1984) Nash, R.G. and. M.L. Beall, Jr. 1980. Distribution of sllvex, 2,4-D and TCDD applied to turf 1n chambers and field plots. J. Agrlc. Food Chem. 28: 614-623. (Cited 1n U.S. EPA, 1984) Rappe, C., S. Marklund, P.A. Bergqvlst and P. Hansson. 1983. Polychlori nated dioxins, dlbenzofurans and other polychlorinated polynuclear aromatics formed during Incinerator and PCS fires. n: Chlorinated Dioxins and Dlbenzofurans In the Total Environment, Vol. I, L.H. Keith et al., Ed. Butterworth Publishers, Hoolburn, MA. (Cited 1n U.S. EPA, 1984) Thibodeaux, L. 1983. Offsite transport of 2,3,7,8-tetrachlorod1benzo-pdloxln from a production disposal facility. In: Chlorinated Dioxins and Dlbenzofurans 1n the Total Environment, L. Keith, Ed. Butterworth Publishers, Woolburn, MA. p. 1-14. (Cited In U.S. EPA, 1984) 01320 IV--7 01/16/85 Tiernan, T.O. 1983. Analytical chemistry of polychlorinated dlbenzo-p- dloxlns and dlbenzofurans: A review of the current status. In.: Chlorinated Dioxins and Dlbenzofurans In the Total Environment, Vol. I, L.H. Keith et al.f Ed. Butterworth Publishers, Woolburn, HA. p. 211-237. (Cited In U.S. EPA, 1984) . Tiernan, r.O., M.L. Taylor, J.G. Solch, G.F. Van Ness and J.H. Garrett. . 1982. Characterization of toxic components 1n the effluents from a refuse- fired Incinerator. Res. Conserv. 9: 343-354. (Cited In U.S. EPA, 1984) U.S. EPA. '1380. Dioxins. Industrial Environmental Research Lab., U.S. EPA, Cincinnati, OH. EPA 600/2-80-197. (Cited In U.S. EPA, 1984) U.S. EPA. 1982. Environmental. Monitoring at Love Canal, Vol.. I. Office of Research and Development, U.S. EPA. EPA 600/4-82-030a. U.S. EPA. 1984. Ambient Water Quality Criteria for 2,3,7,8-Tetrachlorod1benzo-p-d1ox1n. Prepared by the Office of Water Regulations and Stan dards, Criteria and Standards Division, U.S. EPA, Washington, DC. EPA 440/5-84-007. 01320 IV--8 01/16/85 V. 'HEALTH EFFECTS IN ANIMALS Exper1mF'">.al Animals Acu':e Toxicity. . Lethal Effects -- There have been studies In a variety of species defining the doses necessary to cause death after acute exposure to 2,3,7,8TCDD. A summary of the single dose L D ^ data for 2,3,7,8-TCDD Is present ed 1n Table V-l. The dose that results 1n death varies extensively with species, with the male guinea pig the most sensitive`species tested (L D ^ of 0.6 pg/kg) (Schwetz et al., 1973), and the male hamster the least sensitive species tested (LD^q of 5051 pg/kg) (Henck et al., 1981). The rat and monkey appear to-be the second most sensitive species, with LDggS between 22 and 70 pg/kg (Schwetz et al., 1973; McConnell et al.,_ 1978a), while other species tested (rabbit and mouse) had LDggS between 114 and 283 pg/kg (Sc'nwetz et al.., ..1973; McConnell et al., 1978b; Vos et al., 1974). Schwetz et al. (1973) found male rats more sensitive to 2,3,7,8TCDD, 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 regard 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 00110 TABLE V - l Lethal Oases of 2,3,7,0-TCDD Following Acute Exposure Specles/Straln Sex/No./Group " ` Route/ Vehlcle Dose Tested (ug/kg) Duration of Observation Guinea pigs/ Hartley Guinea pigs/ Hartley Guinea pigs/ Hartley H/NR H/HR H/9 gavage/corn' oll-cione (9:1) gavage/corn oll-acetone (9:1) gavage/ corn ol HR - 2 -8 weeks NR 2 -8 weeks HR 30 days Guinea pigs/ Hartley F/ 6 Guinea pigs/ Hartley F/6 Rats/ Sherman Rats/ Sherman Rats/SpragueDawley Rats/SpragueOawley M/S-10 F/HR H/6 F/6 gavage/ corn ol gavage/ methyl cellulose gavage/corn oll-acetone (9:1) gavage/corn oll-acetone (9:1) l.p./olive otl l.p./olive ol 0 .1 O.S 2.5 12.5 20.0 0 .1 0.5 2.5 12.5 20.0 a 16 32 63 HR HR HR 42 days 12 days 2 -8 weeks 2 -8 weeks 20 days 20 days LO50 (ng/kg) Coiments Reference 0 .6 (0.4-0.9)* Time to' death was 5-34 days, the 2,3,7,8-TCOD was 91X pure Schwetx et al., 1973 2 .1 (1.5-3)* Time to death was 9-42 days, the 2,3,7,8-TCDO was 99X pure Schwetz et al., 1973 2 j 2.5 (1.2-5.4. 9SX confidence) Median 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 |ig/kg group, with SOX mortality by day 42 McConnell et al., 1978b Sllkworth et al., 1982 19 . (15-23, 95X confidence) Time to first death was 12 days In the 20.0 iig/kit group, with 67X mortality by day 42 Sllkworth et al., 1982 22 Time to death was 9-27 days, the Schwetz et al., 2,3,7,8-TCDO was 91X pure 1973 45 (30-66)* 60 25 Time to death was 13-43 days, the 2,3,7,8-TCGD was 91X pure LD50 (ug/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 ng/kg Schwetz et al., 1973 Beatty et al., 1978 Beatty et al., 1978 V-2 08/11/84 I TABLE V-1 (cont.) OOTIO V-3 08/11/84 Spectes/Straln Sex/No./Group Route/ Vehicle Dose Tested (wg/kg) Duration of Observation Honkey/rhesus H1ce/C57B1 F/3 H/14 H1ce/C57B1 H/9 gavage/ corn oil gavage/corn oil-acetone (9:11 0 70 350 0 100 150 200 gavage/ corn oil NR >35 days 60 days 30 days LD50 (ng/kg) <70 114 283.7 1 M1ce/C578i/10 H/5 M1ce/C57B1/10 F/5 H1ce/C57B1/6J H1ce/D8A/2J mce/B6D2Fi/J H/NR H/NR H/NR gauge/ arachli oil gauge/ arachls nil l.p./olive oil l.p./olive oil l.p./olive oil 85 107 135 170 213 05 107 135 170 213 269 338 426 536 NR NR NR 45 days 45 days 30 days 30 days 30 days 146 >450 132 620 .300 Connients 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 25X 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 95X confidence limits of 111-211 yg/kg. Most 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 yg/kg Smith et al.. 1981 BG02Fi/J mice are the offspring of C57B1/6J and DBA/2J. The 0GD2]/J mice are heteroxygous at the Ah locus. No ccHnient 6as1 ew1cz et al., 1983a,b 6as1 ew1cz et al., 1983a,b Gaslewlcz et al., 19B3a,b TADLE V-l (coni.) 00110 V-4 09^18/84 Specles/Straln Sex/No./Group Rabbits/ New Zealand Rabbits/ New Zealand MM/NR MAF/5 Rabbits/ New Zealand MM/NR Hamster/ golden Syrian M/6 Hamster/ golden Syrian MAF/5-6 Hamster/ golden Syrian H/5 Oogs/beagle H/2 Oogs/beagle F/2 Route/ Vehicle Dose Tested (ng/kgj gavage/corn oll-acetone (9:1) i.p./ corn oil dermal/ acetone gavage/corn oll-acetone (9:1) I.p./ olive oil gavage/ olive oil gavage/corn oll-acetone (9:1) gavage/corn oll-acetone (9:1) NR 32 63 126 2S2 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 Duration of Observation 2-0 weeks 4 weeks 3 weeks 55 days 50 days 50 days 2-8 weeks 2-0 weeks 1050 (vg/kg) 115 (30-345)* NR 275 (142-531)* 5051 (3076-18,407, 95X confidence) >3000 1157 NA NA Comments Time to death was 6-39 days, the 2,3,7,8-TCDD was 91X 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 Reference Schweiz et al., 1973 Schweiz et al., 1973 Schweiz et al., 1973 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 pg/kg group Henck et al., 1901 Significant, dose-related decrease In thymus weight starting at 500 pg/kg, only 2 deaths occurred out of 11 hamsters in the 3000 pg/kg group. ?eath generally occurred between 24 and 45 days, decrease In bw above 2000 pg/kg, proliferative ileitis with mild to severe inflammation All animals died Olson et al., 1900b Olson et al., 1900b Schweiz et al., 1973 All animals survived Schweiz et al., 1973 The number In parentheses appears to Indicate the range of lethal doses; however, the article did not specify what these nuijuiers represented. I.p. = Intraperitoneal; NR Not reported; NA Not applicable t 25 pg/kg, 6 weekly doses of 5 pg/kg, or 30 dally doses of 1 pg/kg were all the threshold dose for observing a decrease .1n body weight. Other endpoints, Including lethality, decrease 1n thymus weight, and a no effect level for body weight change 1n rats, mice and guinea pigs in general 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 103-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 LDjQ 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 in 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 pg/kg, the weight loss was blphaslc (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 some of the weight loss Induced by 2,3,7,8-TCDD; however, there was no protection from the lethal effects of 2,3,7,8-TCDD. In yet another study, Seefeld and Peterson (1983) suggest that a reduction 1n food Intake caused by 2,3,7,8-TCDD is 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 pg/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 2i3,7,8-TCDD groups. The mortality In- the 25 and 50 pg/kg groups was 33 and 75%, respectively, while 1n the corresponding pair-fed groups the mortality was 0 and 15%. The authors proposed a hypoth esis that 2,3,7,8-TCDD lowers a regulated level or "set-point" for body weight control 1n 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-TCDD. Groups of female Fischer 344 rats administered 2,3,7,8-TCDD (20 pg/kg) and main tained on low (3.5%), normal (26%) or high (55%) 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 In treated animals was 10-08 g less than that In the respective control rats. Dietary protein also had no effect on preventing or enhancing the 2,3,7,8-TCDD Induced thymic atrophy. Although weight loss and thymic atrophy were present 1n 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-TCDO (Grelg et al., 1973, Gupta et al., 1973). Hemorrhages were observed In a number of organs Including the adrenal gland, urinary bladder, GI 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 Uver. 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 2,3,7,8-TCOD, 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 1n the liver of guinea pigs treated with 2,3,7,8-TCOD. Groups consisting of 4-6 female Hartley guinea pigs were treated with 2,3,7,8-TCDD at doses of 0.0, 0.1, 0.5, 2.5, 12.5 or 20 jig/kg, and 1 male guinea pig each was treated with a 00110 V-7 08/11/84 dose of 0.1 or 0.5 pg/kg. The 2,3,7,8-TCDD was administered by gavage as an aqueous suspension 1n C.75J4 methyl cellulose and surviving animals were killed 42 days after treatment. A second group of guinea pigs (6 males and 6- 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 U v e r 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 hyalin-like cytoplasmic Inclusion bodies were observed. Even though there was no dose-response relationship for these Uver 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 LDgg for female guinea pigs was determined 1n this study, and reported by Sllkworth et al. (1982), to be 2.5 or 19 jig/kg bw depending on whether the compound was administered by gavage 1n 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 1n the liver, as compared with guinea pigs. An early report by 8uu-Ho1 et al. (1972) described alterations 1n the architecture of the Uv e r of rats within 5 days of receiving a low dose of 2,3,7,8-TCDD (10 pg/kg by 1.p. Injection). At higher oral doses of 100 or 50 pg/kg, which killed 43 00110 V-8 09/18/84 and 7% of the animals, respectively, Gupta et al. (1973) also observed marked distortion of liver architecture in rats; however, only mild regener ative changes of the liver were observed at the sublethal dose of 5 pg/kg administered weekly for 6 weeks. Liver toxicity appeared to develop slowly 1n the rat with no change 1n 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 pg/kg (Grelg et al., 1973). Bilirubin levels were, however, markedly elevated from 0.33 pg/100 ma, 1n control animals to 10.97 pg/100 ma. 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-treatment). 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 11p1d 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 ra'ts that died from 2,3,7,8-TCDD exposure, there were extensive hemorrhages of the heart, liver, 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 al., 1974). Death 1n mice was frequently attributed to terminal hermorrhages (Vos et al.. 1974). In monkeys exposed to lethal levals of 2,3,7,8-TCDD, McConnell et al. (1978a) reported clinical and histologic signs of .oxldty, some of which were similar to those already described for other species. Severe thymic atrophy and edema occurred 1n treated animals, as well as extensive weight loss that could account for up to 38% of the body mass. As 1n guinea pigs, liver 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 summarized the toxic response of various species to 2,3,7,8-TCDO (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 LD50 for nine congeners of PCDD following a single treatment by gavage' In mice and guinea pigs. A comparison of the LDg0 expressed as pmol/kg body weight Is presented In Table V-3. The limited data suggest that con geners containing chlorine 1n 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 Cowb Rat Mouse Rabb1tb Ch1ckenb Hamster Pig Hyperplasia and/or metaplasia Gastric mucus Intestinal mucosa Urinary tract B11e duct and/or gall bladder Lung: focal alveolar - Skin Hypoplasia, Atrophy or Necrosis Thymus Bone marrow Testicle Other Liver lesions Porphyria Edema f ft ft t* + + 0 1- . 0 If 0 0 + + % + 0 0 + i+ *, *d 0 0 ++ 0 0 0 ff 0 ++ * + 0+ ++ 0 ff 0 0 *+ f + +t +0 ff f References: Monkey (McConnell et al., 1978b; Norback and Allen, 1973; Allen et al., 1977); guinea pig (McConnell et al., 1978b; McConnell, 1900; Moore et al., 1979; Turner and Collins, 1903); cow (McConnell, 1980); rat (McConnell, 1900; Koclba et aT., 1970a,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). ' Responses followed exposure to 2,3,7,8-TCDD or structurally related chlorinated aromatic hydrocarbons. ^Symbols: 0, lesion not observed; +, lesion observed (number of denote severity); , lesion observed to a very limited extent; blank, no evidence reported 1n literature. ^Skln lesions In cattle are observed, but they differ from the skin lesions observed 1n other species. Adapted from Poland and Knutson, 1902 , 09/18/84 TABLE V-3 Estimated Single Oral LDso_30 Values for PCDOs3 Chlorination of PCDOs 2.8 2.3,7 2.3,7,8 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 Guinea Pigs (pmol/kg)b >1180 120.41 0.006 0.009 3.15 0.185 0.178-0.255c 0.153-0.255c >1.400 aSource: Adapted from McConnell et al., 1978b bSpearman-Karber method cEst1mated range due to variability In replicates MR = Mot reported ' Mice (pmol/kg)b NR >10 y 0.88 0.94 >14 2.11 3.19 . >3.67 NR 00110 V-12 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 1n lethality. Although the congeners vary 1n effective dose between mice and guinea pigs, the relative order of toxicity of these congeners did not change. Also, similar effects of toxicity were observed for all congeners as described above for 2,3,7,8-TCDD when the comparison was made within a single species. Effects on the Liver -- The histological and ultrastructural changes 1n the Uver Induced by oral exposure to 2,3,7,8-TCDD 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-TCDD at 0.0, 5 and 25 pg/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 canallcuH. 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 roigh 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 1.p. administration of 20 pg/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 1n 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 multinucleated 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 zon-s 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 Mg++-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 Mg++-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-TCDD on ATPase activity was not the result of 2,3,7,8-TCDD Induced food deprivation and ljn vitro studies Indicated that the loss of activity was ;not due to the direct Interference of 2,3,7,8-TCDD 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 1.p. Injection of 2,3,7,8-TCDD, Indicating that exposure was actually affecting membrane components (8rewster et al., 1982). Peterson et al. (1979a) did. observe a positive correlation between.the levels of LSM ATPase activity and both 1_n vivo cumulative biliary excretion of ouabain and bile flew (pi/mln/g Uver). Using perfused Uver, 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-16<*-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 excretldn In male CD rats given a single dose of 2,3,7,8-TCDD at 25 or 5 pg/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-l 5 09/18/84 Inverse relationship between 2,3,7,8-TCDD exposure and bile flow, with maximum bile flow observed in the 25 pg/kg dose group at 16 days. Even with this Increased bile flow, however, the cumulative biliary excretion of ICG was decreased 1n a dose-dependent manner with the greatest depression observed 7 and 16 days after the exposure to 2,3,7,8-TCDD. The levels of ICG 1n the plasma and Uver was higher 1n treated animals than In 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-TCDD on the biliary excretion of the organic neutral compound, ouabain, with that of the organic anions phenol-3,6-d1bromophthale1n (DBSP) and sulfobromophthaleln (BSP) In male Holtzman rats. Animals were Intubated with 2,3,7,8-TCDD at doses of 10 or 25 ng/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 1n the high dose group. In the low dose animals there was actually an Increase at days 10 and 25 1n 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, In 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 1n 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 2,3,7,8TGDD-1nduced liver damage similar to the rat, there was reduced blood clear ance of ICG (Seefeld et a!., 1979, 1980). 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 Uver 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 Uver damage were elevated, the :lilstopathology of the Uver was within normal limits. It appears that major effects on biliary excre tion occur only In species tha 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 1n .In, vivo (1 hour pulse) Incorporation of. 3H 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 10 pg/kg followed 1n either 3 or 7 days by the assessment of I1p1d synthesis. At 3 days Incorporation decreased from 258 to 98 dpm/mg Upld 1n 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 1n the synthesis of triglycerides, dlglycerldes 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 lipids 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 In the content of phospholipids, free cholesterol or triglycerides. The fatty changes In the U v e r 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 1n the lipid profile of the liver was attributed to 2,3,7,8-TCDD Induced mobilization of body fat, a decrease 1n lysosomal acid Upa s e (74% decline In this enzyme 10 days after a 50 pg/kg dose of 2,3,7,8-TCDD) and an Increase In U p l d peroxidation as Indicated by a sharp Increase 1n the production of Upofuscln pigments. Porphyria was Initially characterized quantitatively 1n mice by Goldstein et al. (1978). Groups of 12 male C57B1 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 liver 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) 1n 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 In hepatic porphyrins 1n the two high dose groups 3 weeks after a single exposure to 2,3,7,8-TCDD at 0.0, 5, 15, 50 or 75 pg/kg, while only minimal nondose-related changes 1n hepatic porphyrin were observed 1n DBA/2 mice exposed to up to 1200 pg/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-TCDD 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 pg/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-TCDD (Canton1 et al., 1981). Female CD rats were orally administered a weekly dose of 2,3,7,8-TCDD at levels of 0.01, 0.1 and 1.0 pg/kg for 45 weeks. The Initial Increase was observed 1n the high dose group at 3 months, and 1n 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 2,3,7,8-TCDD Induced por phyria, the effects of 2,3,7,8-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-l1m1t1ng enzyme In porphyrin synthesis, was slightly Increased (2-fold) 1n male C57B1 mice 00110 V-19 09/18/84 given 4 weekly doses of 2,3,7,8-TCDD at 25 pg/kg. This dose of 2,3,7,8TCOO Increased Uver 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. (1981) reported a decrease 1n UD activity from -25-7 nmoles/hr/g U v e r 1n male and female C5781 mice 3 weeks after a single oral exposure to 2,3,7,8-TCDD at a dose of 75 pg/kg. No effect of 2,3,7,8-TCDD on UD activity was observed 1n DBA/2 mice which were Insensitive to the Induction of porphyria. A time course of changes 1n 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% 1n DBA/2 mice. .Other factors besides the Increase In -amino levulinic acid synthetase and the decrease 1n UD activity may also partici pate 1n the dramatic Increase 1n Uver porphyrin 1n mice associated with exposure to hear lethal doses cf ?,2,7,8-TCDQ. 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-TCDD, Investigations have been conducted to determine the Influence of 2,3,7,8-TCDD on DNA synthesis 1n the Uver. Grelg et al. (1974) measured the in vivo Incorporation of ^ - t h y midine (1 hour pulse) Into U v e r 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 Dh'A synthesis was measured 24 hours after the operation. i Although 2,3,7,8-TCDD had no effect on in vivo DNA synthesis, similar studies by Conway and Hatsumura (1975) and Dickens et al. (1981) demonstrat ed an Increase In thymidine Incorporation when determined in vitro. Conway and Hatsumura (1975) administered male Sprague-Dawley rats 2,3,7,8-TCDD at a dose of 5 yg/kg followed 1n 10 days by removal of the liver and the 1n vitro determination of DNA synthesis 1n liver slices. Incorporation of thymidine Into the nuclei Increased from 29 cpm/mg 1n control animals, to 45 cpm/mg In 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 Uver slices was Increased 10-fold In 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 1n both 2,3,7,8-TCDD treated and control animals; however, the treated animals had a more rapid and extensive Increase In 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 1n both the treated and control animals was predomi nantly sem1conservat1ve. Further studies are needed to determine the reason for the difference observed between In. vitro and in. vivo measurements of DNA synthesis In the Uver after exposure to 2,3,7,8 TCDD. 00110 V--21 08/11/84 Extensive hepatic necrosis 1n the rabbit may be responsible for death 1n this species (Poland and Knutson, 1982). Besides the effects on the liver of 2,3,7,8-TCDD exposure described above, It Is known that 2,3,7,8-TCDD Is a potent Inducer of microsomal enzymes. These studies will be discussed 1n the Enzyme Induction by TCDD Section, which describes the ability of this xenoblotlc to Induce microsomal enzymes 1n 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 2,3,7,8-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 1n 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 pg/lig, D-glucose was absorbed to a lesser degree at all doses than In control animals. The two low doses produced a dose-related decrease In absorption; however, at doses of >75 pg/kg the decrease was uniform. At a dose of 150 pg/kg, decreased absorption of D-glucose 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 D-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-TCDD had no effect on the absorption of the other compounds Investi gated. In a similar experiment 1n Sprague-Dawley rats, Ball and Chhabra (1981) also observed malabsorption of D-glucose. In this study, however, absorption of hleuc1ne 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-TCDD. ' In contrast to the results observed for i)-glucose, Intestinal Iron transport was shown to be elevated by exposure to 2,3,7,8-TCDD. Manls and K1m (1979a) examined the effect of prior treatment of male Sprague-Dawley rats on the 30-m1nute transport.of S9Fe out of a duodenal loop created by ligating a section of the Intestine In. s1tu. At single 2,3,7,8-TCDD doses of between 22 and 84 pg/kg there was Increased serosal transfer of s*Fe measured 48 hours after .treatment. At doses >42 pg/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 l.p. Injection. In similar experiments calcium transport was decreased, and galactose and prollne transport were unaffected by prior exposure to 2,3,7,8-TCDD. 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-TCDD exposure observed 1n the adjacent distal segment of the Intestine. Increased Iron transport was also observed by Hanls and K1m (1979a) 1n an unidentified strain of mice. Increased 1ror port may be one of the earliest effects of 2,3,7,8-TCDD; however, at present the toxico logic relevance of this transient disturbance 1n Iron transport 1s unknown. 00110 V-23 09/18/34 One of the common gross observations of 2,3,7,8-TCDD toxicity 1s severe edema, suggestive of a breakdown 1n salt and water homeostasis. These observations prompted Investigations to determine the effect of 2,3,7,8-TCDD on the function of the kidney. Pegg et al. (1976) measured renal function IE vltro using renal cortical slices obtained from male Sprague-Dawley rats 3 and 7 days after Intubation with 2,3,7,8-TCDD 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 M-methyln1cot1nam1de Into the cortical slices. Anion accumulation was lower In the high dose group, uhlle cation transport was lower at both dose levels tested. The decrease In anion transport was confirmed In an In, vivo study. AmmonlogenesIs and gluconeogenesls were not affected In 2,3,7,8-TCDD treated rats, even when the animals were made acldotlc, Indicative of no effect on the kidneys' ability to maintain acid base balanc.Also, sodium reabsorption was shown In vivo to be within normal range. Since decreases 1n 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-TCDD were merely a general decrease In 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 only minimally affected by exposure to 2,3,7,8-TCDD, Grelg et al. (1974) demonstrated that pre-exposure to 2,3,7,8TCDD could reduce the ability of the rat kidney to respond to stimuli of DMA sythesls. Folate-stimulated DNA synthesis measured In. vivo in Porten strain rats was decreased between 67 and 25% 1n animals receiving 2,3,7,8-TCDD at a 00110 V-24 08/11/84 dose of 10 vg/kg on day 0-9 before administration of folic acid. No significant difference In folate-stimulated DNA 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-TCDD shortly after treatment with folic acid suggested that 2,3,7,8-TCDD did not directly Interact with cellular DNA, nor Inhibit the protein synthesis necessary to support folate-stimulated DNA synthesis. Similar Inhibitory effects of 2,3,7,8-TCDD were observed when lead acetate was used to stimulate kidney DNA synthesis. The mechanism by which 2,3,7,8-TCDD prevents the kidney from responding to proliferative stimuli 1s not known, although 1t was demonstrated that another agent capable of Inducing microsomal enzymes, 3-methylcholanthrene (3-MC), had similar effects on the kidney. Additionally a number a hematologic and clinical chemistry changes have been observed In the blood of laboratory animals after exposure to 2,3,7,8-. TCDO. Many of these changes, as described by Zlnklet al. (1973), reflect damage to previously described organ systems. In female CD rats given 30 dally doses of 2,3,7,8-TCDD at levels of 0.1, 1.0 or 10 pg/kg, the clini cal chemistry of the serum reflected U v e r 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 1n the high dose animals start ing 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 1n the high-dose animals. Along with these clinical chemistry changes Indica tive of U v e r 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 the 10 and 1 Pg/kg groups, while 1n the 0.1 Pg/kg group a signifi cant decrease was not observed until day 17. Thrombocytopenia was also observed 1n female guinea pigs after 8 weekly oral doses of 2,3,7.8-TCDD at 0.2 Pg/kg, and 1n mice (administered a single dose of 1.0, 10 or 50 pg/kg). In guinea pigs lymphopenia was also observed. Other hematologic changes were attributed to hemoconcentratlon. In a more extensive Investigation of 2,3,7,8-TCDD-1nduced hyperlipidemia In 11,316 Sprague-Dawley rats. Poll et al. (1980) treated animals with a single 1.p. Injection of 2,3,7,8-TCDD at 2 doses of 2.5, 5, 10 and 20 pg/kg. At day 21 after treatment there was adose-related Increase 1n total plasma cholesterol and high density lipoprotein cholesterol, while no change was observed 1n triglycerides or very low and low density lipopro teins (VLOL and LDL, respectively). At a dose of 20 Pg/kg the maximum Increase 1n .H0L 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 1n 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). v-26 08/11/84 / In contrast to rats, male Hartley strain guinea pigs given a single l.p. Injection of 2,3,7,8-TCDD at a dose of 2 jjg/kg had Increased hyperlipid emia characterized by Increases In VLDL and LDL (Swift et al., 1981). In animals sacrificed 7 days after exposure to 2,3,7,8-TCDD, there was an Increase In total serum I1p1d, 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 qualitative changes occurred,reflecting an Increase In the types of fatty acids that were abundant In the adipose tissue of guinea pigs. Anaylsls of lipoproteins revealed a 19-fold Increase In VLDL and a 4-fold Increase In LDL, with no change observed In the levels of HDL. The VLDL was also qualitatively different In the 2,3,7,8-TCDD treated animals, containing less cholesterol ester and an altered C apoprotein. The Importance of these qualitative changes 1s 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 In serum lipid levels to the mechanism of 2.3.7.8- TCDD toxicity needs further study. Elovaara et al. (1977) observed some changes 1n biochemical: of the brain of male Wlstar and heterozygous Sunn rats given a single Intubation of 2.3.7.8- TCDD at a dose of 20 yg/kg. At 7 days post-treatment, there was a small but significant decrease as compared with vehicle treated control animals In both the protein and RNA content of the Wlstar rats, while levels of acid proteinase and DT-d1apnorase (an enzyme Induced by 2,3,7,,8-TCDD In the Uver) had a small but significant Increase 1n the heterozygous Gunn rats. There were no significant changes observed 1n homozygous rats given 00110 V-27 08/11/84 2,3,7,8-TCDD at 20 pg/kg. The authors noted that acid proteinase may participate In chemically Induced degeneration of the brain. Immunological Effects -- During acute toxicity studies with 2,3,7,8TCDD, thymic atrophy was noted as a consistent effect 1n all species that have been Investigated. This finding suggested that 2,3,7,8-TCDD may alter the Immune response, and Initiated 1mmunotox1c1ty studies 1n exposed animals. In guinea pigs treated with 8 weekly oral doses of 2,3,7,8-TCDD (0, C.008, 0.04, 0.2 or 1.0 pg/kg bw), 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 of 10 pg/kg 2,3,7,8-TCDD (Helssberg and Z1nkl, 1973). Increased red blood cell count, decreased platelet count, Increased neutrophil count and Increased packed cell volumes were reported 1n 2,3,7,8-TCDD-exposed rats. _A summary of the data available on the 1mmunotox1c effects of 2,3,7,8-TCDD In animals 1s presented In Table V-4. A review of 1mmunotox1cUy and Immunosuppression was reported by Vos (1977). Vos et al. (1973) Investigated the humoral and cell-mediated Immune response In Hartley guinea pigs, CD rats and B6D2F^ mice. The humoral Itvfliune 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 TABLE V-4 Immunological Effects of 2,3,7,8-TCDD In Animals 00110 V--29 Species/ Strain Sex Exposure Route Dose(s) Duration of Exposure . Hlnlmum Effective Dose Parameter Effect Reference H1ce/D6D2F] H gavage H1ce/C57B1/6 f.K maternally administered (gavage) Nice/ C5TBl/6Jfh H gavage 0, 0.2, 1.0, 5.0, 4 weeks 25.0 |ig/kg bw/week NA 5.0 ug/kg bw/week 5.0 ug/kg bw/week 0, 1.0, 2.0, 5.0, 25.0 ug/kg 4 or 6 weeks (3 or 5 administrations) 1.0 |ig/kg bw/week 25.0 ug/kg bu/week 2.0 |ig/kg bw/week 0, 0.5, 1, 5, 10, 20 |ig/kg bw/week 4 weeks ' 1.0 |ig/kg bw/week j bw thymus weight graft-versushost response thymus weight PHA response skin graft rejection Salmonella Infection j1 1 Hlce/Swlss H gavage 0, 1.5, 5. 15, 50 |ig/kg hv/week 4 weeks H1ce/B(C3Fi f In vitro 0.5, 5.0, 50 ng/ml 5-60 seconds (spleen cells) Hlce/Swlss- F.H maternally Kebs ter administered (diet) 0. 1, 2.5, 5. 10. 20 ppb (dietary) 10 weeks (pre-gestation and 3 weeks post parturition) 1.5 ug/kg bw/week 50 |ig/ml 2.5 ppb 2.5 ppb 5 ppb 1 ppb Hlce/CB H gavage 0, 0.01, 0.1, 1.0, up to B weeks 10.0 |ig/kg bw/ueek NA . 0.01 ug/kg bw/week 1.0 |ig/kg bw/week Hlce/CD H In vitro 10"-10" H single 10 H endotoxin (E. coll) susceptibility protein, DNA, and RNA synthesis antigenic R8C reaction thymic cortex contact sensitivity to DNFB endotoxin (Salmonella! susceptibility Listeria Infection serum Immunoglobln level serum Immunoglobln level lymphocyte blaslogenlc transforma tion no change decreased decreased Vos et al., 1973 decreased decreased prolonged Vos and Hoore, 1974 Increased mortality and decreased time to death Thigpen et al., 1975 Increased Vos et al.. mortality 1976a decreased Luster et al., 1979a,b decreased atrophy decreased Thomas and lllnsdl 11, 1979 Increased mortality no change Increased decreased Sharma and Gehrlng, 1979 Increased Sharma and Gehrlng, 1979 09/18/84 TAOLE V-4 (cont.) . 00110 Species/ Strain Sex Exposure Route Oose(s) Hlce/Swlss- F oral (diet) Hebster 0, 10, 100 ppb Mice/ C57B1/6J H 1-P. 0. 1. 2, 6, 30 ug/kg bw Duration oF Exposure 5 weeks (or more) single Injection Minimum Effective Dose 10 ppb 10 ppb 10 ppb 10 ppb 10 ppb 1 ug/kg I H1ce/B6C3F] H.F aiaternally administered H1ce/C57Bl/6 H t.p. Htce/C57Bl/6 H t.p. Rat/C0 F oral Rat/CO F oral 0, 1.0, S.O, 1S.0 |ig/kg bu/day 4 days during gestation and lactation 1.0 ug/kg bw/day 1.0 ug/kg bw/day 5.0 ug/kg bw/day 0, 0.4, 4.0, 40 tig/kg bw/week 4 weeks - 0, 0.004, 0.04, 0.4 tig/kg bw/ueek 4 weeks 0, 0.2. 1.0, 5.0 ug/kg bw/ueek 6 weeks 4.0 ug/kg bw/ueek 0.4 ug/kg bw/week 0.004 ug/kg bw/week 5.0 ug/kg bw/week 5.0 ug/kg bw/week NA 0, 10 |ig/kg bw/day 10, 14 days 10 ug/kg bw/day 10 ug/kg bw/day 10 ug/kg bw/day Parameter tetanus response antigenic RDC response sensitization to DNFB resistance to Salmonella resistance In Listeria macrophage and natural killer cell activity macrophage and natural killer cell number antibody production L. monocvtoaenes susceptibility PYB6-tumor suscep tibility bone marrow hypocellulartty thymus atrophy cytotoxic T-cell response In vltro-qeneralion of cytotoxic T-cells bw thymus weight tuberculin hyper sensitivity erythrocyte count platelet count neutrophil count Effect Reference decreased Hlnsdlll, decreased et al.. 1980 decreased Increased mortality Increased mortality no change Hantovanl et al., 19B0 decreased decreased Increased Increased Increased Luster et al., I960 Increased Clark et al., decreased 19B1 decreased Clark et al., 1981 decreased decreased no change Vos et al., 1973 Increased decreased Increased Helssberg and Zlnkl, 1973 V-30 09/18/84 OO Species/ Sex Exposure Route O Strain Oose(s) Duration of Exposure Minimum . Effective Dose Parameter Effect Reference Rat/F-344 F.H maternally administered 0, 1.0, 5.0 yg/kg bw/dose 4 or 6 weeks (3 or 5 administrations) 1.0 yg/kg bw/dose 5.0 yg/kg bw/dose 5.0 yg/kg bw/dose 5.0 yg/kg bw/dose 5.0 yg/kg bw/dose NA 'bw and thymus ' weight spleen weight PIIA response graft-versus-host response skin graft rejection pseudorabies ` virus Infection decreased decreased decreased decreased Vos and Hoore, 1974 prolonged no change Rat/F1scher F.H maternally administered INR) NR 4-6 weeks (during ges- ` tatton and neonatally) NR NR Con A and PIIA response oxazolone skin hypersensitivity decreased decreased Hoore and Faith, 1976 Rat/FIscher- F.H Ulstar maternally administered INR) I CO 0. 5 yg/kg bw/dose 3 or 4 applications during gestation and neonatally 5 yg/kg bw/dose 5 yg/kg bw/dose 5 yg/kg bw/dose antibody production to bovine ganna globulin PIIA and Con A response thymus and bw no effect Faith and Luster, 1979 decreased decreased until 135 days Rat/Spraguc- H' l.v. Dauley 0. 1 ng/kg bw single Injection 1 yg/kg bw thymic RNA synthesis thymic RHA polymerase activity decreased decreased Kurl et al., 1902 Guinea pig/ F gavage Hartley 0. 0.00B, 0.04. 0.2, 1.0 yg/fcg bu 0 weeks 0.04 yg/kg bw/week 0.04 yg/kg bw/wcek 0.04 yg/kg bw/week 0.2 yg/kg bw/week bw thymus weight tuberculin hyper# sensitivity ' tetanus antitoxin decreased decreased decreased decreased Vos et al., 1973 H - male; F - female; l.p. - Intraperitoneal l.v. - intravenous; PUA - Phytohemagglutlntn; Con A - Conconavalln A; ABC - red blood cell; DNFB * 2.4-dlnltro O 1-fluorobenzene; NA Nol applicable; NR * Nol reported KD oo 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 1n hamsters treated with 0.04 g 2,3,7,8-TCDD/kg and higher doses. Decreased tetanus antitoxin levels were evident 1n guinea pigs treated with 0.2 pg 2,3,7,8-TCDD/kg, but not at lower dose levels. Vos et al. (1973) also tested the cellmediated Immunity 1n rats exposed to 2,3,7,8-TCDD (0, 0.2, 1.0 or 5.0 pg/kg, once weekly for 6 weeks). M. tuberculosis was Injected Into rats by day 28 of the treatment period, followed by Intradermal hypersensitivity testing on day 42. No changes 1n the thickness of skin were noted 1n 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 pg/kg once weekly for 4 weeks) of the C57B1/6 strain were Injected Into the right footpad of a hybrid recipient mouse (C57B1/6 x DBA-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 C57B1/6 antigens. In this test Vos et al. (1973) noted a significant (p<0.01) dose-related decrease 1n 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 i(p<0,01) 1n hybrid recipient mice receiving spleen cells from mice treated with 5 pg 2.3.7.8- TCDD/kg/week than from donor cells of untreated mice. 00110 V-32 08/11/84 Studies continued 1n an attempt to Identify the mechanism of 2,3,7,8- TCDD-1nduced Immunodeficiency. Rats (F-344) exposed, pre- and postnatally by maternal dosing (1 or 5 pg 2,3,7,8-TCDD/kg administered to dams on days 11 and 18 of gestation and 0, 7 a.d 14 postnatally) had prolonged times until 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 1n..iune response, was actually Increased. ' Since thymus-derived lymphocytes (T-cells) 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-TCDD on host resistance to Infection, a vital measure of Immune response, was tested by Thigpen et al. (1975) In male pathogen-f.ree-mlce (C57Bl/6Jfh). 2,3,7,8-TCDD was adminis tered to mice at 0.5, 1, 5, 10 or 20 pg/kg once .weekly for 4 weeks followed by Inoculation with Salmonella bern 2 days after the final 2,3,7,8TCDD administration. Mortality rates and "time until Infection" were used to determine the Immunological effect of 2,3,7,8-TCDD. A significant {p<0.05} Increase 1n mortality and decrease 1n time of Infection were noted 1n groups treated with 1 pg/kg or higher doses of 2,3,7,8-TCDD when compared with controls. 2.3.7,8-TCDD at 0.5 pg/kg did not alter these parameters and was regarded as a no effect level. The Immune-resistance of mice to S. bern 1s therefore reduced by treatment with 1 pg 2,3,7,8TCDD/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 . coll endotoxin (Vos et al., 1978a). Injection of 250 pg of endotoxin to mice pretreated with 0, 1.5, 5 and 15 pg 2,3,7,8-TCDD/kg resulted 1n 0/5, 1/5, 6/6 and 6/6 deaths, respectively. Mice pretreated with 15 and 50 pg 2,3,7,8-TCDD/kg andInjected with 10 pg 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 pg of endotoxin was administered, while 10 pg of endotoxin was sufficient to cause similar mortality (2/5) 1n mice treated with 50 pg 2,3,7,8-TCDD/kg. The Immunocomptence of 5-week-old offspring of Swlss-Uebster mice fed diets containing 1, 2.5, 5, 10 or 20 ppb 2,3,7,8-TCDD was tested by several means (Thomas and H1nsd1Tl, i979). The number of cells reactive to anti genic RBC, differential white blood cell counts, organ weights, hlstopathologles, hypersensitivity to 2,4-d1n1tro-l-fluorobenzene (DNFB) and the resistance to . coll lipopolysaccharide (LPS), Listeria monocytogenes and Salmonella typh1mur1um 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 postparturlt1on. Young mice being tested for 1mmunotox1c1ty were therefore exposed to 2.3.7.8-TCDD only in utero and through lactation. The typical decrease 1n thymus weight was noted 1n mice exposed to 2.5 and 5.0 ppb but was not evident 1n the 1.0 ppb group. A decrease 1n the number of plaque-forraing cells (PFC) reactive to sheep RBCs war significantly reduced 1n the 2.5 and 5.0 ppb 2,3,7,8-TCDQ-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 anti-RCD antibodies,however, was not lower In 2,3,7,8-TCDD-exposed groups when compared with controls. A decrease In the skin hypersensitivity to ONFB following sensitization was noted In all 2 ,3 ,7 ,8-TCOD-treated groups (only the 5 ppb group was statistically reduced from controls). 2,3,7,8TCDO caused an Increased susceptibility (Increased mortality level) to S. tvph1mur1um In a dose-related fashion. The response to E. coll. IPS and L. monocytogenes was not different from controls. 2,3,7,8-TCDD exposure did not alter the response of lymphocytes (Band T-cells) In vitro, to Con A, nor was mitogen-induced lymphocyte proliferation affected (Thomas and Hlnsdlll, 1979). ' Similar findings were reported In F1scher/W1star rats exposed to 2.3.7.8- TCDD during gestat1on~(T8th 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- TCDD-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 B6C3F1 mice, exposed to prenatal (maternal dosing on day 14 of gestation) and postnatal (days 1, 7 and 14 after birth) doses of 0, 1.0, 5.0 or 15.0 pg/kg 2,3,7,8-TCDD, were studied for Itnmunotoxlc effects and host susceptibility (Luster et al., 1980). At the 15.0 pg 2,3,7,8-TCDD/kg dose level, 70* of the neonates died with overt toxic effects (decreased body weight, liver weight, spleen weight and thymus weight). Bone marrow hypoce.lularlty and depressed macro'phages-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 In the 2,3,7,8-TCDD-exposed neonates. Death occurred In 73 and 40* of the L. monocytogenes Inoculated (1.2x10* viable organisms) mice In the 5.0 and 1.0 pg/kg dose groups, respectively, compared with 28* of controls. Tumor development occurred In 44, 60 and 22* of the neonates Inoculated with 5xl04 tumor cells from the 5.0 pg 2,3,7,8-TCDD/kg, 1.0 pg 2,3,7,8-TCDD/kg and control groups, respectively. Hlnsdlll et al. (1980) reported that 2,3,7,8-TCDD administered in the diet of 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 B-globul1ns. 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 1 tero and neonatally 1s not more crucial than In other periods. Vos and Moore (1974) had previously reported 00110 V-36 09/18/84 that 1-month-old mice were more sensitive to 2,3,7,8-TCDD than were 4-monthold mice (C57B1/6). Decreased body weight and thymus weight and spleen cell response to PHA were evident at lower doses In 1-month-old mice than 1n 4-month-old mice. The effect of single l.p. doses of 2,3,7,8-TCDD (1, 2, 6 and 30 pg/kg) on peritoneal macrophage and splenic natural killer cell function 1n 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-TCDD-treated animals, however, was reduced when compared with untreated controls. Harked hypocellularlty noted In the bone marrow of 2,3,7,8-TCDD-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-TCDD-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 (Grelg et al., 1973). The actual mechanism of 2,3,7,8-TCDD Immunotoxlclty 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 corticosteron levels in guinea pigs exposed to 2,3,7,8-TCDO 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. Indirect 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-TCDO treatment. Thymosin administered in conjunction with 2,3,7,8-TCDD did not protect mice from the typical 2,3,7,8-TCDD-induced immunotoxic 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-TCDD affects the supply or synthesis of thymic hormones which could lead to the observed immunosuppression. 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-TCDD exposure in female F-344 rats. Adrenalectomy and exogenous growth hormone had no preventative action on thymic involution. Hypophysectomized rats showed advanced thymic atrophy. Sharma and Behring (1979) noted that 2,3,7,8-TCDD caused stimulation of lymphocyte transformation to blast form cells (mitotically active precurs ors) when no mitogens were present in the culture system. This represents a phenomenon similar to actual antigenic challenge. At low doses (0.01 and 0.1 tig 2,3,7,8-TCDD/kg/week for up to 8 weeks), serum immunoglobulin levels were elevated in male CD-I mice. Larger doses of 2,3,7,8-TCDD (1.0 00110 V-38 08/11/84 and 10 pg/kg/week) resulted 1n a decrease In the serum Immunoglobulin 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- TCDD-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 cell's was not altered, since no change 1n response to bovine gamma globulin (requires T-helper cell cooperation) was noted 1n HI star/Fischer and Fischer rats exposed to 2,3,7,8-TCDD... In. vitro. 2,3,7,8-TCDD (100 ng/ma) sup pressed DNA, RNA and protein, synthesis In splenic lymphoid cells from B6C3F.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-TCDD. T-lymphocytes were more susceptible to 2.3.7.8-TCDD, measured by specific mitogen binding assays, than B-lymphocytes. These authors (Luster et al., 1979a) suggested that 2,3,7,8-TCDD may b1pd directly to the lymphocyte cell membrane and alter Its function. Faith and Luster (1979) reported that lymphocytes from the spleen, thymus, bone marr.ow and lymph nodes of Fischer rats exposed to 2,3,7,8-TCDD showed abnormal homing patterns within the body. 2,3,7,8-TCDD 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 In 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-tocell recognition, causing Immunosuppression and thymic atrophy. Clark et al. (1981) reported that 2,3,7,8-TCDD treatment (0.4, 4.0, 40 jg/kg weekly for 4 weeks by l.p. Injection) caused functional Impairment of cytotoxic T-cells 1n 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-TCDD treatment Impaired the generation of cyto toxic T-cells by the spleen (at doses as low as 0.004 pg/kg when detected 1n vitro) but did not appear directly toxic to the cytotoxic T-cells. At present, the mechanism .of Immunosuppression caused by 2,3,7,8-TCDD 1s 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-TCDD was required to suppress cytotoxic T-cells 1n D8A/2 mice as compared with C5681/6 mice. This Indicates that susceptibility to 2,3,7,8TCDD 1mmunotox1c1ty segregates with the Ah locus which 1s consistent with a receptor mediated mechanism. The receptor mediated mechanism was further supported by the susceptibility of the C5781/6 x D8A/2J hybrid mouse to 2.3.7.8- TCDD suppression of the cytotoxic T-cells which 1s again consistent with the dominant Inheritance of Ah (Nagarkattl et al., 1984). 00110 V-40 08/11/84 Few reports are available 1n which the Immunological effects of 2,3,7,8TCDD exposure were studied 1n humans. Regglanl (1980) reported that the 1mmunocapab1l1ty of 17 people, ranging In age from 3-60 years, who had been exposed to 2,3,7,8-TCQD, was normal 1n all cases. In a survey of 41 workers exposed to 2,3,7,8-TCDD, Ward (1982) measured 1mmunoglob1n 3, A, M, 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-TCDD-lnduced chloracne In this group of workers, there was a significant Increase in the proportion of cases with reduced IgO and IgN. It was suggested that the 2,3,7,8-TCDD-exposed group had a reduced Immune capability and a deficiency In T-cell and B-cell cooperation. The Immunotoxlclty of 2,3,7,8-TCDD 1n humans cannot be properly assessed because of the pauc.ity of data recorded soon after exposure. The most prominent effects In animals (1.e ., humoral responses) were not measured In humans. Enzyme Induction by TCDD -- ,> In Cell Cultures. Although 2,3,7,8-TCDD 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; Niwa et al., 1975; Bradlaw et al., 1976; Malik and Owens, 1977; Malik et al., 1979; Bradlaw et al., 1980). This enzyme Induction 1s so sensitive that It has been proposed as a bio assay for detecting planar polychlorinated organic compounds (Bradlaw et al., 1975, Bradlaw and Casterline, 1979; Niwa et al., 1975). 00110 V--41 09/18/84 Kourl et al. (1974) found that 2,3,7,8-TCDD Induced aromatic hydrocarbon hydroxylase (AHH) activity 1n 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-HC. Nlwa et al. (1975) compared AHH Induction by 2,3,7,8-TCDD among cell cultures (H-4-II-E, VERO, HTC, LB82, MA, Hepa-1, TRL2, ERL-2, NRKE and Chang). ED5Q values ranged from 0.12 nH In the Hepa-1 cell line to >100 nM 1n the VERO and HTC cell lines. 2,3,7,8-TCDD did not Induce AHH activity 1n LB82 colls. The respon siveness of AHH Induction to 2,3,7,8-TCDD was 250-9C0 times greater than to 3-HC. 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 to enzyme Induction by 2,3,7,8-TCDD 1s thus similar to the effects seen Vn vivo. The Inductive effect of 2,3,7,8-TCDO was blocked by actlnomyrln _Q. and cycloheximide. Implying that Induction Involved the sythesls of new nRNA and protein. Enzyme Induction by 2,3,7,8-TCDD, therefore, Involves an Initial RNA synthesis and continuous protein synthesis (Hallk and Owens, 1977; Hallk et al., 1979). In all of these studies, there was no correlation between cytotoxicity and enzyme Induction. This Implies that, despite the correlation In. vivo, there may be no direct connection betweeen enzyme Induction and the toxicity of 2,3,7,8-TCDO. ' In H1ce and Rats. The effects of 2,3,7,8-TCDD on enzyme activity 1n 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 1n 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-demethylat1on in 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 oral dose of 25 pg 2,3,7,8-TCDD/kg bw. The suppression of N-demethylase activity was seen only in adult animals. In 10-day-old rats, 2,3,7,8-TCDD had an inductive effect on this activity. The inductive effects of 2,3,7,8-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 1n the liver, kidney, lung, small intestine and testes of male Wistar rats. Honooxygenase act1v1ty_.was stimulated in the liver, lung and kidney, but not in any other tissue investigated. UDP 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 in any of the tissues studied, although stimulation of hepatic glutathione S-transferase has been reported by other investigators (Hanis and Apap, 1979). Enzyme induction has also been reported 1n rat mammary gland (Rikans et al., 1979), mouse testes (Hattison and Thorgelrsson, 1978), and rat prostate gland (Lee and Suzuki, 1980), but the rat adrenal gland is apparently insensitive to inductive effects of 2,3,7,8-TCDD (Guenthner et al., 1979). 00110 V-43 09/18/84 In the liver of rats and mice, 2,3,7,8-TCDD affects a wide range of enzymatic activities, Including DT-d1aphorase (Beatty and Neal, 1976a,b), bilirubin catabolism (Kap1tuln1k and Ostrow. 1978), ornithine decarboxylase (Potter et al., 1982), 7-ethoxycourjr1n O-demethylase (Greenlee and Poland, 1978), glutathione S-transferase (Baars et al., 1978; Manls and Apap, 1979), aldehyde dehydrogenase (Lindahl et al., 1978; Oeltrlch et al., 1977), uroporphyrinogen decarboxylase (Jones and Sweeney, 1977.) -aminolevulinic acid synthetase (Goldstein et al., 1982a; Woods', 1973), UOP-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 _an.d 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 (K1tch1n 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 Is quite different from that observed In rats and mice (Hook et al., 1975a). The only changes 1n hepatic enzyme activities observed were suppression of benzpyrene hydroxylase and benzphetamlne N-demethylase. In the same study, biphenyl 4-hydroxylase was Induced In the lung and benzpyrene hydroxylase was Induced 1n the kidney. In a similar study, a hepatotoxlc dose of 2,3,7,8-TCDD (30 pg/kg) failed to alter prostaglandin synthetase activity In hepatic or renal tissue (Kohl 1 and Goldstein, 1981). In a series of studies, Johnson and Muller-Eberhard (1977a,b,c,d), Johnson et al. (1979), Norman et al. (1978), L1em et al. (1980) and Oees.et al. (1982) Isolated a series of cytochromes P-450 from rabbit Uver mlcrosomes. These cytochromes were Immunologically distinct, functioned 1n dif ferent catalytic pathways, and responded differently to Induction by poly cyclic aromatic hydrocarbons. 2,3,7,8-TCOO was found to Induce two cyto chromes, 'designated as form 4 and form 6. . Form 4 1s the major cytochrome Induced 1n adult rabbit Uver by 2,3,7,8-TCOO; however, form 6 1s the major cytochrome Induced 1n newborn rabbit liver (Norman et al., lS78b), adult rabbit lung, and adult rabbit kidney (L1em et al., 1980; Oees et al., 1982). Other Species. The guinea pig, the species most sensitive to the toxic effects of 2,3,7,8-TCOO, 1s similar to the rabbit 1n Its response to 2,3,7,8-TCOO. Biphenyl 4-hydroxylase was Induced 1n the Uver, lung and kidney, biphenyl 2-hydroxylase was suppressed 1n the Uver, and benzpyrene hydroxylase was Induced 1n the kidney (Hook et al., 1975b). Testicular microsomal cytochrome P-450 content was depressed following a single oral dose of 1 pg/kg, reaching 52% of controls by 1 day and remaining at this 00110 V-45 09/18/84 level for 9 days (Tofilon et al., 1980). Testicular microsomal herne levels and 6-am1nolevul1n1c acid synthetase activity were unaffected by this treatment. In contrast to the rat, 2,3,7,8-TCDD did not Induce DT-d1aphorase 1n brain, spleen, kidney, lung, heart or Uver of male guinea pigs {Beatty and Neal, 1978). Aryl hydrocarbon hydroxylase and a-amlnolevullnlc acid synthetase 1n the ck embryo have been reported to be e'xtremely sensitive to the Inductive effects of 2,3,7,8-TCDD {Poland and Glover, 1973a,b), with maximal Induction occurring with 155 pmoles/egg. This Induction 1s relatively long lasting, with 70JC 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 sys'temlc toxic effects of subchronic exposure to 2,3,7,8-TCDD In rodents. Also, one sem1-controlled study evaluated the toxic effects to rabbits after confine ment to an area containing soil contaminated with 2,3,7,8-TCDD. No Informa tion was found In 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 HxCDDs in 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 pg/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 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-TCDD. During the study there were five treatment-related deaths 1n the high dose group females, with three occurring during treatment and two 1n the post-treatment period. In male animals only two deaths occurred 1n the post-treatment period 1n the high dose group. Both the male and female rats of the 0.1 and 1.0 jig/kg groups had depressed body weight; however, greater relative depression of body weight was observed In the high dose females. Other changes such as Increases 1n bilirubin concentrations, urinary coproporjjhyr1n excretion, and changes 1n relative thymus or Uver-to-body weight ratio occurred 1n the two high-dose female groups, but only 1n the 1.0 pg/kg male group. Although male rats had significantly decreased hematologic values (packed cell volume, RBC count and hemoglobin) 1n 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 In the female rats. No specific data were provided, however, to support this last conclusion. After necropsy, gross examination revealed subcutaneous edema, a decrease 1n the size of testes and uteri, and a decrease 1n the number of corpora lutea. Histologic examination revealed Involution of the thymus, 00110 V-47 08/11/84 decreased number of thymocytes, and focal necrosis and pigment accumulation 1n the liver. These observations were made only 1n 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 liver In the group fed 0.1 pg/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 1n a second animal there was severe anemia, suggesting possible Involvement of the hematopoietic system near the time of death. Liver toxicity was the only effect of treatment observed during histo logic examination of rats (Osborne-Hendel) and mice (B6C3F^) 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 In corn o1l-acetone (9:1) twice a week at doses for rats of 0.0, 0.5, 1, 2, 4 and 8 pg/kg/week, and for mice at doses of 0.0, 1, 2, 5, 10 and 20 pg/kg/week. Deaths occurred at the two h1gh-dcse levels 1n rats, with 4 females 1n the 8 pg/kg/week and 1 In the 4 pg/kg/week group dying, while only 2 male rats 1n the 4 pg/kg/week group died. Deaths were accompanied by severe toxic hepatitis. Hepatic lesions were observed In all other; rats examined In groups administered 1-8 pg/kg/week; however, not all animals 1n each group were submitted to necropsy. Normal liver histology was observed In 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 1n mice, with a single death occurring 1n each sex at the high-exposure level, along with reports of hepatic lesions on histologic examination. In contrast to rats, female mice were less sensitive to the hepatotoxlc effect of 2,2,7,8-TCDD than were the male mice. Hepatic lesions were observed 1n 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, It appeared that sex differences In the sensitivity to the toxic effects of 2,3,7,8-TCDO 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 developmen-t 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 o11-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 monitored 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 1n male rats rd a decrease 1n body weight gain 1n the high-dose female rats, the only effect of exposure to 2,3,7,8-TCDO was histologic changes 1n the liver. Liver pathology was normal 1n 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 lipid droplets 1n a few centrllobular hepatocytes to 11p1d droplets In all centrllobular hepatocytes with extension Into the mldzonal hepatocytes. No clear dose-response pattern was observed 1n this study; however, 1t did appear that the severity of fatty changes was greater 1n male rats. During the recovery period, fatty changes progressively decreased 1n severity, but were still present In some treated animals 12 weeks after cessation of exposure. Other histologic changes observed In the liver predominantly In the animals killed at 28 weeks Included necrosis, Increased nuclear size, subtle distortion of liver architecture, and hyperchromatic 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 U v e r was the most sensitive organ to the toxic effect of 2,3,7,8.-.TC0D, and although recovery occurred after termination of treatment, the recovery process was slow. The recovery time was also demonstrated to be long 1n a subchronic study by Goldstein et al. (1982b) of 2,3,7,8-TCDO Induced porphyria. Groups of 8 female Sprague-Dawley rats were given 2,3,7,8-TCDD In corn oil-acetone (7:1) weekly by gavage for 16 weeks at doses of 0.0, 0.01, 0.1 or 10.0 pg/kg/ week and killed 1 week after the last treatment. Additional groups of rats received doses of 0.0 or 1.0 pg/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 pg/kg/week. After 16 weeks of exposure to 2,3,7,8-TCDD, Uver porphyrins were elevated -1000-fold 1n 7 of 8 animals receiving 1.0 pg/kg/week, but only 1 of 8 animals In the 0.1 00110 V-50 08/11/84 yg/kg/week group had elevated porphyrin levels. No effect was observed 1n the low-dose animals. After a 6-month recovery period the porphyrin level In animals exposed to 1 ug/kg/week was still 100-fcId higher than values 1n the control group. A similar pattern was observed for urinary excretion of uroporphyrin. The rate limiting enzyme 1n heme synthesis, -aminolevu 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 glucu- ronyl 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-TCOD at a dose of 1.0 pg/kg/week was not sufficient for complete reversal of 2,3,7,8-TCDD-lnduced porphyria. . In addition to the above laboratory studies, Strlk and de W1t (1980) attempted to Investigate the toxicologic effect on rabbits of exposure to a natural environment that was contaminated with 2,3,7,8-TCOD. Groups of 20 female rabbits and 1 male rabbit were housed for 5 months In pens, located In five separate areas, on soil that had been contaminated with 2,3,7,8TCOD. The soil had been cleaned by replacement or cultivation before Initi ation of the study. The ievels of 2,3,7,8-TCOD before cleaning were from 0.8-23.2 jjg/m3; however, the levels of contamination after cleaning were not determined. At 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-glucarlc-acld. 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. 00110 V--51 08/11/84 Information on the subchronic toxicity of HxCOD was provided in a pre liminary range-finding study for a chronic bioassay conducted by NTP (198Gd) on a 1-2 mixture of 1,2,3,6,7,8- and.1,2,3,7,8,9-HxCDD. Osborne-Hendel rats and B6C3F.J mice in groups of 10 males and 10 females were administered the HxCOD mixture in corn oil-acetone (9:1) by gavage twice a week for 13 weeks. The total weekly doses given rats were 0.0, 2.5, 5, 10, 50 and 100 jig/kg, while mice received 0.0, 1.25, 2.5, 5. 10 and 50 yg/kg. At week 10 of the study, the body weight in rats was decreased in a dose-related manner to a maximum of -20% in the high-dose group. In mice, body weight was also decreased 10-20% in the treated animals; however, there appeared 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 hepatotoxicity occurring at doses of 5 and 10 yg/kg/week for maleand female rats, respectively, and at 10 yg/kg/week for both sexes of mice. At higher exposures, splenic hyperplasia and cortical atrophy of the thymus were also detected in rats. In rats it was unclear whether the low-dose animals were free of any patho logic findings or none were subjectedto necropsy. In mice it was stated that no changes were observed in males exposed to 2,3,7,8-TCDO at 1.25 yg/kg/week or in females exposed to 1.25 or 2.5 yg/kg/week. Although the data are limi-ted, it appears that the same target organs are sensitive to the toxic effects of both 2,3,7,8-TCDO and this mixture of HxCDO. In addition, a second subchronic range finding study conducted by NTP (1980c) evaluated the dermal toxicity of the above mixture of HxCOD. Groups of 10 male and 10 female Swiss-Webster mice were treated by dermal applica tion 3 times/week for 13 weeks. The doses used were from 0.01-50 yg/ G0110 V-52 09/18/84 application with the test compound dissolved in acetone. There was 100% mortality in the 25 and 50 pg/application groups and 80% mortality in the 10 pg/application group. On histologic .xamination, there were signs of liver damage at the lowest dose tested in both sexes; however, the incidence and degree of damage were not well correlated to the dose applied. Chronic Toxicity. In rats and mice the toxic effects of chronic exposure to 2,3,7,8-TCDD are summarized in Table V-5. These studies were predominately designed to assess the carcinogenicity of 2,3,7,8-TCDD and the observations of non-neoplastic systemic toxicity are, therefore, limited. Van Miller et al. (1977a,b) fed groups of 10'male Sprague-Dawley rats diets containing 1, 5, 50, 500, 1000, 5000, 50,000, 500,000 or 1,000,000 ppt 2,3,7,8.-TCDD (10-3 pg/kg. diet)., for 78 weeks in order to .determine .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 pg 2,3,7,8-TCDD/kg bw/week, respec tively. All animals that received diets containing >1000 ppt (>0.4 pg/kg bw/week) were dead by the end of the study (95 weeks). It appeared that diets containing >1000 ppt (>0.4 pg/kg bw/week) 2,3,7,8-TCDD definitely increased mortality. The three highest dietary Tevels (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 sizes and the high mortality in the control group (6 of 10). In the groups receiving <5000 ppt, weight gain was significantly depressed only in the 00110 V-53 09/18/84 TABLE V-5 Effects of Chronic Exposure to 2,3,7,8-TCDD In Laboratory Rodents 00110 Specles/Straln Sex/No. Oose Rat/SpragueOauley male/10 0.0 ppt Treatment Schedule Ouratlon of Study Parameters Monitored NA 95 weeks survival male/10 1 ppt contlnous In diet for 78 weeks 95 weeks> survival male/10 5 ppt continuous In diet for 78 weeks 95 weeks , survival male/10 SO ppt continuous In diet for 78 wreks 95 weeks ; survival male/10 male/10 500 ppt continuous In diet for 78 weeks 95 weeks 1000 and 5000 ppt continuous In diet for 78 weeks 95 weeks survival 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 SOX survived until 95 weeks, the first death occurred at week 86 60X survived until 95 weeks, the first death occurred at week 33 60X survived until 95 weeks, the first death occurred- at week 69 5OX 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 Hiller et al., 1977a,b V-54 08/11/84 lABLC V-5 (coni.) 00110 Spedes/Straln Sex/No. Dose Rat/SpragueDawley N and F/ -2193 ppt 50 and 50 (0.1 |ig/kg/day) H and F/ -20B ppt 50 and 50 (0.01 ug/kg/day) H and F/ -22 ppt (0.001 50 and 50 pg/kg/day) Treatment Schedule Duration of Study Parameters Honttored Effects of Treatment* 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, ii.-atology, urine analyses, and clinical chemistry - Cumulative mortality. Increased (F); Body weight gain. decreased (H,F); Red blood ceil count, decreased (H,F); Packed celi volume, decreased (H,F); Hemoglobin, decreased (H.F); Reticulocytes, Increased (H.F); Milite blood ceii count, decreased (F); Serum glutamic pyruvic transaminase, Increased (F); G-Glutamyl transferase, Increased (F); Alkaline phosphatase, Increased (F); Urinary coproporphyrln, Increased (F); Urinary uroporphyrin. Increased (F); Urinary deita-amtnolevultntc a d d , Increased hepatic degeneration, Increased (H.F) extensive hlstopathology, hematology, urine analyses and clinical chemistry Urinary coproporphyrln. Increased (F); Urinary uroprophyrIn, Increased (F); Hepatic degeneration, Increased (H.F) extensive hlstopathology, urine analyses and clinical chemistry No differences from values obtained from control animals K o d b a et al., 1978a, 1979 V-55 09/18/84 TABLE V-S (cont.) oono V-56 Specles/Straln Sex/No. Oose Treatment Schedule Duration of Study Rat/OsborneMendel N and F/ 75 and 75 N and F/ SO and SO 0.0 pg/kg/week 0.5 pg/kg/week N and F/ 0.05 pg/kg/week SO and SO N and F/ 0.01 pg/kg/week 50 and 50 H1ce/B6C3F1 * Nlce/Swlss N and F/ 75 and 75 N and F/ 50 and 50 H and F/ SO and SO N and F/ 50 and SO H/30 0.0 pg/kg/week 0.5 pg/kg/week (N); 2.0 pg/kg/ week (F > 0.05 pg/kg/week (K); 0.2 pg/kg/ week (F) 0.01 pg/kg/week (It); 0.04 pg/kg/ week. (F) 0.0 pg/kg/week HA 105 weeks administered by gavage biweekly for 104 weeks 107 weeks administered by gavage biweekly For. 104 weeks 107 weeks administered by gavage biweekly for 104 weeks 107 weeks . NA 105-106 weeks administered by gavage biweekly For 104 weeks administered by gavage biweekly for 104 weeks administered by gavage biweekly for 104 weeks NA 107 weeks 107 weeks 107 weeks 580 days H/44 H/44 H/43 0.007 pg/kg/week 0.7 pg/kg/week 7.0 pg/kg/week administered by gavage weekly for 1 year administered by gavage weekly for 1 year administered by gavage weekly for 1 year `Results of statistical analysis were not provided. HA Not applicable 649 days 633 days 424 days 08/11/8A Parameters Mon Ha re d extensive hlstopathology extensive hlstopathology extensive hlstopathology extensive htslopathology extensive htstopathology extensive hlstopathology extensive hlstopathology extensive htstopathology histology on all organs histology on all organs histology on all organs histology on all organs Effects of Treatment* Toxic hepatitis; 0/74 (M), 0/75 (f) Toxic hepatitis; 14/50 (It), 32/50 (f) Toxic hepatitis; 0/50 (H), 1/50 (F) Toxic hepatitis; 1/50 (M), 0/59 |F) Toxic hepatitis; 1/73 (M), 0/73 (F) Toxic hepatitis; 44/50 (H), 34/47 (F) Toxic hepatitis; 3/49 (M), 2/40 (F) Toxic hepatitis; 5/44 (It), 1/50 (F) Oermatills and amyloidosis; 0/30 Dermatitis and amyloidosis; 5/44 Dermatitis and amyloidosis; 10/44 Early mortality, dermatitis and amyloidosis; 17/43 Reference NTP, 1900a MTP, 1900a Toth et a!., 1970, 1979 5000 ppt group. The only histopathologic changes reported 1n these groups were neoplastic changes (see Carcinogenicity). Kodba cc al. (1978a, 1979) maintained male and female Sprague-Dawley rats (50/sex/dose) on diets containing levels of 2,3,7,8-TCDD that resulted 1n doses of 0.001, 0.01 or. 0.1 yg/kg/day. Increased mortality was observed In the high-dose female,. In the groups receiving 0.01 or 0.1 vgAg/day, treatment-related changes were observed In hematologic, clini cal chemistry and urinary analysis values. Urinary excretion rates of coproporphyrin and uroporphyrin were Increased 1n these groups. Histologic examination revealed degenerative, necrotic and Inflammatory changes 1n the 11ver. The NOEL 1n this study was 0.001 yg/kg bw/day. Toxic hepatitis (Upldosis .and hydropic degeneration of the cytoplasm of the hepatocytes) was observed In both sexes of Osborne-Hendel rats receiving 2,3,7,8-TCDD by gavage 1n corn o1l:acetone (9:1) (0.25 or 0.025 yg/kg bw), tw1ce/week for 104 weeks (NTP, 1980a). No other non-neoplast1c lesions were observed, even though extensive histological examinations were performed. This study demonstrated a N0AEL of 0.05 yg/kg bw/week for hepatitis. In this same study, B6C3F1 mice were treated biweekly for 104 weeks with 2,3,7,8-TCDD 1n corn o1l:acetone (9:1). Hales 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 observed 1n control and treated groups, but appeared to be significantly elevated only 1n the high dose groups (NTP, 1980a). .. 00110 V-57 09/18/84 In another study, Toth ei al. (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. Amyloidosis of the kidney, spleen and'liver, and dermatitis were observed 1n all three treatment groups. This suggests a LOAEL of 0.007 pg/kg bw/week In mice 1n this study, but a NOAEL was not established. Some Information on the toxicity of chronic dermal exposure can be obtained from the NTP (1980b) dermal carcinogenicity study In Swlss-Hebster mice. Thirty males were treated with 0.01 yg 2,3,7e8-TCDD/appl1cat1on and 30 females were treated with 0.005 jig/appUcatlon, 3 t1mes/week for 104 weeks. Vehicle-treated and untreated controls were Included 1n the experi 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 TCDD, guinea pigs and monkeys have few or no histopatho logic changes 1n the liver, whereas rats and mice have extensive histopatho logic effects 1n this organ. Gupta et al. (1973) and Grieg et al. (1973) observed marked distortion of Uver architecture In rats given 50 or 100 v9 2,3,7,8-TCDD/kg, with marked necrosis of hepatocytes. The livers of mice given lethal doses of 2,3,7,8-TCDD also had necrosis (Vos et al., 1974). According to Jones and Grieg (1975), centnlobular necrosis, bile duct proliferation and I1p1d accumulation were more severe 1n mice than 1n rats. Livers of mice contained excess amounts of porphyrin (McConnell et al., 1978a). 00110 V-58 08/11/84 Light microscopic, ultrastructural and hlstochemlcal changes 1n 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) administered 0.0, 5.0 or 25.0 yg 2,3,7,8-TCOD/kg bw by gavage to groups of 30 male rats. The rats were sacrificed 1, 3, 6, 9, 16 or 28 days after treatment. Major ultrastructural changes occurred 1n the cells near the bile canallcuH 1n both treatment groups. A dose-related Increase In the ' smooth and rough endoplasmic reticulum was observed starting on day 3, reaching a maximum on days 6-9, and returned to normal by day 28. Jones and Butler (1974) administered 200 yg 2,3,7,8-TCOD/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 prolif erative changes 1n the l.lvec.: Degenerating 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 multlnucleated cells, was prevalent by week 9. Jones (1975) gave 23 male Porton rats a single gavage dose of 200 yg 2,3,7,8-TCOD/kg In arachls oil, and 7 control rats received arachls oil alone. Loss of ATPase activity along the canalicular borders and Increased activity In the sinusoids In the livers of rats were observed 3 days follow 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 ATPase activity 1n hepatocyte plasma membranes. Liver surface membranes were Isolated from male Holtzman rats 00110 V-59 09/18/84 using a modified discontinuous sucrose gradient method on days 2, 10, 20 or 40 post-treatment. Both doses resulted in similar depression of Na/K-ATPase on days 2-40; however, Mg++-ATPase was depressed to this extent only in the high-dose groups. In the low-dose group, Mg++-ATPase activity was decreased on day 20, but returned to normal levels by day 40 post-treatment. 2,3,7,8-TCDO did not inhibit ATPase activity in vitro, nor did the decrease in ATPase activity correlate with 2,3,7,8-TCDO induced food deprivation. There was, however, a correlation between liver surface membrane activity, bile flow and biliary excretion of ouabain in vivo. In similar experiments, Hwang (1973) reported that bile flow was stimulated, but indocyanine green excretion was inhibited, in male CD rats following treatment yith 5 or 25 jig 2,3,7,8-TCD0/kg bw by gavage. Peterson et al. (1979b) ,,investigated the relationship between ATPase activity and biliary excretion 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 pregnenolone-16-a-carbonitrile, indicating that this ATPase activity was not directly involved in ouabain transport. The ability of 2,3,7,8-TCUD to inhibit biliary excretion has been demon strated to correlate with the species sensitivity to the hepatotoxic effects of 2,3,7,8-TCDD (Seefeld et al., 1979, 1980). Yang et al. (1977) reported that the effect of 2,3,7,8-TCDD on biliary transport is compound dependent. In these studies, ouabain excretion, a neutral compound, and bile flow were inhibited, while the excretion of the organic anions phenol-3,6-dibromophthalein and sulfobromophthalein were essentially unchanged. 00110 V-60 09/18/84 Cunningham and Williams (1972) detected a decrease in in vivo lipid formation in the liver of male Wistar rats receiving 10 pg 2,3,7,8-TCDD/kg bw, based on [3H]sodium 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 following treat ment with 50 pg 2,3,7,8-TCDD/kg bw. Levels of free fatty acids and cholesterol enters were increased while levels of phospholipids, free cholesterol and triglycerides remained constant.When a sublethal dose, 10 pg/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. Goldstein et al. (1978) determined that 4 weekly doses of 25 pg 2,3,7,8-TCDD/kg bw or higher would result in Increased hepatic porpuyrin levels in male C57B1 mice. Smith et al. (1981) found that strain differ ences in sensitivity between C57B1 and D8A/2 mice given a single gavage dose of 75 pg 2,3,7,8-TCDD/kg correlated with the ability of 2,3,7,8-TCDD to Induce increases in hepatic porphyrins; however, there was no correlation between toxicity and Increased porphyrins between sexes within strains. Rats are less sensitive to 2,3,7,8-TCDD Induced porphyria, with Increases In porphyrin levels occurring only after subchronic! exposure (>0-l01 mg/kg/week by gavage for 6 months) (Cantonl et al., 1981). The major factors involved in 2,3,7,8-TCDD-lnduced porphyria have been Identified as an Increase in -aminolevulinic acid synthetase (Goldstein et al., 1978) and a decrease In uroporphyrinogen decarboxylase activity (Smith et al., 1981; Sweeney and Jones, 1978). 00110 V--61 09/1B/84 A number of groups investigated the effect of 2,3,7,8-TCDD on hepatic DNA synthesis. Grieg et al. (1974) measured the incorporation of [*H]thymidine 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 [*H]thymidine incorporation into DNA in liver slices obtained from rats which had been pretreated with 5 yg 2,3,7,8-TCDO/kg bw. 2,3,7,8-TCDD 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 acute toxicity studies suggested that 2,3,7,8-TCDD may alter the immune response and prompted investigations of Immunotoxicity, summarized in Table V-4. The results suggest that 2,3,7,8-TCDD 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. Pretreatment with 0.04 yg 2,3,7,8-TCDD/kg bw/week reduced the development of skin hypersensitivity to Mycobacterium tuberculosis in guinea pigs; no effect on this parameter was seen in rats. Tetanus antitoxin levels were reduced in guinea pigs receiving 0.2 yg 2,3,7,8-TCDD/kg bw/week. 2,3,7,8TCDD has also been demonstrated to inhibit cell-mediated immunity in "graftversus-host* experiments utilizing spleen cells from mice which had received 4 weekly oral doses of 0, 0.2, 1.0 or 5.0 yg 2,3,7,8-TCDD/kg bw (Vos et al., 1973). 00110 V-62 09/18/84 Vos and Moore (1974) and Moore and Vos (1974) administered 1 or 5 pg 2,3,7,8-TCDO/kg bw to female F-344 rats on days 11 and 18 of gestation and days 0, 7 and 14 postpartum. This treatment resulted In prolonged times until graft rejection, decreased spleen cell graft-versus-host activity and decreased binding response to phytohemogglutlnln, but an Increased humoral Immune response to conconavalln A 1n the pups. Thigpen et al. (1975) determined the effect of 2,3,7,8-TCDD on host resistance to Injection by Salmonella bern In male SPF C57Bl/6jfh mice, an effect largely mediated by thymus-derived lymphocytes. Pretreatnent with >1 pg/kg bw/week by gavage resulted 1n an Increase 1n mortality and a decrease 1n time until Infection. The NOAEL was determined to be 0.5 pg/kg bw. Pretreatment with 2,3.7,8-TCDO has also been demonstrated to enhance the susceptibility of jn.1ce to E. coll endo.toxln (Vos et al., 1978a). Thomas and H1nsd1ll (1979) maintained female Swlss-Webster mice on diets containing 1, 2.5, 5, 10 or 20 ppb 2,3,7,8-TCDD (1, 2.5, 5, 10 or 20 pg/kg diet) from 4 weeks before mating until 3 weeks postpartum. Thymus weight was decreased 1n the pups from groups receiving >2.5 ppb, along with a decreased number of plaque-forming cells reactive to sheep red blood cells and an Increased susceptibility to S. typh1mur1um. This treatment did not, however, lower the humoral content of ant1-RBC antibodies, affect the response to E. coll LPS or L. monocytogenes, affect nitrogen-induced lympho cyte proliferation, or alter the response of 8- and T-cells to conconcanvaI1n A In. vitro. Similar results have been reported 1n Fisher/Wlstar rats (Faith and Luster, 1979; Luster et al., 1980). 00110 V-63 09/18/84 Hinsdi11 et al. (1980) found that feeding Swiss-Webs ter mice diets con taining 100 ppb (100 pg/kg diet) 2,3,7,8-TCDD for 5 weeks Increased serum 8-globul1ns, but suppressed total serum protein, Y-globul1n and albumin. A decreased Immune response to tetanus toxoid, sheep red blood cells, S. typhlmuMum and L. monocytogenes and a lowered contact sensitivity to DNFB were noted at dietary concentrations as low as 10 ppb (10 pg/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. Mantovanl et al. (1980) administered single l.p. doses of 1, 2, 6 or 30 p 9 2,3,7,8-TCDD/kg bw to C57B1/6J mice. This treatment resulted 1n a decreased total number of.macrophages and splenic natural killer calls, but. did not affect the cytostatic or cytocldal activity of the remaining cells. McConnell et al. (1978a) suggested that the decrease 1n peripheral cell counts may be the result of the hypocellularUy seen 1n the bone marrow of ' 2,3,7,8-TCDD-treated mice. Other Organ Systems. One of the characteristic effects of 2,3,7,8TCDD 1s the loss of body weight. Because decreased food consumption 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-TCDD on Intestinal absorption 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 pg 2,3,7,8-TC00/kg bw decreased 00110 V-64. 09/18/84 the In. vitro intestinal absorption of D-glucose, but did not affect absorp tion of D-galactose, L-argenine or L-histidine. 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 Sprague-Oawley rats. Manis and Kim (1979a,b) found that 22-84 Pg 2,3,7,8-TCDD stimulated intestinal transport of iron in male Sprague-Dawley rats and in an unidenti fied strain of mice. Gavage administration was more effective than i.p. injection. This stimulation occurred in the duodenum, but the distal segment of the intestine was unaffected. In parallel experiments, calcium transport was decreased and galactose and proline 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 pg 2,3,7,8-TCDO on the in, vitro function of renal cortical slices obtained from male Sprague-Dawley rats 3 or 7 days following intubation. Anion transport, as measured by accumulation of p-aminohippuric acid, was decreased by N-methyl-nicotinamide -accumulation, at both dose levels. In the in vivo studies, sodium reabsorption was within the normal range and ) ammoniogenesis and gluconeogenesis were unaffected, even when the rats were made acidotic. The authors concluded that the observed decrease in kidney function was a res.ult of the generally poor condition of the treated animals and not a direct effect of 2,3,7,8-TCDO. 00110 V-65 09/18/84 Grieg et al. (1974) found that Intraperltoneal administration of 10 2.3.7.8- TCDD/kg bw Inhibited the DNA synthesis stimulated by folate (250 mg folic ac1d/kg) or lead acetate (40 mg Pb+^/kg) In the kidneys of male and female Porton 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 mlcrosome. Z1nkl et al. (1973) observed a number of hematologic clinical chemistry changes In female CD rats given 30 dally doses of 0.1, 1.0 or 10 jig 2.3.7.8- TCDD/kg bw; however, they concluded that these changes simply reflected damage to other organs, such as the liver, and hemoconcentratlon. The only other major effect observed was thrombocytopenia, which was also observed In mice and female guinea pigs. Lymphopenia was observed In guinea pigs that received a single dose of 1, 10 or 50 pg/kg bw. . Poll et al. (1980) Investigated the Induction of hyperlipidemia In male Sprague-Dawley rats following a single 1.p. Injection of 2.5, 5, 10 or 20 jig 2,3,78-TCDD/kg bw. There was a dose-related Increase 1n total plasma cholesterol and high density lipoprotein cholesterol by 21 days post treatment, but triglycerides and low and very low density lipoproteins were unaffected. In contrast, hyperlipidemia 1n male Hartley guinea pigs given a single 1.p. Injection of 2 jig/kg bw was characterized by Increases In low and very low density lipoproteins. Other Effects Carcinogenicity. 2,3,7,8-TCDD has been tested for carcinogenicity 1n rats and mice by administering the compound In the diet and by gavage. 00110 V-66 09/18/84 Also, the tumor incidence in native nice inhabiting an area with heavy exposure to the herbicide Agent Orange has been assessed and compared with micefrom an uncontamlnated habitat. The results of these bioassays are summarized in Table V-6. Along with studies using the oral route, 2,3,7,8TCDD has been tested for tumorigenlcity by dermal application (Table V-7). Using the skin two-stage tumorigenlcity model, 2,3,7,8-TCDD has been tested for promoting and initiating activity as veil as anticardnogenic 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 carcinogens. In a limited study, Van Miller (1977a,b) maintained small groups of male' Sprague-Dawley rats on diets containing 2,3,7,8-TCDO. 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-TCDD 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 pg/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 were observed for an additional 17 weeks before and until sacrific 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 09/18/84 TABLE V-6 Carcinogenicity Bioassays of 2,3,7,8-TCDD Administration by the Oral Route 00110 V-68 Exposure Duration Route Specles/Straln Sex Dose or Exposure of Duration of Study Treatment Vehicle Tumor Type Tumor Incidence p Value Reference Gavage Gavage Gavage rats/ Csborne-Hendel H rats/ Osborne-Hendel F mlce/B6C3F] . N 0.0 ug/kg/week 0.01 ug/kg/week 0.05 |ig/kg/week 0.5 ug/kg/week 0.0 iig/kg/ueek 0.01 yg/kg/week 0.05 |ig/kg/week 0.5 ug/kg/week 0.0 u9/kg/week 0.01 u9/kg/week 0.05 |i9/kg/week 0.5 ug/kg/week 104 weeks 105 weeks corn oilacetone (9:1) folllcular-cell adenomas of the thyroid, carcinoma of the thyroid 104 weeks 107 weeks corn oilacetone (9:1) folllcular-cell adenomas of the thyroid, carcinoma of the thyroid 104 Weeks 107 weeks corn oilacetone (9:1) folllcular-cell adenomas of the thyroid, carcinoma of the thyroid 104 weeks 107 weeks corn oilacetone (9:1) folllcular-cell adenomas of the thyroid, carcinoma of the thyroid 104 weeks 105 weeks corn oilacetone (9:1) neoplastic nodule of the liver, hepatocellular carcinoma of the liver 104 weeks 107 weeks corn oilacetone (9:1) neoplastic nodule of the liver, hepatocellular carcinoma of the liver 104 weeks 107 weeks corn ollacetone (9:1) neoplastic nodule of the liver, hepatocellular "carcinoma of the liver 104 weeks 107 weeks corn oil- . acetone (9:1) neoplastic nodule of the liver, hepatocellular carcinoma of the liver 104 weeks 105 weeks corn oil- . hepatocellular carcinoma acetone (9:1) 104 weeks 107 weeks corn oil- hepatocellular carcinoma acetone (9:1) 104 weeks 107 wfeeks corn oil- ' hepatocellular carcinoma acetone (9:1) 104 weeks 107 weeks c o m oil- hepatocellular carcinoma acetone (9:1 ) 1/69 0/69 5/40 0/48 6/50 2/50 10/50 1/50 5/75 0/75 1/49 0/49 3/50 0/50 12/49 3/49 8/73 9/49 8/49 17/50 -0.006 -0.042 >0.021 >0.001 <0.001 NS NS -0.006 -0.002 NS NS >0.002 NTP, 1980a NTP, 1980a NTP, 1980a 09/18/84 TABLE V-6 (cant.) 00110 V-69 Exposure Duration Ouratlon Route Spectes/Straln Sex Dose or Exposure of of Study Treatment Vehicle Tumor Type Tumor Incidence p Value Reference Gavage raUe/BCSFi F 0.0 ng/kg/ueek 104 weeks 105 weeks corn oil- . hepatocellular carcinoma. acetone (9:1) Follicular-cell adenomas of the thyroid 1/73 0/69 >0.008 >0.016 NIP, 1980a 0.04 ng/kg/week 104 weeks 107 weeks corn oilacetone (9:1) hepatocellular carcinoma, follicular-cell adenomas of the thyroid 2/50 3/50 NS NS Oral '* 0 .2 ng/kg/woek 2.0 ng/kg/ueek . rat/ H 0.0 ppb Sprague-Oawley 0.001 ppb 104 weeks 104 weeks 78 weeks 70 weeks 107 weeks 107 weeks 95 weeks 95 weeks corn oilacetone (9:1) corn ptlacetorte (9:1) In diet In diet hepatocellular carcinoma, follicular-cell adenomas of the thyroid hepatocellular carcinoma, follicular-cell adenomas of the thyroid all tumors3 all tumors3 2/40 1/47 6/47 5/46 0/10 . 0/10 NS NS >0.014 >0.009 NR NR Van Hiller et al., 1977a,b 0.005 ppb 70 weeks 95 weeks In diet all tumors3 5/10 NR 0.05 ppb 78 weeks 95 weeks In diet all tumors3 3/10 NR 0.5 ppb 70 weeks 95 weeks In diet all tumors3 4/10 NR l.S ppb 70 weeks 95 weeks In diet all tumors3 4/10 NR 5.0 ppb 70 weeks 95 weeks In diet all tumors3 7/10 NR Oral rat/ H 0 .0 ng/kg/day Sprague-Oauley 105 weeks 105 weeks In diet squamous cell carcinoma of the hard palate, smiamous cell carcinoma of the tongue, adenoma of the adrenal cortex 0/05 0/05 0/85 NS NS NS Koclba et al., 1978a,b 0 .0 0 1 ng/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 0/50 1/50 0/50 NS NS NS 09/18/84 TABLE V-6 (coni.) 00110 Exposure Duration Duration Route Specles/Stratn Sex Dose or Exposure of of Study Treatment Vehicle Oral rat/ N 0.01 pg/kg/day Sprague-Dawley 105 weeks 105 weeks In diet 0.1 pg/kg/day 105 weeks 105 wyeks In diet Oral rat/ F 0.0 pg/kg/day Sprague-Dawley 105 weeks 105 weeks I 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/ F 0.1 pg/kg/day Sprague-Dawley 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 0/50 1/50 2/50 squamous cell carcinoma of the hard palate, squamous cell carcinoma of the tongue. adenoma of the adrenal cortex 4/50 3/50 5/50 hepatocellular carcinoma, squamous cell carcinoma of the tongue, squamous cell carcinoma of the lung 0/86 0/B6 0/B6 hepatocellular carcinoma, squamous cell carcinoma of 1 0/*50 the tongue, 0/50 squamous cell carcinoma of the lung 0/50 hepatocellular carcinoma. squamous cell carcinoma of the tongue, ' squamous cell carcinoma of the lung 2/50 1/50 0/50 hepatocellular carcinoma. squamous cell carcinoma of the tongue, squamous cell carcinoma of the lung 11/49 4/49 7/49 NS NS NS <0.05 <0.05 <0.05 NS NS NS NS NS NS NS NS NS <0.05 <0.05 <0.05 Koclba el al.. 197Ba,b Koclba et al.. 1978a,b Koclba et al., 197Ba,b V--70 09/18/84 09/18/84 V--71 00110 1ABLC V-6 (cont.) Exposure Ouratlon Route Specles/Straln Sex Dose or Exposure of Ouratlon of Study Treatment Vehicle Tumor Type Gavage mice/ Svlss/H/Rlop Oral mice/ Peromvscus noltonotus N 0.0 pg/kg/week 365 days 0.007 pg/kg/week 365 days 0.7 pg/kg/week 365 days 7.0 pg/kg/week 365 days H.f 0.0012 pg/kg/day HA SOD days 649 days 633 days 424 days NA sunflower oil sunflower oil sunflower oil sunflower oil , contaminated soil ' liver tumors1* liver tumors^ liver tumorsb liver tumors** liver 0.0 pg/kg/day NJk NA contaminated liver soil aNo single target organ for cancer was outstanding. includes hepatomas and hepatocellular carcinomas. NR Not reported NS . Not significant Tumor Incidence p Value Reference 7/30 NS 13/44 NS 21/44 <0.01 13/43 NS 0/15 NS 0/15 NS Toth et al., 1979 Cockerham et al.. 1900 TABLE V-7 Carcinogenicity Bioassays of 2,3,7PB-TCDD Administered by the Dermal Route 00110 V-72 Species Sex Doseb Duration of Exposure Target Organ Tumor Type Tumor Incidence Mice M 0.01 yg/appl1cat1on 104 weeks Integumentary fibrosarcoma 6/20 system 0.0 pg/appl1cation (vehicle control) 104 weeks i Integumentary system fibrosarcoma 3/42 0.0 iig/appllcation * (untreated control) NA Integumentary system fibrosarcoma 0/28 F 0.005 yg/app11cation 104 weeks Integumentary fibrosarcoma 8/20 system 0.0 yg/app11cation (vehicle control) 0.0 yg/app11cat1on (untreated control) 104 weeks NA Integumentary system Integumentary system fibrosarcoma fibrosarcoma 2/41 1/27 aSource: NTP, 1980b . ^The compound was applied 3 t1mes/week 1n 100 yi of acetone. NA = Not applicable 09/18/84 All animals in groups maintained on diets containing 1-1000 ppb of 2,3,7,8-TCDD 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-TCDD 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 in 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 in the diagnosis of neoplasms. Various benign and malignant tumors were found in each treatment group. No tumors were observed, in the controls (Table V-8). . . Statistically significant increases of squamous cell tumors of the lungs and neoplastic nodules of the liver were observed in rats ingesting 5 ppb TCDD (Tables V-9 and V--10). In addition, two animals in the 5 ppb dose group and one animal in the 1 ppb dose group had liver cholangiocarcinomas, which are rare in Sprague-Dawley rats. These results provide evidence of a carcinogenic effect. The observation of no tumors of any kind in the controls is 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 TABLE V-8 2,3,7,8-TCDD Intake and Mortality In Hale Sprague-Dawley Rats3 Doseb (ppb) 0.0 0.001 0.005 0.05 0.5 1 5 Weekly Dose/Rat (>.g/kg bw) 0.0003 0.001 0.01 0.1 ...... 0.4 2.0 Week of First Death 68 86 33 69 17 31 31 Number of Rats Dead at 95th Week 6/10 2/10 4/10 4/10 5/10 (60%) (20*) (40%) (40%) (50%) 10/10 (100%) 10/10 (100%) aSource: Adapted from Van Miller et al.f 1977a,b bRats at 50, 500 and 1000 ppb dose levels were all dead within 4 weeks. 00110 V-74 09/25/84 TABLE V-9 Benign and Malignant Tumors In Rats Ingesting 2,3,7,8-TC00a 6oseb Benign Malignant Number of Tumors Number of Rats W1 th 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 3* 3/10 (30%) 49 4/10 (40%)h 1 ppb 0 4 51 4/10 (40%) 5 ppb 8 2 ioJ 7/10 (70%) a Source: Adapted from Van Miller et al. 1377a,b Rats at dose levels of 50, 500 and 1000 ppb were all dead within 4 weeks. c40 male rats used as controls for another study, received at the same time and kept under Identical conditions, did not have neoplasms when killed at 18 months. ' dl rat had ear duct carcinoma and lymphocytic leukemia 1 adenocarcinoma (kidney) 1 malignant histiocytoma (retroperitoneal) 1 angiosarcoma (skin) 1 Leydlg cell adenoma (testis) e3 rats died with aplastic anemia ^1 fibrosarcoma (muscle) 1 squamous cell tumor (skin) 1 astrocytoma (brain) , 91 fibroma (striated muscle) 1 carcinoma (skin) 1 adenocarcinoma (kidney) 1 sclerosing seminoma (testis) . hl rat had a severe U v e r Infarction *1 rat cholanglocardnoma and malignant histiocytomas (retroperitoneal) 1 angiosarcoma (skin) 1 glioblastoma (brain) 1 malignant histiocytoma (retroperitoneal) 3l rat had squamous cell tumor (lung) and neoplastic nodule (liver) 2 cholanglocardnomas and neoplastic nodules (liver) 3 squamous cell tumors (lung) - 1 neoplastic nodule 00110 V-75 09/18/84 TABLE V-10 Liver Tumors in Rats Ingesting 2,3,7,8-TCDDa Dose (ppb) Neoplastic Nodules Cholanglocard nomas Squamous Cell Tumors of the Lungs 0 0/10 (0%) 0/10 (0%) 1 0/10 (0%) 1/10 (10%) 5 4/10 (401i) 2/10 (20%)b ps0.043 0/10 0/10 4/10 (40%) p=G.043 aSource: Adapted from Van Hiller 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 Koclba et al. (1978a,b), was more extensive. Groups of 100 Sprague-Dawley 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-TCDD 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 1n 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 1n the high dose female rats, and lower body weights 1n 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) 1n both sexes at the 0.1 yg/kg/day dose, and urinary coproporphyrln and uroporphryln were Increasec1 (p<0.05) In females receiving 0.01 and 0.1 yg/kg/day. Statistically significant increases In tumors at several sites occurred 1n both male and female rats In the high dose group and 11ver lesions, Including hepatocellular neoplas tic nodules and lung lesions Including focal alveolar hyperplasia 1n m1ddosed females. Tumors of the hard palate, tongue and adrenal cortex were Increased In high-dosed males; tumors of the Uver, 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 1n the hepatocellular carcinoma Incidence 1n male animals. Other tumors commonly observed In male and female rats were significantly (p<0.05) decreased In the treated animals (Table V-l1). There was no Increase 1ri the neoplastic lesions 1n low-dosed males and females. In a study to determine the carcinogenic potential of the herbicide 2,4,5-trlchlorophenoxyethanol (2,4,5-TCPE), Toth et al. (1978, 1979) tested both this compound containing the known contaminant 2,3,7,8-TCDD and 2,3,7,8-TCDD alone by gavage In 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 vg/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 1n 0.5% carboxymethylcellulcse once a week for 1 year. Control groups administered each vehicle alone were Included Iti the study for comparison. Following the treatment period, th 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 1n either the 2,3,7,8-TCDD or Uic 2,4,5-TCPE/2,3,7,8-TCDD treat ment groups compared with the appropriate vehicle control were tumors of the Uver. These tumors consisted of both benign hepatomas and hepatocellular carcinomas. In the 2,3,7,8-TCDD treated mice, the Incidence of Uver tumors 01330 V-78 09/18/84 01330 TABLE V-ll Tumors That Here Significantly Decreased 1n Rats Following Exposure to 2,3,7,8-TCDDa V-79 Sex Tumor Type 0.0 pg/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 Pheochromocytoma of the adrenal H Subcutaneous flbroadenoma/f1broma/11poma F Benign tumors of the uterus F Benign tumors of the mammary gland F Carcinoma of the mammary gland F Pituitary adenoma 14/85 28/05 10/85 28/86 73/06 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 1n 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 con trol animals. The high dose group had a shorter lifespan, 424 days as com pared with 588 days 1n the control group, and this may have affected tumor yield. The tumor Incidence for mice receiving J.4.5-TCPE containing 2.3.7.8- TCDD 1s presented 1n Table V--12. The authors concluded that both 2,4,5-TCPE and 2,3,7,8-TCDD were carcinogenic since an Increased Incidence of U v e r tumors was observed 1n groups receiving 2,4,5-TCPE even though the 2.3.7.8- TCDD level 1n one group was 0.007 yg/kg/day, which was shown to be nontumor1genic 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 receiving the mixture 1s difficult to assess as a result of the i> of .twovehicles (sunflower oil and carboxymethylcellulose) with drastically differ ent physical properties. Under the National Toxicology Program, 2,3,7,8-TCDD has been tested for carcinogenicity by oral administration In Osborne-Hendel rats and B6C3F1 mice (NTP, 1980a) and by dermal application to Swiss-Webster mice (NTP, 1980b). 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 tr1chlorod1benzo-p-d1ox1n and PeCOO, with HxCDO 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 09/18/84 TABLE V-12 Tumor Incidence In Mice Treated with '2,4,5-TCPE Contaminated with 2t3,7,8-TC0Da 01330 Group TCPEb (mg/kg) Treatment Number Effective of Tumor TCDD Vehicle0 Sex Number of Bearing (tig/kg) (mg/kg) HUe Nice Liver (X) I Number of Animals with Tumors of: Lung Lymphomas Other Organs Average Lifespan V-81 1 67.0 2 70.0 3 4 7.0 5 7.0 6 0.7 7 8 9 10 11 12 -- 0.112 (1.6 ppm) 0.007 (0.1 ppm) control 0.07 (10 ppm) 0.0007 (0.1 ppm) 0.007 (0.1 ppm) control 7.0 0.7 0.007 -- 50 50 50 50 50 50 10 10 10 10 N 88 F 83 69 61 . H Sh F 96 H 93 F 84 78 59 63 57 N 93 F 96 N 94 F 93 N 97 F 94 79 60 77 71 78 64 K . 96 ' F 04 H 96 F 91 74 55 78 57 H 43 44 H, 44 N 38 * 27 36 39 27 aSource: Toth et al., 1979 bTCPE - Trlchlorophenoxy ethanol cCarboxymelhyl cellulose In groups 1-8, sunflower oil In groups 9-12. dp<lX ....... ep<0.1X 42d (18) 7 (8) 57e (58) 9 (9) 24 (26) 4 (5) 25 (27) 10 (10) 23 (21) 0 (9) 24 (25) 5 (5) 32 (33) 4 (5) 32 (33) 4 (4) 13 (30) 21 (48) 13 (29) 7 (18) 50 52 10 39 44 41 38 30 50 42 51 38 44 38 30 31 11 IB 27 15 . 7 15 11 15 8 23 18 19 23 36 20 22 14 18 22 24 6 12 10 6 16 595 25 552 16 571 23 582 17 577 13 639 22 641 19 589 17 660 21 590 17 643 21 566 22 615 17 565 15 651 19 549 7 424 4 633 6 649 7 588 09/18/84 In the oral study, (NtP, 1980a) groups of 50 male and 50 female rats received 2,3,7,8-TCDD by gavage In corn o1l:acetone (9:1) 2 days/week for I- 104 weeks. The weekly dose of 2,3,7,8-TCDD was 0.01, 0.05 or 0.5 j<g/kg bw. The male mice received Identical oral doses of test compound on the above schedule, while female mice received weekly doses of 0.04, 0.2 and 2.0 pg/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 chronic study the only overt sign of toxicity 1n rats was a slightly lower body weight 1n the high dose male and female animals after 55 and 45 weeks of treatment, respectively. Survival among control and treated groups was not significantly -different. Histologic examination revealed that toxic hepatitis was common 1n the high dosed animals, with the Uver 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 1n both Uver 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 1n the control, low, medium and high dose groups, respectively. .The Incidence of hepatic neoplastic nodules 1n 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->Arm1tage test (p=0.005 and p=0.001 for males and females, respectively); however, the Incidence of these tumors was 01330 V-82 09/18/B4 significantly (p=0.001) higher than controls only in 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 follicular-cell adenomas, adrenal adenomas or carcinomas and subcutaneous adenomas in 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 Bonferroni Inequality, criteria. The only tumors considered by.the NTP to be related to exposure to 2,3,7,8-TCDD were the thyroid tumors in male rats and the liver tumors in female rats. As observed in the rats, administration of 2,3,7,8-TCDO by gavage produced no overt signs of toxicity in mice with the only non-neoplastic histopathologic effect being toxic hepatitis. Neoplastic lesions that demonstrated both a significant dose-related trend and a greater incidence in the high dose animals Included hepatocellular adenomas or carcinomas in both male and female mice and thyroid follicular adenomas in female mice. The incidence of liver tumors was 8/73, 9/49, 8/49 and 17/50 in males and 1/73, 2/50, 2/48 and 6/47 in females, while the incidence of thyroid tumors in 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 subcutaneous fibromas in female mice and alveolar/bronchiolar adenomas or carcinomas of the lung in male mice were significant by either the CochranArmitage test or the Fisher exact test but not both, and did not meet the Bonferroni inequality criteria for overall significance. Under test condi tions it was concluded by the NTP (1980a) that 2,3,7,8-TCDD was carcinogenic to both male and female B6C3F1 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-TCDD levels of 150 ppt at the surface. Measured levels of 2,3,7,8-TCDD in the liver of beach mice from the contaminated area was determined to be 1300 ppt in males and 960 ppt In females. Detection of 2,3,7,8-TCDD in the liver .indicates that the compound was absorbed; however, since seeds in the area did not contain 2,3,7,0-TCDD it was believed that the animals Ingested the compound from contaminated dust while grooming. In the 10 male and 5 female animals cap tured in the contaminated area, there were no histopathologic differences, including neoplastic lesions, observed in the liver as compared with 9 male and 6 female beach mice captured in a noncontaminated area. The only observed difference in the two groups of mice was a statistically signifi cant (95% confidence) increase In liver to body weight ratios. The authors back-calculated from the 2,3,7,8-TCDD levels of the liver and .estimated a daily 2,3,7,8-TCDD dose of 0.0012 pg/kg bw. It was noted that this expo sure was much lower than the exposure used in laboratory studies to produce tumors. 01330 V-84 09/18/84 23,7,8-TCDD iKTP, 1980b) has been tested 1n mice for tumor 1genic poten tial by dermal application. This study was conducted under the NTP and the description of the chemicals used was the same as previously presented 1n the discussion of NTP (1980a). Groups of 30 male and 30 female Swiss-Webster mice were treated with 100 of a solution of the test compound 1n acetone 3 t1mes/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-TCDD used resulted 1n a dose of 0.01 pg/appl1cat1ori In male and 0.005 pg/app11cation 1n female mice. Subchronic toxicity studies used to define the dose levels for the chronic bioassay Indicated that all the doses used resulted 1n some liver damage but no Increase 1n mortality. 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, 1980b), there was no treatment-related difference In body weight of either sex between exposed animals and control groups; however, male mice treated with 2,3,7,8-TCDD had a significant shortening of lifespan (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). Nontumorlgenic hepatic lesions were observed 1n treated female mice, but not 1n 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 09/18/84 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=0.007) 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 1n untreated and vehicle control groups was similar. Using the mouse skin two-stage tumorlgenesls model, 2,3,7c8-TCDD has been tested for 1n1tat1ng and promoting activity. In thiT model, an Initia tor 1s a chemical that 1s applied to the skin for a very limited time under a dose schedule which will not result 1n tumor formation during the course of the study. Treatment with the Initiator, however, 1s 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, 1n 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. D1Giovanni et al. (1977) tested 2,3,7,3-TCDD, which was 98.6% pure, for 1n1tat1ng activity on the skin of female CD-I mice. Groups of 30 animals received an Initial single dermal dose of 2,3,7,8-TCDD of 2 vg/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 TABLE V-13 Assessment of the Initiation and Promotion Activity of 2,3,7,8-TCDD In Laboratory .Initials 01330 V-87 Specles/Straln ' Number of Sex Animals/Group Initiator/Dose Prcraoter/Dose H1ce/CD-1 Nice/ Swlss-Uebster F F H N H Nice/ Swlss-Uebster F F F N1ce/C0-1 F F F 30 30 45 30 30 45 30 30 30 30 30 2,3,7,8-TCOO/ 2 |ig/mouse DHBA/ 2.56 pg/mouse none TPA/5 iig/appllcatlon, 2 tlmes/week for 32 weeks TPA/5 iig/appllcatlon, 2 tlmes/week for 32 weeks none none QHBA/50 g (single appllcatlon) none 2.3.T.B-TCDD/0.001 vg/ application, 3 tlmes/week for 104 weeks ; 2,3,7,8-TCDD/0.001 vg/ application, 3 tlmes/week for 104 weeks none none 2,3,7,8-TCDO/0.005 yg/ application, 3 tlmes/week for 104 weeks DHBA/50 n'g ' (single ' appllcatlon) 2,3,7,B-TCDO/0.005 yg/ application, 3 tlmes/week ,for 104 weeks none 2,3,7,B-TCDD/0.1 yg/ application, 2 tlmes/week for 30 weeks DHBA/200 nntol (single appllcatlon) 2,3,7,B-TCOO/0.1 yg/ appllcatlon, 2 tlmes/week for 30 weeks 0HBA/200 nmol (single appllcatlon) TPA/2 yg/appllcatlon, 2 tlmes/week for 30 weeks Tumor Type X or llicldence of Animals Utth Tumors Reference dermal papilloma dermal papilloma 14 63 DIGIovannt et al.. 1977 dermal papilloma dermal papilloma 3/42 6/2B NIP, 1980c dermal papilloma 5/30 dermal papilloma dermal papilloma 2/41 8/27 dermal papilloma dermal papilloma dermal papilloma 6/29 O.OX o.ox Berry et al., 1978, 1979 dermal papilloma 9 09/18/84 TABLE V-13 (coni.) Specles/Straln . Number oF Sex Anlisali/Group Inttlator/Oose Proraoter/Oose Hlce/HRS/J (hr/*) F F F Htce/HRS/J (hr/hr) F F F F F Rats/ Charles River F F 20 0HDA/O.2 pmal none (single application) 20 none 2.3.7,8 TCOO/blweekly application oF SO ng/ application for B weeks followed by 20 ng/applt- catlon for 17 weeks 20 0HBA/0.2 pfflol 2,3,7,8-TCDD/blweekly (single application of SO ng/ application) application for 8 weeks followed by 20 ng/ application for 17 weeks. 20 0HBA/0.2 pmol ; IPA/blweekly application (single of 2 pg/mouse application) 20 DHBA/0.2 pmol none (single application) 20 none t 2,3,7,8-TCDD/blweekly application of 50 ng/ application for B weeks , followed by 20 ng/appll- catlon for 17 weeks 20 DHBA/0.2 vmol 2,3,7,8-TCDD/blweekly (single application of 50 ng/ application application for-8 weeks fallowed by 20 ng/appll- catlon for 17 weeks 20 BHDA/0.2 pmol TPA/blweekly application (single of 2 pg/mouse application) 4 DEN/10 mg/kg none 4 none 2.3.7.B-TC0D/O.14 pg/kg twice a week for 28 weeks* Tumor Type X or Ipcldence of Animals Kith Tumors Reference dermal papilloma 0/20 Poland et al., 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 -70X 0/4 0/4 Pitot et al.. i960 TABLE V-13 (coni.) 01330 Spectes/Stratn ' Number of Sex Anlmals/Group Inttlator/Dose Promoter/Dose Tumor Type Rats/ Charles River F F F 5 none 2.3.7.B-TCDD/1.4 jig/kg twice a week for 28 weeks* 5 DEN/10 mg/kg 2.3.7.8-TC0D/O.14 |ig/kg twice a week for 2B weeks* 7 DEN/10 mg/kg 2,3,7,B-TCDD/1.4 ^g/kg twice a week for 28 weeks* `The 2,3,7,6-TCOD was administered by subcutaneous Injection. hepatocellular carcinoma hepatocellular carcinoma hepatocellular carcinoma X or Incidence of Animals Ulth Tumors Reference D/S Pitot et al.. 1980 0/5 5/7 V-89 . 09/18/84 2.3.7.8- TCDD exposure was chosen from the ED^q for Induction of aryl hydrocarbon hydroxylase activity, and this level resulted In the death of 1/3 of the animals 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 cr 14% with an average of 0.1 paplllomas/mouse. This was 1n comparison with animals Initiated with dlmethylbenzathracene (DMBA), a potent 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 1n the study for comparison. The tumor promoting activity of 2,3,7,8-TCDD was Investigated, along with the complete carcinogenicity 1n the NTP (1980b) bioassay. In the tumor promotion study, groups of 30..mice of each1 sex were Initiated by a single dermal application of 50 pg of the tumor Initiator (DMBA). A week after the Initiation dose, the male mice received applications of 0.001 pg 3 times weekly and the female received 0.005 pg of 2,3,7,8-TCDD for 104 weeks. The skin tumor Incidence In both mle and female mice exposed to 2.3.7.8- TCDD plus DMA or the same level of 2,3,7,8-TCDD alone were nearly Identical with respective tumor Incidence 1n males of 5/30 and 6/28, and Incidences in females of 8/29 and 8/27. Similar results were obtained by Berry et al. (1978, 1979) 1n which 30 female CD-I mice were Initiated with a single 200 nmol application of DMBA followed by twice weekly applications of 0.1 pg of 2,3,7,8-TCDO for 30 weeks. At the termination of promotion, there were no dermal papillomas 1n either the 0MBA plus 2,3,7,8-TCDO group or the 2,3,7,8-TCDD group. In an additional group of mice 1n1t1r'id with DMBA followed by promotion twice a week with 2 pg of the known promoter 01330 V-90 09/18/84 {TPA) there was, however, a 92% Incidence of skin papillomas. The average number of tumors/mouse was 8.1. In the mouse skin two-stage tumorlgenesls model, 2,3,7,8-TCDD did not demonstrate tumor promoting activity. Poland et al. (1982) described studies which Indicate that genetic differences 1n mice affect the tumor promoting capacity of 2,3,7,8-TCDD 1n the skin two-stage tumorlgenesls model. Both 2,3,7,8-TCDD and TPA vfere compared for tumor promoting activity In DBA-1n1t1ated HRS/J mice that were either heterozygous (hr/+) or homozygous (hr/hr) for the recessive "hair less" trait. Promotion with biweekly applications of 2 pg of TPA for 25 weeks resulted 1n papilloma Incidences of 100 and 70% In (hr/+) and (hr/hr) mice, respectively. Promotion of DMBA-1n1t1ated (hr/+) mice with 2,3,.7,8TCDD (50 ng/appl1cation for 8 weeks followed by 20 ng/appl1cat1on) did not result 1n the formation of tumors, while promotion of (hr/hr). m1qe resulted 1n both the same Incidence and multiplicity of tumors as observed In TPApromoted mice. With either DMBA or N-n1trosoguan1dine (MNNG)-1n1t1ated (hr/hr) mice, the effective dose of 2,3,7,8-TCDD v/as -100-fold less than TPA on molar basis. Histologic examination of the skin showed that TPA produced both acute Inflammation and hyperplasia 1n (hr/+) and (hr/hr) mice, while 2,3,7,8-TCDD produced hyperplasia and hyperkeratosis only 1n (hr/hr) mice with no Inflammatory response. The lack of a 2,3,7,8-TCDD-1nduced Inflanmatory response suggested to the authors that 2,3,7,8-TCDD-promoted skin papillomas 1n (hr/hr) mice by a mechanism different from TPA. Pitot et al. (1980) Investigated the ability of 2,3,7,8-TCDD to act as a promoter for the hepatocardnogen dlethylnUrosam'.ne (DEN). Female CharlesR1ver rats were given DEN at a single 1ntragastr1c dose of 10 mg/kg during 01330 V--91 09/18/84 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 vg of 2,3,7,8-TCOD/kg bw for 28 weeks. Additional groups of partially hepatectomized animals received only the initiation treatment or the promotion treatment. No tumors were observed 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 hepatocellular carcinomas were observed 1n rats exposed to DEN followed by dietary administration of phnobarbital, a promoter of liver carcinogenesis. Foci of cells with altered enzyme pat ' 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 phenobarbi- tol Although this study- is-limited by the small size of the experimental, groups, the authors concluded that 2,3,7,8-TCDD was a promoter for DEN- initiated hepatocarcinogenesis 1n rats. Investigations have also been conducted on the affects of prior or simultaneous treatment with 2,3,7,8-TCDD on the subsequent development of skin tumors by chemical carcinogens. When 2,3,7,8-TCDD (0.1 yg) was administered simultaneously with DMBA (200 nmol) to the backs cf CD-I mice in ^ single initiation dose, the skin papilloma incidence following promo tion with TPA was nearly the same as when DMBA alone was used as the initia tor (D1G1ovann1 et al., 1977). Although simultaneous exposure to 2,3,7,8TCDD and DMBA did not appreciably affect tumor yield, Berry et al. (1979) demonstrated a marked 93% decrease In the incidence of DMBA initiated tumors when CD-I mice were pretreated 3 days before DMBA initiation with 1 vg/ 01330 V-92 09/18/84 mouse of 2,3,7,8-TCDD. The time of treatment with 2,3,7,8-TCDD in relation to Initiation was shown to be critical in the antitumorigenic effect of 2,3,7,8-TCDD (Berry et al., 1979; Digiovanni 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 day after DMBA Initiation ha'd no effect on tumor yield. There was some indication of an inverse relationship between the pretreatment (3 days beforeDMBA initiation) dose of 2,3,7.8-TCBD and the incidence of tumors. Dosesof 2,3,7,8-TCDD of 0.0, 0.01, 0.1 and 2 pg/mouse resulted In decreases 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 pretreated with 1 pg of 2,3,7,8-TCDD .3 days before, initiation by DMBA. Kouri et al. (1978) reported that i.p. injection of 1-100 mg 2,3,7,8-TCDD/kg preceded by MCA but not trioctanoin raised the carcinogenic index in B6 but notD2mice. The term 'carcinogenic index' was defined by Kcuri et al. (1978)as percentage of tumor Incidence 8 months after treatment divided by the average latency in days multiplied by 100. D161ovanni et al. (1980) investigated the antitumoriqenic effect of 2,3,7,8-TCDD in CD-I mice with chemical carcinogens other than DMBA. As observed with DMBA, exposure to 2,3,7,8-TCDD 3 days before Initiation with either benzo(a)pyrene (BaP) or 3-MC resulted in a decrease in tumor yield as compared with acetone pretreated animals, while pretreatment with 2,3,7,8TCDD 5 minutes before or 1 day after Initiations was ineffective in changing the tumor yield. The maximum decrease in tumor production was 86 and 57%, 01330 V-93 09/18/84 respectively, for BaP and 3-MC Initiated mice. A different temporal rela tionship was observed 1n the ability of 2,3,7,8-TCOD to Inhibit tumor forma tion by BaP-d1ol-epox1de as compared with the previously studied polyaroma tic hydrocarbons (PAH). When 2,3,7,8-TCDD was applied 3 days or 5 minutes before, or 1 day after Initiation with BaP-d1ol-epox1de, decreases 1n tumor yield were 31.5, 49 and 39%, respectively. Examination of PAH metabolism 1n ths skin of mice treated with 2,3,7,8-TCOD showed a 21-fold Increase 1n aryl hydrocarbon hydroxylase (AHH) activity 72 hours after treatment (D1G1ovann1 * et al., 1980). The 1_n v1 tro metabolism of OMBA by dermal homogenates from 2.3.7.8- TCOD-treated mice Indicated both qualitative and quantitative changes In metabolism (1.e., changes 1n both rates of metabolism and rela tive types of metabolites formed) (Cohen et al., 1979; D1G1ovann1 et al., 1979b; Berry et al., 1979). The similarity 1n the time frame of AHH Induc tion and the antitumorlgenlc-effect of pretreatment with 2,3,7,8-TCDD sug gested that the antitumorlgenlc properties of 2,3,7,8-TCDD resulted from 2.3.7.8- TCDD-Induced alteration 1n the metabolism of the Initiating chemi cal. Although metabolic change was a possible mechanism for the Inhibition of DMBA, 3-MC and BaP Initiation; the ability of 2,3,7,8-TCDD to Inhibit tumor yield when administered 1 day after Initiation with BaP-d1ol-epox1de (which does not require metabolic activation) Indicated to D1G1ovann1 et al. (1980) that more than one mechanism may participate 1n the antlcardnogenlc effect of 2,3,7,8-TCDD. Mutagenicity. Short-term In, vitro test systems have been developed to assess the biologic, toxic and genotoxlc effects of chemicals. These assays t have proven to be useful Indicators of potential activity of diverse Indus trial chemicals, a broad range of drugs and xenoblotlcs, carcinogens and 01330 V-94 09/18/84 crude environmental extracts. The most widely used short-term test system, the Ames test for bacterial mutagenesis, employs several strains of Salmo nella typhimurium 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 al., 1982). Mutagenicity assays in microorganisms have been used to assess the genotoxic 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). Hussain et al. (1972) exposed S. typhimurium histidine-dependent strains TA1530 and TA1532 in liquid suspension to 2,3,7,8-TCDO followed by plating into selective medium to observe reversion to prototypes. No increase in the reversion rate was. observed with strain TA1530 at exposure levels of 1 and 10 pg/m2.. These exposures resulted in cell survivals of 90 and <1%, respectively. In strain TA1532, increased reversion frequency was not observed at 2,3,7,8-TCOD concentrations of 2-3 pg/mi, which resulted in a 0-50% decrease in survival; however, at 2,3,7,8-TCDD levels that resulted in a 99% decrease 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-TCOO sample studied in this paper was the Food and DrugAdministration, and its reported purity was 99%. Also, Seiler (1973)' observed a positive mutagenic response in a spot test of 2,3,7,8-TCOD per formed in the absence of a metabolic activation system. However, the purity of the sample studied was not provided. In tester strains G46 and TA1530, 01330 V-95 09/18/84 01330 TABLE V--14 The Results of Mutagenicity Assays for 2,3,7,8-TCDD In Salmonella typhlmurlum V-96 Type of Assay Strains of Salmonella tvphlmurluw S-9 TA98 TA1530 TA1535 TA1537 TA1538 TA1S32 TA19S0 TA1975 TA1978 646 TA160 TA1531 TA1534 Reference Spot test /- NT NT 00 8 8 NT NT NT NT NT NT NT McCann. 1978 Plate /- NT NT 80 0 8 NT NT NT NT NT NT NT McCann. 1978 Incorporation Plate /- 8 Incorporation* 8 0 8 8 8 8 l 8 8 8 8 NT NT 611bert et al.. 1980 fluctuation test /- 8 0 8 ' 8 0 8 8 8 0 8 0 NT NT Gilbert et al., 1980 Spot test - NT 0 NT NT NT NT NT NT 8 . NT QR QR Seller, 1973 Plate 8 NT 08 0 NT NT NT NT NT 0 NT NT Geiger and Neal, 1981 Incorporation Plate - NT NT NT 8 Incorporation NT NT NtT NT NT NT NT NT NT Geiger and Neal, 1981 Suspension assay Suspension assay - NT 8 NT NT ! NT NT NT NT NT NT NT NT Hussain et al.. 1972 /r 0 NT 9 08 NT ,, NT NT NT NT NT 8 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 the ratio of revertants/108 cells In the treated plates divided by spon taneous revertants/10" cells was <1. In strains TA1531 and TA1534, the ratio was between 1 and 2, which was considered a 'doubtful" mutagenic 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, dlethylsulfate, 2-am1nopur1ne and 2-amlnofluorene, produced ratios of 2 to 5, <1 and 5 to 10, respectively, In strain TA1532. In both the study by Hussain et 'al. (1972) and the study by Seller (1973), 2,3,7,8-TCDD produced a positive mutagenic response only 1n the S. typh1mur1um strain TA1532, which Is sensitive to frameshlft mutagens. Hussain et al. (1972) also performed a mutagenicity test of 2,3,7,8-TCDD 1n two other microbial test systems. A positive response was observed 1n Escherichia coll Sd-4 as indicated by a reversion to streptomycin Independ-. ence. In this assay, cells were treated 1n suspension for 1 hour with 2,3,7,8-TCDD at 0.5-4 pg/mi, The greatest mutation frequency (256 mutants x 10"8, as compared with the control frequency of 2.2 mutants x 10"8} occurred at a dose level of 2 pg/mi.. The absolute number of colonles/plate was 7 for the control and 46 for the treated plate. The dose of 2 pg/mi. caused an 89% decrease 1n cell survival. In the second test system, the ability of 2,3,7,8-TCDD to Increase prophage Induction 1n E.. co11 K-39 cells was examined. The vehicle control, DHSO, Inhibited prophage Induction as compared with the untreated controls, while the most effective dose level of 2,3,7,8-TCDD (0.5 pg/mi) resulted In an Increased prophage 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 01330 V -97 09/18/84 capable of causing Increases 1n the reverse mutation rate 1n . coil Sd-4 and that 2,3,7,8-TCDD had a weak ability to Induce prophage 1n E.. coll K-39 cells. . The studies that followed these two early reports of Hussain et al. (1972) and Seller (1973) failed to detect mutagenic activity of 2,3,7,8-TCDD 1n S. typhlmurlum. Wassom et al. (1978) cited a personal communication from McCann (1978), which reported that 2,3,7,8-TCDD was Inactive In both the spot test and plate Incorporation assay with S. typhlmurlum strains TA1532, TA1S35, TA1537 and TA1S38. Doses and other experimental protocols were not mentioned except that the tests were performed both with and without metabo- 11c activation. Gilbert et al. (1980) reported that 2,3,7,8-TCDD gave "substantially negative results" with S. typhlmurlum stra'-v lt TA100, TA1530, TA1535, TA1537, -TA1S38, G46, TA1532, TA1950, TA1 at: TA1978. Both the standard plate Incorporation assay and the bacter>. .'luctuatlon 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 assay. It Is difficult to assess possible reasons for the conflicting results between the earlier studies and these later mutagenicity assays, since Information on experimental conditions was limited 1n the negative studies. In an attempt to resolve the conflicting results and observe a mutagenic response, Geiger and Neal (1981) tested 2,3,7,8-TCDD 1n the standard plate Incorporation assay using S-S prepared from different sources. In order to I 01330 V-98 09/18/84 maximize the amount of compound tested, dioxane, 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 $-9 used in these assays was prepared from the livers of Aroclor 1254 pre treated male Sprague-Dawley rats and male Golden Syrian hamsters, and from 2.3.7.8- TCDD induced male hamsters. In all assays at 2,3,7,8-TCDD concen trations of 0.2, 2, 5 or 20 yg/plate, and regardless of the source of the S-9, there was no observed mutagenic 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 direct descendent of strain TA1532, for mutagenic activity in the absence of S-9. Again, no increase in the number of revertants was observed. In assays either with or without S-9, positiye 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 positive evidence of carcinogenicity. Mutagenic effects of 2,3,7,8-TCDD in yeast were observed by Bronzetti et al. (1983). Positive results for reversion and gene conversion were obtained in vitro and in the host-mediated assay. The in vitro experiments yielded small dose-related increases in trp+ convertants and 1lv+ revertants. An SI0 metabolic activation system was required. Exposure of 01330 V-99 / 09/18/84 the yeast to 2,3,7,8-TCDD at the highest level tested (10 yg/mi.) result ed In 1654 survival and yielded 4-fold Increases 1n reversion and gene conversion. In the host-mediated 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 mi. of a yeast culture (4 x 10 cells) was Instilled retroorbitally. Four hours later, the U v e r and kidneys were removed and the yeast cells 1n these organs were assayed for mutagenic responses. Increases (4- to 6-fold) 1n reversion and gene conversion were observed 1n yeast cells obtained from the livers and kidneys. . The toxic response of the animals to an exposure of 25 yg/kg was not described 1n this report. The positive results described In this paper suggest that 2,3,7,8-TCDD 1s mutagenic 1n yeast, but more definitive studies are needed .before a firm conclusion can be drawn. . Hay (1982) has found that 2,3,7,8-TCDD dissolved 1n DMS0 transformed baby hamster kidney cells (BHK) In vitro. The dioxin Isomers 2,8-d1chloroand 1,3,7-tr1chlorod1benzo-g.-d1ox1n also transformed BHK cells, but the response was weak. The unchlorinated d1benzo--d1ox1n and the fully chlori nated octachlorod1benzo--d1ox1n were both negative In the BHK assay (l.e., there was no cell transformation). More recently, Rogers et al. (1982) reported that 2,3,7,8-TCDD Induced mutations 1n the excess thymidine, th1oguanlne and methotrexate selective systems 1n L5178Y mouse lymphoma cells 1n culture. The National Toxicology Program (NTP) (Zelger, 1983) provided data on 2.3.7.8- TCDD from four assay systems: the S. tvphlmurlum (strains TA98, 01330 V-100 09/18/84 TA1, TA1535 and TA1537) histidine reversion assay, the sex-linked reces sive lethal test in Drosophila. and cytogenetic studies (sister chromatid exchange and chromosome aberrations} in Chinese hamster ovary cells. Nega 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. l vitro reactions of 2,3,7,8-TCDD with bacteriophage QB RNA were eval 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 pg/mi of 2,3,7,8-TCDD. At all- concentrations 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 authors suggested that 2,3,7,8-TCDD inactivity in this assay indicated that 2,3,7,8-TCDD was an intercalating agent, and hence would require double stranded DNA in order to Interact. The data presented in this study, however, were insufficient to support this conjecture. In. vivo binding of radiolabeled 2,3,7,8-TCDD to liver macromolecules was studied in Sprague-Dawley rats by Poland and Glover (1979). Both male and female animals were administered [1,6-*H]2.3,7,8-TCDD i.p. at a dose of 7.5 pg/kg. This dose corresponded to a tritium level of 0.87 mC1/kg. The 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 01330 V-101 09/18/84 label bound to protein, RNA and DNA was determined. The greatest non extractable binding of labeled 2,3,7,8-TCDO occurred to protein; however, the amount of label bound was small and only amounted to 0.03-0.1% of the total radioactivity administered. The total amount of label associated with RNA and ONA 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-TCDO binding. As a result of the extremely low levels of radio activity associated with RNA and DNA, 1t 1s uncertain whether 2,3,7,8-TCDO truly binds covalently to these macromolecules 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 aber rations In the bone marrow of male rats were reported In an abstract by Green and Moreland (1975). -In the Initial experiment, no Increase- 1n chromosomal aberration was observed after five dally gavage treatments at a 2.3.7.8- TCDD dose of 10 pg/kg. In the second portion of this study, rats were exposed by a single Intraperltoneal Injection of 2,3,7,8-TCDD at 5, 10 or 15 pg/kg or a single gavage treatment at 20 pg/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 aberrations was observed, except In the positive control group exposed to trlethylenemelamlne. In a later report, a small but significant Increase In chromosomal aberrations was observed 1n the bone marrow cells of male and female Osborne-Mendel rats (Green et al., 1977). 8one marrow cells for cytogenetic analysis were obtained from Osborne-Mendel rats used In a range-finding 01330 V-102 09/18/84 study preliminary to a chronic bioassay (Green et al., 1977). The animals 1n groups of 8 males and 8 females received twice weekly Intubations of 2,3,7,8-TCOD at respective doses of 0.25, 1.0, 2.0 and 4.0, or 0.25, 0.5, 2,o and 4.0 pg/kg for 13 weeks. Because 1t wav not required for the range-finding study, a control group was not Included. Bone marrow cells were analyzed for abnormalities and cells 1n mitosis 1n the animals ,that survived to the end of the study (4-8 anlmals/group). The only significant Increases 1n chromosomal aberrations 1n comparison with the low dose group were In males at 2 and 4 Pg/kg and females at 4 pg/kg. The greatest Incidence observed was 4.65 of the cells with chromosomal breaks 1n 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-TCDO toxicity, makes-the conclusion from this study.that 2,3,7,8TCDO produced chromosomal breaks tenuous. A similar weak response wa* observed by Loprleno et al. (1982) 1n male and female CD-I mice which received an 1.p. Injection of 2,3,7,8-TCDO at a dose of 10 pg/kg. At 96 hours post-treatment, there was a significant (p<0.01) Increase 1n 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 chromo somes. The study was continued with a more extensive experiment using CD-C0BS female rats. ' The rats were treated weekly by gavage (vehicle acetone-corn oil 1:6) at doses of 0, 0.01, 0.10 or 1.00 pg/kg for 45 weeks. Analysis of bone marrow cells for chromosomal aberrations 24 hours after the last treatment failed to detect any significant increases. 01330 V-103 09/18/84 Czcizel and Klraly (1976) reported an Increased Incidence (p<0.001) of chromatid-type and unstable chromosome aberrations 1n the peripheral lympho cytes of workers exposed to the herbicides 2,4,5-trlchlorophenoxyethanol (2,4,5-TCPE) and Bumlnol. The 2,3,7,8-TCOD 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 1n the lymphocytes of 15 soldiers exposed to Agent Orange. The expo 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 2,3,7,8-TCDD. 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 Regg1an1 (1980) and Mottura et al. {1981) have studied Inhab itants 1n Seveso, Italy, exposed to 2,3,7,8-TCOD from an accident 1n a trichlorophenol manufacturing plant. Regg1an1 (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 determine extent of Injury. Although burn-Hke skin lesions In these 17 Individuals Indicated chemical exposure, no Increase 1n chromosomal aberra tions was detected. The methods of performing the analyses and the actual number of aberrations detected were not described. Similar negative results 01330 V-104 09/18/84 were reported in an abstract by Hottura et al. (1981). In this study, sub jects were chosen from the area of heavy contamination following the acci 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 group was not provided. The specimens were examined by three independent laboratories and no laboratory reported an increase in chromosomal aberrations, although there was a significant difference in the . reported scores between laboratories. There 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 chromoso mal aberrations. . DiLernla et al. (1982) conducted additional studies on lymphocytes prepared in 1976 and 1979. from eight persons considered acutely exposed to. 2,3,7,8-TCDD in the Seveso accident, eight ICMESA factory workers (consider ed chronically exposed), and 14 control subjects (eight had chromosome prep arations made in 1976 and six in 1979). Cells were examined for average number of SAs (evidence for functional ribosomal genes), both on a cell basis and for the large acrocentric chromosomes (D group chromosomes). There was no change in the frequency of SAs on a per cell basis in any of the groups as compared to control values, nor in D group chromosomes from acutely exposed subjects examined immediately after the accident. There was, however, a decrease in the average frequency of SAs in group D chromo somes of acutely exposed subjects examined in 1977 and in ICMESA workers at both the 1976 and 1979 examinations. Although the biologic relevance of these observations has not yet been confirmed, DiLernia et al. (1982) observed a similar decrease in SAs after exposure of lymphocytes to 01330 V-105 09/18/84 x-1rrad1at1on. It was concluded that the decrease 1n SAs may have resulted from mutagenic damage to functional nucleolar organizing regions. A limited number of Initial studies on the mutagenicity of 2,3,7,8-TCDD 1n bacteria reported positive results 1n S. tvphlmurlum strain TA1532 1n the absence of a mammalian metabolic activation system (Hussain et al., 1972; Seller, .973). 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 1n mutation rate when 2,3,7,8-TCDD was tested 1n the presence of a mammalian metabolic activation system. In other In vitro assays, 2,3,7,8TCDD has produced a :positive response In reversion to streptomycin Independ ence In E.. coll Sd-4 cells and questionable positive response with prophage Induction 1n E. coll K-39 cells (Hussain et al., 1972). Also, 2,3,7,8-TCDD has been reported to be mutagenic 1n the yeast S. cerevlslae 1n both the In vitro assay with S-10 and the host-mediated assay (Bronzettl et al., 1983). Rogers et al. "(1982) have also reported positive mutagenicity results 1n the mouse lymphoma assay system. In the E. coll studies, the poor survival of the cells or the Interference of the vehicle solvent, DMSO, with the assay makes the evaluation of the studies difficult. With the data available, It 1s not possible to[resolve the conflicting reports on the mutagenic poten 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. Kondorosl et al. (1973) demonstrated that 2,3,7,8TCDD did not react with RNA In. vitro 1n the absence of a metabolic activa tion system. In vivo studies using radiolabeled 2,3,7,8-TCDD Indicated some 01330 V-106 09/18/84 association of non-extractable label with RNA and ONA (Poland and Glover, 1979); however, the level of bound label was very low. Similar marginal data were available on the clastogenic effect of 2,3,7,8-TCDD. Although two in vivo studies in rats (Green and Moreland, 1975; Loprieno et al., 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 aberrations. A similar small increase was observed by Loprieno et al. (1982) following a single i.p. injection of 2,3,7,8-TCDD 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) reported an increase 1rt the number of chromosomal aberrations, while no increase was detected in Individuals exposed to 2,3,7,8-TCDD following an industrial accident in Seveso, Italy (Reggiani, 1980; Mottura et al., 1981). The studies of the clastogenic effect of 2,3,7,8-TCDD were presented with little or no experimental detail to assist in evaluating 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. 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-TCDO. This chemical may be a weak mutagen, but because it is very toxic, the dose range for detecting a positive genetic effect may be very narrow. Therefore, additional experi mentation is necessary before any conclusive determination can be made. Suggested further testing Includes the ability of 2,3,7,8-TCDD to Induce forward mutations in mammalian cells 1n culture, additional yeast and bacterial studies and the sex-linked recessive lethal test in Drosophila. 01330 V-107 09/18/84 Teratogenicity and Reproductive Toxldtv. A number of Investigators have studied the role of 2,3,7,8-TCDD contamination In 2,4,5-T-1nduced teratogenicity (Table V-15). Neubert and Olllmann (1972) Investigated the teratogenicity of purified 2,3,7,8-TCOO, 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 urviietermlned amount of 2,3,7,8-TCOO. The EDjQ for the production of cleft palate by 2,3,7,8-TCDD was determined to be 4.6 yg/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 resulted In hepatic and thymic toxicity, no significant changes were noted In' mating frequency, average 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 al., 1971; Khera and McKinley, 1972). At doses <100 mg/kg bw/day of contam inated 2,4,5-T, the only skeletal effect appeared to be delayed ossification (Emerson et al., 1970, 1971; Sparschu et al., 1971b). The teratogenicity of purified samples of 2,3,7,8-TCDD Is presented 1n Table V-16. Courtney and Moore (1971) administered subcutaneous Injections of 1 or 3 ,,g of purified 2,3,7,8-TCDD/kg bw/day to CD-I, DBA/2J and C5781/6J mice on days 6.-15 of gestation. This treatment had no effect on m non 'TABLE V-15 Studies on the Potential Tefatogenlc.Effects of 2,3.7.8-TCDO Contaminated 2.4,5-T 01330 V-109 Species/ Strain Vehicle Nice/ NHRI Rape-seed oil Rape-seed oil Rape-seed oil Rape-seed oil Hice/ NHRI NR Hice/ CO-1 Corn oil: acetone (9:11 Form of 2.4.5-T TCDO Level Dally Dose ---------------- -------------------------------------------------------------------------------------------- -------------- i ----------------------------------------------------------------- Treatment Observation . Oays Day Haternal Response Fetal Response Reference acid <0.02 ppm (Sample A) 0, 15, 30, 45. 60, 90. and 120 mg/kg 6-15 acid 0.05*0.02 ppm (Sample B) 30, 60 and 90 mg/kg 6-15 acid VR 90 mg/kg (Sample CJ 6pl5 butyl ester acid NR 0.05*0.02 ppm 12 and 17 mg/kg . t 20, 35, 60, 90 and 130 mg/kg 6-15 6-15 acid <0.05 ppm 115 mg/kg JO-15 IB No toxic effects: Significant increases In Neubert and Olllmann, decreased maternal the Incidence of cleft 1972 weight at doses of palates at doses above 90 mg/kg and greater 30 mg/kg (see text for additional details). < Significantly decreased (p<0.005) fetal weight at all dose levels. IB Ho toxic effects: I decreased maternal Increases In the Incidence of cleft palate at 60 and weight at 90 mg/kg 90 mg/kg; significant decrease In fetal uelght (p<0.005) at all dose levels IB No toxic effects increase In the Incidence but decreased of cleft palate; significant maternal weight (p<0.005) decrease In fetal weight 10 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) 10 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 09/18/84 to CO o Species/ Strain Vehicle Form of 2,4.S-T TCDO Level TABLE V-15 (coni.) Dally Dose Treatment Observation Days Day Halernal Response Fetal Response Reference Mice/ C5B1/6 or Hlce/ AKR lloneyzwater (1:1) or DHSO acid 30 ppm 21.5, 46.4 and 113 mg/kg 6-14 Hlce/ AKR Honeywater acid (1:1) 30 ppm 113 mg/kg 6-15 Rats/ Gavage/ Sprague- hydroxy- <I Dawley (groups propyl methyl of 25 cellulose rats) acid <3.5 ppm 1. 3, 6. 12 or 24 mg/kg/ day 6-15 Rats/ Distar Gavage/ aqueous gelatin or c o m oil acid <0.5 mg/kg . 25, SO. 100 ' or ISO mg/kg/ day 6-15 3 6avage/ aqueous butyl ester <0.5 mg/kg SO or 150 mg/kg/day 6-15 x> gelatin or corn oil 0* IB 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 Courtney et al., 1970a,b 19 . Increase In llver-to-body ( weight ratio Significant (p<0.05) Increases In the Incidence cf cleft palate and fetal mortality Courtney et al., 1970a,b 20 No effect on body A slight but statistically Emerson et al., weight and no significant (p<0.05) 1970, 1971 M.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., 1971 dose group; the only effect noted was an Increase In the Incidence of Sth par tially ossified sternebrae 22 Some maternal mor tality and decreased body At 100 or ISO mg/kg, decreased fetal.weight. Increased fetal mortality Khera and HcKtnley, 1972; Khera et al., 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 tuo lower below dose levels 22 NR No significant effect on fetal mortality, fetal weight, or the Incidence of anomalies Khera and HcKtnley, 1972; Khera et al.. 1971 T A B V--15 (coni.) 01330 V-lll Species/ Strain Vehicle Form of 2,4,5-T TCOO Level Dally Oose Treatment Observation Days Oay Maternal Response fetal Response Reference Rats/ lloltzman Gavage/ 1:1 solu tion of honey and water acid 30 ppm 4.6. 10.0, and 46.4 mg/kg/day 10-15 Rats/CD 6avage/ 1SX sucrose solution acid 0.5 ppm 10.0, 21.5, 46.4 and 00.0 mg/kg/ day 6-15 Rats/ Strain not speci fied Gavage/ methocel Gavage/ methocel acid 0.5 ppm 50 mg/kg 6-15 acid 0.5 ppm , 100 mg/kg ' 6-10 Syr1an hamsters/ Hesocrtcetus euratus Gavage/ acetone, c o m oil, and carboxymethyl cellulose In ratio of 1:5.8: 10 acid <0.1-4.5 ppm 20, 40. 80 and 100 mg/kg 6-10 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 .ystlc kidneys In treated groups Courtney et al., 1970a,b 20 Reduced maternal Increase In the Incidence weight gain at the of kidney anomalies but t 2 higher dose no Increase In cleft levels (p<0.05) palate and Increased Uver-to-body weight ratio at the highest dose level (p<0.05) Courtney and Hoore, 1971 NS No effect on mor No significant effect on Sparschu et al., 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 ip<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 e Not specified; NR > Not reported 09/18/84 09/18/84 V-112 01330 Specles/Straln Vehicle3 House/C0-1 mouse/DBA/2J mouse/C5781/6J House/C57Bl/6 House/CD-1 0HS0b Acetone:3 corn oil (l:3i 0NS0b or corn oil House/CF-1 House/NHRI corn oil3 : acetone (98:2) rape-seed3 oil Rat/CD DHS0b Rat/SpragueDawley corn oil3/ acetone TABLE V-6 Studies on the Potential Teratogenic and Reproductive Effects of 2,3,7,8-TCOD Compound Dally Dose Treatment Observation Days Oay Maternal Response ^ ----------- 1---------Fetal Response Reference 2,3,7,8-TCOD 1, 3 ng/kg 6-15 17c or IB Increased liver/ body weight ratio cleft palate, kid Courtney and Moore, ney anomalies^ 1971 2,3,7,8-TCOD 1, 3 ng/kg 10-13 or 10 18c none reported cleft palate, kid Moore et al., 1973 ney anomalies'1 2,3,7,8-TCOD 2,3,7,8-TCOD 2.3.7,8-TCDD 2,3,7,0-TCDD 2,3,7,8-TCOD 25, 50, 10, 200, 400 ng/kg 7-16 0.001, 0.01, 0.1, 1.0, 3.0 ng/kg : 6-15 ' 0.3, 3.0, 4.5, ?.o ng/kg 6-15 r 0. 0.5. 2.0 ng/kg 0. 0.03, 0.125, 0.5, 2.0 and B.O ng/kg ,, 6-15. 9 and 10, or 13 and 14 6-15 10e 1. let 18 20c . 20' Increased liver/ body weight ratio none reported no effect observed none reported vaginal hemorrhage at 2.0 and 8.0 ng/kg cleft palate, hydronephrolic kidneys, hydrocephalus, open eyes, edema, peUchlae cleft palate, dilated renal pelvis fetocldal at the high dose, cleft palate at doses at or above 3 ng/kg kidney malforma tions at both dose levels Intestinal hemorrhage at 0.125 and 0.5 ng/kg, fetal death of higher doses, subcutaneous edema Courtney, 1976 Smith et al., 1976 Neubert and Olllmann, 1972 Courtney and Moore, 1971 Sparschu et al., 1971a T A B U V-16 (coni.) 01330 V-113 Specles/Straln Vehicle* ' Compound Dally Dose Treatment Observation Days Day Maternal Response Fetal Response Reference Rat/Ulstar corn oil*/ anlsole Rat/SpragueDawley corn oil*/ acetone (9:1) Rat/SpragueDawley diet Rabbit/ New Zealand Monkey/rhesus corn oil*/ acetone (9:1) diet Honkey/rhesus diet 2,3,7.8-TCDD 2.3.7.B-TCDD 2.3.7.B-TCD0 2,3,7,0-TCDD 2,3,7,6-rTCDO 0. 2,3,7,B-TC00 0.0, 0.125, 0. 25, 0.5, 1. 2. 4. B. 16 pg/kg 6-15 0.0, 0.125, 0.5, 2.0 pg/kg 1-3 0.001, 0.01 and 0.1 pg/kg" throughout ' gestation 0.0, 0.1, 0.25, O'.5 and 1 pg/kg 6-15 8.6 pg/kg/day ' 7 months before and during gestation 55.7 pg/kg/day 7 months before and during gestation 22 2V . 1 postparturltlon 28 at term at term maternal toxicity observed at or above 1 pg/kg Increased fetal death observed at or above 1 pg/kg, subcutaneous edema and hemorrhages In the 0.25-2.0 pg/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 pg/kg group, cystic kidneys and dilated renal pel vis occured In the 2'pg/kg group Glavlnl et al., 1982a low fertility at 0.01 and 0.1 pg/kg, decreased body weight at 0.01 and 0.1 pg/kg, dilated renal pelvis low survival at 0.01 and 0.1 pg/kg, decreased body weight at 0.01, slight dilated renal pelvis at 0.001 pg/kg Murray el al., 1979 maternal toxicity at doses of 0.25 pg/kg and above Increases In extra Glavlnl at al., 1982b ribs and total soft `' tissue anomalies 6/8 conceived, nor mal serum estradiol and progesterone 3/8 rofmal births Allen et al., 1979 3/B conceived, de creased serum estra diol and progester one I/O normal births Allen et al., 1979 Administration was by gavage. Administration was by subcutaneous Injection. cF1rst day of gestation designated day zero. . Aldney anomalies were not specifically defined. eF1rst day of gestation designated day one. fThe high dose level (0.1 pg/kg/day) was discontinued because of very low fertility In adults. 09/18/84 fetal mortality; however, cleft palate and unspecified kidney anomalies were found at all dose levels 1n all strains, with C57B1/6J being the most sensi tive strain. Moore et al. (1973) administered oral doses of 2,3,7,8-TCDD (1 or 3 pg/kg/day) to C57B1/6 mice by gavage on days 10-13 of gestation. Cleft palate and hydronephrosis were observed 1n both dose groups. These kidney lesions were apparently reversible, since very few Utters (1/14) that were cross-nursed on control mothers had pups with kidney abnormalities. In contrast, kidney lesions were found 1n 4 of 14 control Utters that were nursed on 2,3,7,8-TCDD treated mice. Neubert and Olllman (1972) and Neubert et al. (1973) administered dally oral doses of 0.3, 3.0, 4.5 .or. 9.0 pg/kg bw to NMRI mice on days 6-15 of gestation. Extensive resorption (6 of 9 litters completely resorbed) occur red In.the high-dose group. Cleft palate, but not kidney abnormalities, were observed at a dose of 3.0 pg/kg bw/day or higher, with 0.3 pg/kg bw/day being the N0AEL 1n this study. A single dose of 45 pg/kg bw could produce cleft palate 1f given before day 13 of gestation, with maximum Incidences occurring when the dose was given on day 8 or 11. Courtney (1976) compared the effectiveness of oral and subcutaneous administration 1n CD-I mice. Subcutaneous administration was found to pro duce a greater teratogenic response at a lower dose than did oral administration. 01330 V-114 09/18/84 Smith et al. (1976) determined the minimum effective oral dose (MED) for producing teratogenic effects in CF-1 mice to be 1 pg 2,3,7,8-TCDD/kg bw/day. The NOAEL in this study was 0.1 pg/kg bw/day. Courtney and Hoore (1971} determined the teratogenic potential of subcutaneously Injected 2,3,7,8-TCDD (0.5 or 2 pg/kg bw/day) in CD rats. The compound was administered in dimethylsulfoxide on days 6-15, 9-10 or 13-14 of gestation. The only developmental anomalies observed were kidney malformations 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) administered 0.03, 0.125, 0.5, 2.0 or 8.0 pg 2,3,7,8-TCDD/kg/day by gavage_.to Sprague-Dawley rats on days 6-15 of yestation. 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 (1S73) intubated groups of 7-15 pregnant Wistar rats with 0.125, 0.25, 0.5. 1, 2, 4, 8 or 16 pg 2,3,7,8-TCDD/kg bw/day on days 6-15 of gestation. Severe fetotoxic effects were observed at doses of 1 pg/kg bw/day or higher, with no live fetuses found in the groups exposed to 4, 8 or 16 pg/kg bw/day. No anomalies were observed in the group receiving 0.125 pg/kg bw/day. In the intermediate dose groups, 0.25-2.0 pg/kg bw/day, a number of anomalies, including subcutaneous edema of the head and neck and hemorrhages in the intestine, brain, and subcutaneous 01330 V--115 09/18/84 tissue, were observed. When the dams were allowed to Utter and wean the pups, none of the pups 1n groups receiving >1 pg/kg bw/day survived until weaning. Fostering pups from dams exposed to 1 pg/kg bw/day to control dams did not appreciably Increase survival (36/42 died). G1av1n1 et al. (1982a, 1983) administered 0, 0.125, 0.5 or 2.0 pg 2.3.7.8- TCDD/kg/day by gavage to Sprague-Dawley rats on days 1-3 days of gestation or to female CRCD rats dally for 2 weeks before mating. In the groups dosed on days 1-3 of gestation, fetal weight was significantly decreased 1n the 0.5 and 2.0 pg/kg bw/day groups, but no statistically significant Increases In malformations were noted. When adult female rats were treated for 2 weeks before mating, an Increased number of cystic kid neys and dilated renal pelvis were observed 1n the pups1n the high dose group. In these studies, 0.125 pg/kg bw/day was the NOEL for both mater nal toxicity and adverse effects on the fetus. The reproductive effects of 2,3,7,8-TCDD were also studied in a 3-generatlon study using Sprague-Dawley rats (Hurray et al., 1979). Throughout the study, animals were continuously maintained on diets providing doses of 0 , 0.001, 0.01 or 0.1 pg 2,3,7,8-TCDD/kg/day. The parental group (fQ ) was maintained for 90 days on the test diets prior to mating. The f rats were mated twice, producing the filial generations (f. and f.D ). Selected f1Q and f., rats were mated at -130 days of age to produce the f2 and f3 Utters, respectively. In later generations, the high dose group (0.1 pg 2,3,7,8-TCDD/kg/day) was discontinued because few offspring were produced 1n this group. At the Intermediate dose (0.01 pg/kg/day), 2.3.7.8- TCDD caused lower body weight 1n exposed rats of both sexes (f1 and f2). At the low dose, no toxic effects were discerned. 01330 V-116 02/07/85 Fertility was greatly reduced 1n the f generation exposed to 0.1 pg 2,3,7,8-TCDD/kg/day. At 0.01 pg 2,3,7,8-TCDD/kg/day, fertility was significantly (P<0.05) reduced 1n the f-j and f2 rats. Fertility 1n rats (of any generation) exposed to 0.001 pg 2,3,7,8-TCDD/kg/day was not different from that of control rats. Decreases 1n Utter size were noted 1n the f ^ group exposed to 0.1 pg/kg/day and the f2 and ^3 Utters exposed at 0.01 pg/kg/day. Statistically significant decreases 1n fetal survival throughout gestation were noted 1n f2 and fg Utters of the 0.01 pg 2,3,7,8-TCDD/kg/day exposed dams. At 0.001 pg 2,3,7,8-TCDD/kg/ day, a decreased gestational survival was reported for the f2 Utters, but not for other generations. Decreased neonatal survival was noted among f1A and f2 pups exposed to 0.01 pg 2,3,7,8-TCDD/kg/day, but not among f1B or fg pups. Postnatal body weights of the f2 and fg Utters at 0.01 pg 2,3,7,8-TCDD/kg/day were significantly depressed. At the low dose (0.001 pg 2,3,7,8-TCDD/kg/day), necropsy of 21-day-old pups revealed a statistically significant (P<0.05) Increase 1n dilated renal pelvis 1n the f.| generation. Subsequent generations at this dose level or any at the Intermediate dose (0.01 pg 2,3,7,8-TCDD/kg/day) did not have a significant Increase 1n this abnormality. Significantly decreased thymus weight and Increased Uv e r weight were reported 1n the fg generation, but not 1n ne f.j 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 0.1. pg/kg/day Impaired reproduction among rats, and NOAELs were! associated with 0.001 pg 2,3,7,8-TCDD/kg/day. 01330 V-117 02/07/85 Nisbet and Paxton (1982) reevaluated the primary data of Hurray et al. (1979) using different statistical methods. From this rvaluation 1t was concluded that 2,3,7,8-TCDD significantly reduced the gestational Index, decreased fetal weight, and Increased U v e r to body weight ratios and the Incidence ofdilated renal pelvis 1n both lower dose rroups. Nisbet and Paxton (1982) concluded that the dose of 0.001 pg/kg/day. was not a NOAEL 1n this study. The FIFRA Scientific Advisory Panel has also reviewed the data from this three generation study and concluded thatthe effects observed at the 0.001 pg/kg dose were not consistent enough between the different generations to consider them treatment-related (U.S. EPA, 1979b). Although the panel considered the data suggestive of an embryotoxlc effect, they concluded that 0.001 pg/kg represented a NOEL. Subsequently, EPA did further evaluation of Hurray et al. (1979) data and arrived at a conclusion that 0.001 pg/kg represents a LOAEL (U.S. EPA, 1984a). Berry et al. (1976, 1977) treated Sprague-Dawley rats on day 17 of gestation with 0.2, 0.5, 2.5 or 6 pg 2,3,7,8-TCDD/kg bw 1ntraper1toneally. Haxlmal Induction of fetal hepatic aryl hydrocarbon hydroxylase (AHH) acti vity (measured by fluorometrlc methods) and N-hydroxylat1on of FAA (measured by autoradiography) was observed at a dose of 2.5 pg/kg bw. At lower doses these effects were not observed 1n either the fetus or the dams. Electron microscopic examination revealed cellular necrosis, Increased glycogen and rough endoplasmic reticulum and swollen mitochondria 1n the Uver at dose levels that Induced AHH activity. Induction of epoxide hydratase was also observed In the lungs (ratio 2,3,7,8-TCDD/control=2.85), kidney (ratio 2,3,7,8-TCDD/control=l.06) and skin (ratio 2,3,7,8-TCDD/ con t r o l . 62). 01330 V-118 02/07/85 Nlsbet and Paxton (1982) reevaluated the primary data of Murray et al. (1979) using different statistical methods. From this rvaluation 1t was concluded that 2,3,7,8-TCDO significantly reduced the gestational Index, decreased fetal weight, and Increased U v e r to body weight ratios and the Incidence of dilated renal pelvis 1n both lower dose groups. Nlsbet and Paxton (1982) concluded that the dose of 0.001 yg/kg/day was not a NOAEL 1n 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. EPA, 1979b). Although the panel considered the data suggestive of an embryotoxlc effect, they concluded that 0.001 yg/kg represented a NOEL. Berry et al. (1976, 1977) treated Sprague-Dawley rats on day 17 of gestation with 0.2, 0.5, 2.5 or 6 yg 2,3,7,8-TCDO/kg bw 1ntraper1toneally. Maximal Induction of fetal hepatic aryl hydrocarbon hydroxylase (AHH) acti vity (measured by fluorometrlc methods) and N-hydroxylat1on of FAA (measured by autoradiography) was observed at a dose of 2.5 yg/kg bw. At lower doses these effects were not observed 1n either the fetus or the dams. Electron microscopic examination revealed cellular necrosis, Increased glycogen and rough endoplasmic reticulum and swollen mitochondria 1n the U v e r at dose levels that Induced AHH activity. Induction of epoxide hydra- tase was also observed 1n the lungs (ratio 2,3,7,8-TCOO/contro ), kidney (ratio 2,3,7,8-TCDD/controUl.06) and skin (ratio 2,3,7,8-TC con t r o l . 62). 01330 V-118 02/07/85 Lucler and McDaniel (1979) intubated CD rats with 0 or 3 pg 2,3,7,8TCDD/kg on days 5, 10 or 16 of gestation. They measured the activity of fetal and newborn hepatic microsomal benzo[a]pyrene hydroxylase and p-n1trophenol glucuronlde formation on gestation day 21, postnatal day 8 and post natal day 21. The BaP hydroxylase activity of controls on gestation day 21, treated rats on gestation day 21, controls on postnatal day 8 , treated rats on postnatal day 8 , controls on postnatal day 21 and treated rats on post 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/m1n/mg, respectively. The rate of p-nitrophenyl glu curonlde formation at the above intervals was 13.4, 14.4, 28.4, 99.5 (P<0.01), 23.1 and 164.2 (p<0.01) nmol/min/mg, respectively. Glavini et al. (1982b) administered 0.1, 0.25, 0.5 or 1 pg 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 pg/kg bw/day. There were Increases in abortions and resorptions at doses of 0.25 or 0.5 pg/kg bw/day, with no live fetuses found in the 1.0 pg/kg bw/day dose groups. Extra ribs were found 1n all dose groups. Hydronephro sis was a common finding 1n all groups, but the increase in treated groups over control values was not statistically significant. 2,3,7,8-TCDD also induced fetal U v e r microsomal enzymes 1n New Zealand rabbits.' (Norman et al., 1978). The BaP hydroxylase activity of the Uv e r 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 con trols, adult controls, treated newborns and treated adults was 0 .3 , 1 .8 , 1.6 and 3.7 nmol/mg protein, respectively. 01330 V-119 02/07/85 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 ani mals consumed a total dose of 1.8 and 11.7 yg 2,3,7,8-TCDD, respectively. The animals were bred at 7 months of treatment resulting 1n pregnancy 1n 6 of 8 females 1n the low-dose group and 3 of 8 1n the high-dose group. They were continued on treatment during pregnancy. In both groups, two-th1rds of the pregnancies ended 1n spontaneous abortions. There were no reported malformations 1n the three surviving Infants. All of the controls (one group of 8 and another of unspecified size) conceived and gave birth to normal offpsrlng. McNulty (1978) reported a dose-related Increase 1n spontaneous abortions 1n rhesus monkeys given oral doses of 0.0, 0.2, 1 or 5 yg/kg bw 3 t1mes/week for 3 weeks starting -20 days preconception. The group sizes (2-4 animals) were too small for adequate statistical analysis. Summary There are wide variations 1n the species sensitivity to the acute toxi city of 2,3,7,8-TCDD. LDggS range 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 al., 1980b; Henck et al., 1981). The toxic manifestations seem to be the same whether the compound 1s given as a single oral dose or as a limited number of multiple treatments, with death occurring from 5-45 days post treatment. Lethal exposures result 1n weight loss, often described as "wasting away," and thymic atrophy. In some species, particularly rats and mice, extensive U v e r damage 1s observed (Gupta et al., 1973). In general, no specific cause of death has been Identified, although extensive hemor rhaging has been Implicated 1n mice (Vos et al., 1974). 01330 V-120 02/07/85 In rats and mice, single high doses produce U v e r necrosis (Jones and Butler, 1974), while lower doses produce fatty changes and proliferation of the endoplasmic reticulum (Fowler et al., 1973). Other effects seen 1n some species Include Induction of microsomal enzymes, degeneration of plasma membranes with loss of ATPase activity, a decreased ability to excrete some xenoblotlcs 1n the bile, porphyria, altered gastrointestinal absorption of some nutrients and decreased blood cellularlty. 2,3,7,8-TCDD 1s an Immunotoxln, predominately affecting cell-mediated Immunity. Hypersensitivity, adverse effects on the thymus and Increased sensitivity to antigens have demonstrated the Immunctoxlc potential of 2,3,7,8-TCDD. In rats and mice, the U v e r appears to- be the most sensitive organ following chronic or subchronic exposure. Hepatotox1c1ty 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 1n this report, the NOAEL of 0.01 pg/kg bw/day (Koclba et al., 1976) and 0.07 pg/kg bw/day (NTP, . 1980a) have been reported for rats. A NOAEL of 0.29 pg/kg bw/day was evident for female mice and a L0EL of 0.14 pg/kg bw/day for male mice 1n the NTP (1980a) study. A NOAEL of 0.001 pg/kg bw/day, a NOAEL of 0.05 pg/kg bw/day, and a frank effect level (FEL) of 0.1 pg/kg bw/day have been reported for rats following chronic exposure (Koclba et al., 1978a,b, 1979; NTP, 1980a). Toth et al. (1978, 1979) observed toxic effects 1n mice at doses as low as 0.007 pg/kg bw/week. 01330 V-121 In a preliminary study by Van Miller (1977a,b), 2,3,7,8-TCDD was tested for carcinogenicity following oral administration to rats. Increases In the Incidence of total tumors was observed In some groups; however, the group sizes, ~10 anlmals/group, were too small for an assessment of a treatmentrelated response. In a second, more extensive study by Koclba 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 yg/kg/day. In the high dose group, both male and female animals had sig nificant Increases In site specific tumors. The target organs and tumor types 1n male animals were squamous cell carcinomas of the tongue and hard palate, and adenomas of the adrenal cortex; 1n 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 dally dose of 0.1 yg/kg produce Increased tumor Incidences 1n both male and female rats. Though the Increase was not significant, these tumor types were also found 1n lower dose groups. Under the National Toxicology Program, 2,3,7,8-TCDD was tested for carcinogenicity In rats following administration by gavage (NTP, 1980a). 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 adenomas or carcinomas of the thyroid 1n male animals, and neoplastic nodules or hepatocellular carcinomas of the Uver 1n female animals. Th Incidence of these tumors was significantly greater than control In the high dose groups and the Incidence of both tumors showed a positive dose-related trend. Under the conditions of this assay, 2,3,7,8TCDD was concluded * - .e carcinogenic 1n both male and female rats. 01330 V-122 02/07/85 Further studies 1n mice exposed by gavage have provided support for the carcinogenicity of 2,3,7,8-TCDD. Toth et al. (1979) exposed male mice to 2,3,7,8-TCDD at doses of 0.0, 0.007, 0.7 and 7.0 pg/kg week 1n a study to determine whether 2,4,5-TCPE, Its contaminant 2,3,7,8-TCDD, or both were carcinogens. At the 0.7 ug/kg/week level there was a significantly Increased Incidence of Uver tumors. Liver tumors were not significantly Increased 1n the high dose group; however, early mortality 1n this group from high doses may have precluded observ1ngrlate developing tumors. Simi larly Increased Incidences of U v e r tumors were observed In the NTP (1980a) study In the high dose male mice exposed to 0.5 pg/kg/week and In the high dose female mice exposed to 2 pg/kg/week of 2,3,7,8-TCDD by gavage. Female mice also had an Increased Incidence of follicular-cell adenomas of the thyroid. In both studies, 2,3,7,8-TCDD was carcinogenic to mice with effective doses ranging between 0.5 and 2 pg/kg/day depending on sex and the Individual study. The mouse skin two-stage tumor1gen1c1ty model has also been used to test the carcinogenic potential of 2,3,7,8-TCDD. Following long-term dermal application 3 tlmes/week of 2,3,7,8-TCDD at levels of 0.01 and 0.005 pg/ application to male and female mice, respectively, there was an Increased Incidence of skin tumors only In female mice (NTP, 1980b). Along with the Indication that 2,3,7,8-TCDD was a complete carcinogen 1n this system, D1G1ovann1 et al. (1977) reported that 2,3,7,8-TCDD was also a tumor Initia tor In mouse skin. The ability of 2,3,7,8-TCDD to Initiate, however, has yet to be confirmed since appropriate vehicle and promotion-only control groups were not Included. Attempts to demonstrate tumor promoting activity with 2,3,7,8-TCDD on mouse skin have produced negative results 1n some 01330 V-123 02/07/85 assays (NTP, 1980b; Berry et al., 1978, 1979); however, Poland et al. (1982) reported that 2,3,7,8-TCDD was a tumor promoter when tested on the skin of mice homozygous for the "hairless" trait but not 1n mice heterozygous for this recessive trait. Pitot et al. (1980) also reported that 2,3,7,8-TCDD was a promoter for DEN-1n1t1ated hepatocardnogenesls 1n rats following parenteral administration of the compounds. On mouse skin, 2,3,7,8-TCDD was a complete carcinogen and possibly a tumor Initiator, while no tumor promot ing activity could be attributed to 2,3,7,8-TCDD 1n the assays. In rat U v e r Initiated with DEN, 2,3,7,8-TCDD was a tumor promoter. In studies of the Interaction of 2,3,7,8-TCDD with other chemical carci nogens, Kourl 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 DMBA, however, did not affect tumor yield (D1G1ovann1 et al., 1977). Simi larly, no effect was observed when 2,3,7,8-TCDD was administered either Immediately before (5 minutes) or 1 day after DMBA Initiation (Berry et al., 1979; D1G1ovann1 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 anticarclnogenlc action. Although 1-5 days prior exposure to 2,3,7,8-TCDD Inhibited tumor Initiation by BaP, 3-MC and BaPd1ol-epox1de, 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 (D1G1ovann1 et al., 1980). The Increased AHH activity resulting from 2,3,7,8-TCDD exposure may account for the anticarclnogenlc activity by altering the metabolism of the Initiating compound; however, D1G1ovann1 et ai. (1980) suggest that the Inhibition of the Initiating 01330 V-l 24 02/07/85 activity of BaP-d1ol-epox1de 1 day after Initiation Indicates that more than one mechanism participates 1n the antlcardnogenlc activity of 2,3,7,8-TCDO. Early reports Indicated that 2,3,7,8-TCDO was mutagenic 1n S. typhlmurlum strain TA1532 (Hussain et al., 1972; Seller, 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). 2,3,7,8TCDD has been reported to be mutagenic to E. coll 1n vitro (Hussain et al., 19'2 ) and to S^. cerevl s1ae 1n vitro, and In the host-mediated assay (Bronzettl et al., 1980). Covalent Interactions with nucleic acids are minimal 1f they occur at all (Kondorosl et al., 1973; Poland and Glover, 1979). Only marginal effects have been observed on the Incidence of chromo somal aberrations 1n vivo (Green and Moreland, 1975; Green et al., 19?7). 2,3,7,8-TCDD has been demonstrated 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. With a MED of 1 vg/kg/day, 2,3,7,8-TCDD 1s the most potent terato gen known. At higher doses, 2,3,7,8-TCDD has a marked fetotoxlc effect, as measured by decreased fetal weight and Increased fetal toxicity. Hemor rhagic GI tract has been associated with 2,3,7,8-TCDD fetal toxicity. In rats, 1t has also been consistently observed that 2,3,7,8-TCDD produced teratogenic and fetotoxlc responses 1n 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 vg/kg/day. In addition, there 1s some evidence that 2,3,7,8-TCDD can 01330 V-125 02/07/85 Induce microsomal enzymes 1n the fetus exposed In. utero. and this Induction 1s accompanied by damage to the fine structure of the U v e r 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 1n mice, hemor rhagic GI tracts have been observed 1n rat fetuses exposed In. utero to 2,3,7,8-TCDD. Rabbits and monkeys are also susceptible to the fetotoxlc 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 fetotoxlclty. 01330 V-126 02/07/85 VI. HEALTH EFFECTS IN HUMANS Clinical Case Studies Acute exposure to 2,3,7,8-TCDD results in nausea and vomiting, headache, and irritation of the eyes, skin and respiratory tract. The initial skin reaction is a cutaneous reaction resembling 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, is 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; Pass! et al., 1981). Most of the documented acute exposures to 2,3,7,8-TCDD have been the result of chemical Industry accidents involving 2,4,5-trlchlorophenoxyacetic acid, which 1s contaminated with 2,3,7,8-TCDD. According to Holmstedt (1980), the first cases of chloracne associated with exposure to dioxins occurred following an explosion 1n.a chemical plant producing 2,4.5-T in 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 H p i d levels and prolonged prothrombin times. Residual chloracne, pe ripheral neuropathy, fatigue and severe aches and pains persisted for up tc 2 years. Holmstedt (1980) and May (1973) reviewed reports of three other indus trial explosions: a BASF factory in Ludwigshafen, Germany in 1953; the Coalite and Chemicals plant In England in 1968; and a 2,4,5-T producing factory in Amsterdam 1n 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 were per formed on 14 of the -90 workers 1n the Coalite and Chemicals plant at the time of the explosion. Eleven of the 14 had altered Uv e r function, altered hematological parameters or glucosurla. An accident at the ICMESA plant at Seveso., Italy, 1n 1976, resulted 1n the exposure of at least 8655 workers and nearby residents when a reactor vessel used 1n manufacturing 2,4,5-T exploded (Garatt1n1, 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; G1anott1, 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-TCDD on children and adults at Seveso (Pocchlarl et al., 1979; Boerl, 1978; Chlapplr.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 blastogenlc 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 1n adults have not revealed any reduction 1n Immunocapablllty (Reggianl, 1980; Hay, 1982). Caramaschl et al. (1981) reported an Increase 1n the frequency of head aches, eye Irritation, gastrointestinal tract symptoms and abnormal y-GT, 01340 VI-2 09/18/84 serum GPT and aminolevulinic acid levels 1n children, living 1n the Seveso area, who developed chloracne. Increased urinary glucarlc acid levels, Indicative of Increased microsomal enzyme activity, were found 1n children 3 years after the accident {Ideo et al., 1982). Six children dermally exposed to contaminated soil {30 ppm, 30 mg/kg soil) In horse arenas 1n Eastern Missouri developed headaches, skin lesions and polyarthralgla (Kimbrough et al., 1977). In the most* severe case, epVstaxIs and lethargy were reported. Numbness of the extremities,' skin rashes and Irritation, liver dysfunc 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 as con taminants 1n Agent Orange, 1n- veterans and residents of Vietnam. The rela tionship between exposure to 2,3,7,8-TCDD and the development of these symp toms Is, as yet, unknown (Holden, 1979; Bogen, 1979). Stevens (1981) estimated the cumulative minimum toxic dose of 2,3,7,8- TCDD 1n man to be 0.1 pg/kg, based on analogy to 2,3,7,8-tetrachTjrodl- benzo-2-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 contaminated 2,4,5-T, have been performed. In two of the studies Involving exposure during the Seveso Incident, no Increase 1n the Incidence of chromosomal aberrations was observed as com 01340 VI-3 08/10/84 pared with nonexposed controls (Reggiani, 1980; Hottura et al., 1981). In a third such study, changes suggestive of a mutation in functional nucleolar organizing regions of lymphocyte chromosomes were observed {OiLernia et al., 1982), but this assay has yet to be validated. Workers exposed to the herbicides 2,4,5-trichlorophenoxy-ethanol (2,4,5-TCPE) and Buminol, contami nated with 2,3,7,8-TCDO, were found to have an increased incidence (p<0.001) of chromatid-type and unstable chromosomal aberrations in peripheral lympho cytes (Czeizel and Kiraly, 1976). No Increased incidences of chromosomal aberrations were observed in soldiers exposed 10 years previously to Agent Orange, as compared with unexposed subjects (Mulcahy, 1980). With the exception of the Jiulcahy (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 Vietnam veterans. The cancers (including 1 lung, 3 kidney and 2-3 in testes) were attributed to 2,3,7,8-TCDO exposure during "Agent Orange" (a mixture of 2,4-0, 2,4,5-T and 30-50 ppm 2,3,7,8-TCDO) sprayings in 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 soft tissue sarcoma have been reported among chemi cal Industry workers in 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 Selikoff, 1981). Honchar and Halperin (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 fourth case of malignant fibrous histiocytoma In a phenoxy acid chemical worker. For a similar Industrial settl.ig, except that 2,3,7,8-TCDD levels were <1 ppm, Ott et al. (1980) reported no Increase 1n 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, dl- and trlchlorophenols, 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 1n the production of 2,4,5-sodium trlchlorophenoxyacetate and trlchlorophennxyacetate butylester (PazderovaVejlupkova et al., 1981) and phenoxy acid herbicide (Singer et al., 1982). Dioxin contaminants were suspected to be the causative agents 1n these cases. 01340 71-5 09/18/84 A positive association between 2,4,5-T exposures and Increases In 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 1u human populations In 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 In this type of epidemiologic Investigation, as well as the difficulties In 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-TCDD 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 1n humans. Based on a report of a high Incidence of abortions In 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, 1f spontaneous abortion rates evidenced seasonal variation 1n 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, Is "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. tPA 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 U<ban area; (2) there was a statistically signifi cant seasonal cycle 1n the abortion Index 1n each of the areas with a period of -4 months. In particular there was an outstanding peak 1n the study area 1n June; and (3) there was a statistically significant correlation between the Spontaneous Abortion Rate Index and spray patterns 1n the study area when a lag-time of 2 or 3 months was Included. The U.S. EPA concluded how ever, "This analysis 1s a correlational analysis, and correlation does not necessarily mean causation." H1lby et al. (1980), 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 1n human risk assessment. The Alsea II study has also been, reviewed by a panel of epidemiologists who, 1n 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 1n 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 U.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 effort. 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/effect correlations were noted, and the general increase with time in the incidence of facial clefts was attributed to better reporting procedures; however, there does not have to be a direct correspondence of malformations in human beings and experimental animals. Of the four reports available from New Zealand (Dept, of Health, New Zealand, 1980; McQueen et al., 1977; Hanify 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 insuffi- 01340 VI-8 08/10/84 d e n t evidence to Implicate 2,4,5-T spraying as a causative factor. Even 1f the spraying had been Implicated, a lack of information on 2,3,7,8-TCDD 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. The study by McQueen et al. (1977) is not published in the open litera ture but 1s summarized by Milby 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 is no evidence to impli cate 2,4,5-T as a causal factor 1n human birth defects.'" No additional details are provided. Hanify 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 in 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. During 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 abnrrmallties, 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 ant! 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, however, no statistically significant association where 1.0 was used as the fractional removal rate. Smith et al. (1982a) Investigated the outcome of pregnancy 1n families of professional 2,4,5-T applicators and agricultural contractors 1n 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 89% 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 C.89 (0.61-1.30% 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. The two reports from Australia (Aldred, 1978; Field and Kerr, 1979) also present apparently conflicting results. The report by Aldred (1978) 1s not published 1n the open literature, but the following summary 1s taken from 01340 VI-10 / 08/12/84 Milby et al. (1980): "The report concluded that birth defects 1n a group of babies born 1n 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 In 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 2,3,7,8TCOD 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 reportingneural-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 1n all of Australia with the Incidence of defects 1n one area of Australia 1s 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 U p , cleft palate, spina bifida, anencephalus and cystic kidney disease 1n all of Hungary between 1976 and 1980 with 2,4,5-T use In 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 (5554) 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 1n Hungary had risen from 46,000 kg 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-TCOD exposure, 2,3,7,8-TCDO 1s also an Inadvertent contaminant of 2,4,5-trlchlorophenol (TCP). Chronic exposure to 2,3,7,8-TCOD may occur during the manufacture of TCP and high level acute exposure to 2,3,7,8-TCDO has occurred after an accident 1n July 1976, at the ICMESA TCP chemical factory 1n 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-TCDO 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-TCDO released was not known, the reported cases of chloracne, a symptom of acute exposure to 2,3,7.8-TCDD, Indicated that exposure to 2,3,7,8-TCDD had occurred. Some preliminary results are available from epidemiologic studies of reproductive events 1n the 1nhab1ta;:Lj of Seveso, and recently a study has become available on the reproductive history of men employed 1n the chemical manufacturing Industry with possible chronic exposure to 2,3,7,8TCOD (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 been reported for inhabitants in the area around Seveso by Bonacr.orsi et al. (1978), Reggiani (1980) and Bisanti 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 13.05X, 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 in 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-TCDD 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-induced birth defects. In a recent review of the progress of epidemiologic investigations of the Seveso accident, Tognoni and Bonaccorsi (1982) indicated that the data on spontaneous abortions and malformaticn 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-TCDD during chlorophenol produc tion. A total of 930 potentially exposed males were Identified who had worked for >1 month between January 1939 and December 1975 In a Job with 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 1n operations resulted 1n a number of cases of chloracne. The control population was an equal number of male employees not Involved 1n 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 1n 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 1n the exposed category and 1785 conceptions 1n the control category (concep tion that occurred 1n the exposed group before availability of work records Indicating potential exposure to 2,3,7,8-TCDD were placed 1n 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 1f the populations were subdivided by extent of. exposure. It 'js 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 lip were reported to be elevated by 1.9 (90% confidence intervals of 1.0-3.6) in the 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 , Disease Control on the yearly rate of cleft palate alone or cleft lip 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 1975-1981. 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, it 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 in 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 in 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 2,2,7,8-TCDD. 01340 VI-15 08/10/84 Several Investigators have suggested that 2,3,7,8-TCDD is the causative agent of excess liver 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 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 Hardell (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 a<.`.ds 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 1n a cohort study of workers exposed to 2,3,7,8-TCDD 1n a trlchlorophenol 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 N1tro 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 h1s 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 e! al. (1980) 1n a cohort mortality study of 204 employees exposed to 2,4,5-T during Its manufacture from 1950 to 1971, found no soft-tissue sarcomas among 11 deaths that had occurred by 1976. One 1 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 llposarcoma was found), and a study by Ott et al. (1980), Honchar and Halperln (1981) noted 3 (2.9%) soft-tissue sarcomas In a total of 105 deaths, compared roughly to 0.07% deaths due to soft tissue sarcoma expected 1n United States males 20-84 years old, (ICD 171, 8th Revision, 1975)* Indicating an unusual excess of such tumors. The researchers underestimated the results because of the possibllty that some soft-tissue sarcomas may have been coded to categories other than ICO 171. Individually, none of the reported case studies report ed a significant excess of soft-tissue sarcomas. Cook (1981) found an addi tional malignant fibrous histiocytoma after a later review of the medical records from h1s earlier cohort study. Cook, who was familiar with the three earlier cases, noted that frank chloracne occurred previously 1n two cases of the four having a diagnosis of malignant fibrous histiocytoma. A third person diagnosed as having a fibrosarcoma worked 1n a trlchlorophenol*I. Department of Health, Education, and Welfare. U.S. Public Health Service. National Center for Health Statistics of the United States, 1974. Vol. II. Mortality, Part A. 01340 VI-17 09/18/84 (TCP) process area contaminated with 2,3,7,8-VCDD. This individual exhibit ed facial dermatitis but no diagnosis of chloracne. The fourth case (diag nosed as a liposarcoma) was an individual who had been employed 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 cigarette smokers with chloracne caused by 2,3,7,8-TCDD exposure may be subject to an increased risk of fibrous softtissue sarcomas. Hardell 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 saromcas, and that in their subsequent case control study, they found no difference in smoking habits between his cases and controls. ... . Hoses and Selikoff (1981) reported a fifth soft-tissue 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 schwancma) in 1980 at the age of 58. The employee, prior to his death, in a detailed occupational history said that he believed he was exposed to these chemicals while he was a truck driver, hauler and maintenance 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-tis:-;*? sarcomas (the 33-year-old son was diagnosed as having a fibrosarcomatous mesothe lioma, while the 53-year-old father had a liposarcoma): Both were exposed 01340 VI-18 09/18/84 to halogenated phenol derivatives. The author noted that 2,4-d1chlorophenol 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. Sarma and Jacops (1981) reported three cases of thoracic soft-tissue sarcoma 1n 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 1n areas where defoliants were used at the time. One was drenched with the material in a single, spraying. . Bishop and Jones (1981) found two cases of non-Hodgkin's lymphomas of the scalp in a related clinical study of 158 employees of a pentachlorophenol manufacturing plant 1n Wales. Homologues of 2,3,7,8-TCDD occurred as contaminants at up to 300 ppm at Intermediate manufacturing stages and 5 ppm In the final products. Mild, moderate and severe cases of chloracne were seen in 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 1n a group of 158 workers (ICO 200 and 202), although the basis for the computation of expected numbers Is not stated. Olsson and Brandt (1981) noted that of 123 male patients seen at their clinic 1n Sweden with a Tecent 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 exposure to phenoxy acids, and believed their observations were similar to those of Bishop and Jones (1981). The total number of workers with these 11 Inessas who were exposed to phenoxy a d d s or chlorophenols or both -is small, but considering the rarity of this cancer, it is 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 nonmesenchvmal cells. Classification, grading and staging of STSs is difficult because of the capacity of such cells to differentiate into many different tissues. Fairly precise histogenetlc 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, tendosynovial 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 in the ninth and latest revision of the International Classifi- 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" (ICD 171). Lymphosarcomas, retroperitoneal sarcomas and extra skeletal STSs of the bone are coded elsewhere. In some Instances, 1f site 1s mentioned, 1t 1s coded to the site, e.g., leiomyosarcoma of the stomach (ICD 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 Is usual with most carcinogenic chemicals and substances. Unfortu nately, with respect to STSs, tallies of deaths caused by STSs of particular sites arid types are not maintained separately by the vital statistics offices because of their rarity, and therefore, It Is Impossible to derive risk estimates for particular types at given sites. Altogether, -2000 deaths/year can be attributed to STSs 1n the United States, most of which are coded to ICD category 171 for purposes of developing Incidence and mor tality rates for this composite cause. Within ICD 171, Individual types that may be correlated with exposure cannot be Identified. A separate problem that potentially could arise from assigning STSs to multiple ICD 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 ICD codes other than ICD 01340 VI-21 08/10/84 171 are lumped together while expected STSs are based upon ICO 171 only. Thus, action of this sort, especially with respect to cohort studies of Individuals exposed te dioxin-con',alnlng herbicides or chlorophenols or both, could lead to risk estimates that may be biased upward by the Inclusion of STSs In 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 (Hardell, 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 1n 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 1n patients with these tumors as compared with that of STS (Hardell, 1983). Two case-control studies were conducted, the first 1n northern Sweden (referred to as Study A), and the second 1n the southern part of the country (Study B). The exposures to the substances of primary Interest are shown 1n 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=110) 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 Unpublished Information supplied by Harden to EPA (Hardell and Sand- strom, 1979) - bEr1ksson et al., 1979, 1981 .0 1 3 4 0 VI-23 / 08/10/84 TABLE VI-2 . Exposure Frequencies in Two Case-Control Studies of Soft-Tissue Sarcoma Substancc(s) Percent Exposed Studv- A Studv B Cases (n=52) Controls (n=206) Cases {n=l10) Controls (n=219) Phenoxyacetic 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, Hardell and Sandstrom, 1979; Study B, Eriksson et al., 1979, 1981 01340 VI-24 08/10/84 acetic acids to which exposure occurred consisted predominantly of 2,4,5-T and 2,4-D 1n both studies. Exposure to 2,4,5-T 1n the absence of 2,4-D was rarely reported In either study. Exposure to chlorophenols, which contain chlorinated d1benzod1ox1n Impurities (Levin et al., 1976) occurred mostly 1n sawmill work and paper pulp production. Very few persons reported exposure both to phenoxyacetlc acid and Ci.lorophenols 1n these studies. Of the two predominant phenoxyacetlc acids, only 2,4,5-T 1s known to be contaminated with 2,3,7,8-TCDD. In Study B, a relative risk of 4.9 (90% confidence Intervals 1.6-11.1) was found in relation to exposure to phenoxyacetlc acid herbicide other than 2,4,5-T (2,4-D, MCPA, mecoprop, dlchloroprop). Relative risks 1n relation to the three major categories of exposure are shown 1n Table VI-3.* Studies A and 8 Indicate a risk of developing STSs among workers exposed to' phenoxyacetlc acids only, chlorophenols onlyi or phenoxyacetlc 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 unlikely to have resulted by chance alone. . Since little Is known of the etiology of STSs, the consideration of con founding 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 phenoxyacetlc acids only and chlorophenols only, persons exposed to the other categories of substances were excluded. In Study A, the three persons exposed to both chlorophenols and phenoxy acetlc acids were Included In all comparisons. 01340 VI-25 08/10/84 o TABLE VI-3 Relative Risks of Soft-Tissue Sarcoma 1n Relation to Exposure to Phenoxyacetlc Acids and Chlorophenols In 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 Interval0 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-ta11ed test 04/05/84 confounding factors 1n the selection of controls.* Because of the high cor relation between exposure to the substances of Interest and employment 1n agriculture and forestry, a possible alternative hypothesis could be developed that some other unknown factor present 1n these occupations was responsible for the elevated relative risks. To test this hypothesis, 1t 1s possible to calculate the relative risk 1n relation to the phenoxyacetlc acid exposure 1n Study B, 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 1n risk found 1n relation to phenoxyacetlc acid exposure. Because 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 ~tor1es. 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 phenoxyacetlc 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-methcd relative risks for exposure to phenoxyacetlc acids and/or chlorophenols were 6.2 (p<0.001) 1n Study A and 5.1 (p<0.001) 1n .Study B. 01340 VI-27 08/10/84 chlorophenols and comparing them with unexposed persons 1n other occupations was 0.9 (90% confidence Interval0.3-2.4) In Study B. This suggeststhat 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 Impuri ties-- polychlorinated d1benzod1ox1ns and dlbenzofurans. The risk ratio given exposure to phenoxy acids free of polychlorinated d1benzod1ox1ns 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 phenoxya d d s that contain such Impurities, the relative risk was 17.0.A description of the basis for the determination of exposure or nonexposure to dioxins 1s not well presented 1n this study. The authors concluded that exposure to phenoxy acids and chlorophenols "might constitute a risk factor In the development of soft-tissue sarcomas." This risk relates not only to 2,4,5-trlchlorophenoxy acids containing dioxin Impurities, but to other phenoxy acids as well. Some doubt was raised con cerning the possible mlsclasslf1cat1ons of Individuals who were exposed to phenoxy acids free of polychlorinated d1benzod1ox1ns [1.e., 1n particular, "dlchoroprop" 1n the Eriksson et al. (1981) study]. In a recent communica tion from Hardell (1983), Eriksson recalculated risk estimates after * reclassifying dlchoroprop-exposed cases and controls Into the category 0* probable exposure to phenoxy acids contaminated with polychlorinated dlbenzodloxlns 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 acids free of contamination and 10.9 for those exposed to contaminated phenoxy 01240 VI-28 09/18/84 acid. The first estimate was of only borderline significance utilizing the Mietinen 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 1n a 1964 exposure Incident who had chloracne caused by absorption of 2,3,7,8-TCOD. The skin lesions characterizing chloracne ranged from a few comedones on the back of one employee (predating h1s 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 in 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 metastases, and 3) adenocarcinoma. The authors report that all three victims smoked a minimum of one pack of cigar ettes a day for "many years." Not enough Information is provided by the authors to conclude that any of these four deaths were smoking related. Site of tumor is not mentioned in the cancer deaths. Cancer mortality was slightly elevated in 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 Uver 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 Harden 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 In 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 1s 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, 1f 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 Flbrosancoma Llposarcoma 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/B4 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 of 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, 1s not useful 1n assessing the risk of STS from exposure to phenoxy acids 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, Is 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 1n the "first 20 Interviews" 1n 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 1n those cases and controls 1f In fact one did occur possibly for several reasons, (separation of the earlier work history from the latter; purgi ig of earlier employment records, etc). Besides, the "first 2 0 Interviews" where a change could be noted 1s not necessarily representative of the entire cohort In 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 chlorophenols, since appar ently no effort'was made to confirm "potential" exposures. Only differences 1n occupational classification were noted where "potentiali! cases or con trols could have had exposure to the dioxin-containing herbicides. It was oolnted out that the risk estimates noted do not "preclude" the possibility that an association may be found 1n 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., 1982b). The distribution of tumor types differed considerably from the Hardell and Eriksson et al. (1981) study to the Smith et al. (1982b) study. Leio myosarcomas, malignant hlstocytomas, neurogenic sarcomas and myxosarcoma seem to predominate In the Hardell and Eriksson (1981) study, whereas fibro sarcomas and llposarcomas appear prominently 1n the Smith et al. (1982b) study. More attention should be devoted to the study of the distributions of STS types 1n registry data everywhere In order to determine If such variations In the reporting of STS types are random occurrences. . It Is possible that the cancer effect of exposure to phenoxy herbicides may be narrowed to Just certain types of STSs, the predominant ones 1n the Swedish 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 08/10/84 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-k1n 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 cf 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 1n some Instances. The authors concluded 1n 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 phen oxyacetlc acid ranging from 1.3 1n those Individuals who were "probably exposed" for a minimum of 5 days not, 1n the previous 10 years before cancer registration to 1.6 1n 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 rr a relative was Interviewed, the rates Increased to 1.7 (from'1.5) 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, 1t would be of Interest to repeat the above calculations excluding only those with potential exposure t 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 1n 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 1s 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 f* . * * "5 days" and >15 years before Initial cancer registration. As a subtle justification for the finding of no significant risk In workers exposed In phenoxy acids, 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 acid 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 in phenoxyacetic 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 phenoxyacetic acid 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 his documentation of exposure to 2,4,5-T (and 2,4-0) is at least as good as that in the Hardell -study, and that although Hardell 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-TCDD contamination might be lower in New Zealand as opposed to 2,3,7,8-TCOD contamination in the Swedish studies. Although, there is no evidence for it. Smith (1983) still maintains that his study shows that exposure to phenoxyacetic acids may not be associated with STS. Pazderova-Vejlupkova et al. (1981) studied 80 workers involved in the production of 2,4,5-sodium trichlorophenoxyacetate and butylester of trichlorophenoxyacetic acid who subsequently became ill from exposure to 2,3,7,8-TCDD 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 U v e r lesion," light steatosis, periportal fibrosis or activation of Kupffer cells, or nervous system focal damage (peripheral neuron lesion In lower extremetles). Altogether six patients were reported to be deceased during this 10-year period, 2 from bronchogenic carcinoma, 1 from cirrhosis, 1 atherosclerosis preclpue cerebl and 2 In 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. ' Rllhlmakl 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 phenoxyaclds had been used since the 1950s In 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 dating back to the 1950s revealed that these mixtures contained 0.1-0.9 mg of 2,3,7,8-TCOD/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 09/18/34 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 of 1975. By 1980, 144 deaths had occurred vs. 184.0 expected, a deficit of 22% In 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 In 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) 1s probably a consequence of the "healthy worker effect" 1n that only able-bodied and healthy Individuals were selected Into the Industry. The fact that the cohort was assembled 1 i 1972 from records of persons who were exposed as early as 1955 {17 years prior) raises the likelihood that 1n 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 population 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 In 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 In 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 Is too small." It was suggested that more valid conclusions could be made only with the passage of time (Rllhlmakl 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 1960-1969 and 1970-1978, respectively, for white females In Midland. These estimates are based upon 5 deaths and 7 deaths, respectively, and are listed In Table VI-5. No excess risk was reported among white males, however. The i 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 01340 TABLE VI-5 Midland to'inty Soft and Connective Tissue Cancer Deaths 1960-1981 * VI-40 Identification Year of Sex Age Death Type 1961 F 24 Hemanglosarcoma 1963 1964 1958 1969 1970 1970 1974 1976 F 75 Uposarcoma F 51 Leiomyosarcoma F 37 Llposarcoma F 45 Fibrosarcoma Leiomyosarcoma F 59 Kaposi sarcoma F 56 Fibrosarcoma Leiomyosarcoma F 1 Rhabdomyosarcoma F 77 Llposarcoma 1978 F 64 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, U v e r 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 Tvoe of Mallanancv Primary Site Mtastass 1970 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 H 63 Rhabdomyosarcoma Left lower leg Lung and right outer chest wall M 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 Retroperltonal region Hepatic system M 53 Fibrosarcoma Perltloneum Lung, Uver `Source: Michigan Department of Public Health, 19B3b Month and Year Diagnosed 6-76 6-70 3-70 8-61 6-67 1-67 6-64 10-69 7-72 3-75 04/05/84 1970-1979. Midland County is the home of a major chemical company that pro duced phenoxyacetic 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-containing herbicides, the United Kingdom Ministry of Agri culture, Fisheries and Food (1983) concluded that there was no evidence to recommend altering their earlier conclusion that formulations of phenoxy acid herbicides and related *ood preservatives as "presently cleared" are safe and may continue to be used. This report too readily discounts the positive studies of Hardell and Eriksson (1981) as being biased, and it mak?s no reference to the later validity study by Hardell (1981) of his own work utilizing colon cancer controls. In this report Hardell effectively answered these early criticisms that were reiterated by the British in their report. At the same time, the British report ;';'<ears to put undue emphasis on ncnpcsitive 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 sarccma 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 in humans. A separate series of clinical observations at the Department of Oncology in Umea, Sweden (Hardell, 1979), led the researchers to conduct a casecontrol study of malignant lymphoma in relation to phenoxyacetlc acid, chlorophenols, and other organic compounds (Hardell 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 drid styrene. In the published report, the methods and results were incompletely documented, especially the possibility of confounding by exposure t v the organic solvents. r` '' In the update of the earlier 1980 study, Hardell et al. (198.1), 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- t 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, 1f exposures to phenoxy acids are excluded and consideration is given to just chlorophenols (which includes combined exposure to phenoxy acids and chlorophenols), then the relative 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 chlorophenols. 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 expo sure was 8.4 (95% C.I. 4.2-16.9), while that for low-grade exposure equaled 9.2 (95% C.I. 1.6-5.2). If exposure to organic solvents is examined, given that cases and controls exposed to only phenoxy acids or chlorophenols or both were excluded except for combined exposure 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.''U.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 concluded that "exposure to organic solvents, chlorophenols or phenoxy acids constitutes a risk factor for malignant, lymphoma." This latter study is still subject to the same methodological criticisms to which the earlier study was subjected. Chief among those is the pos 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 (Harden et al., 1981). 01340 VI-44 08/10/84 Other research has tentatively suggested that lumberjacks may be at Increased risk of lymphoma (Edllng and Granstam, 1979). The Nlfcro study 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-control study (Hards!1 et al., 1980, 1981) Is con sistent with the two STS studies discussed. On the other hand, the consistency could also reflect an as-yet unidentified consistent flaw 1n all these studies. The two Swedish case control studies on STSs and a later case control study of malignant 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 occupation In a forestry/agrlculture job, secondary to the recall of expo sure to phenoxy acids or chlorophenols or both, the cases and controls were divided Into three groups: those who worked their entire time slqce 1950 In an agrlculture/forestry job, those who worked some time 1n an.agriculture/ forestry job but not exclusively, and the remainder who never worked 1n a forestry/agrlculture job. The study found that the risk ratio was still 8.2 for STS 1n exclusively agrlculture/forestry workers who were exposed to phenoxy acids compared with workers found 1n other occupations having no V apparent exposure to phenoxy acids or chlorophenols. Even when comparing phenoxy acid or chlorophenol exposed agrlcultural/fores try workers exclu sively with nonexposed agrlcultural/forestry workers, the risk ratio was still 7.1. This argument seems to effectively answer questions regarding ` " t. . . - - recall of occupation secondary to exposure. 01340 VI-45 09/18/84 On the other hand, the relative risk remains 5.4 when comparing phenoxy acid or chlorcphenol exposed workers exclusively 1n occupations other than agrlculture/fcrestry with nonexposed workers 1n those same occupations, thus suggesting the presence of either recall bias or still another occupation with potential exposure to phenoxy acids or chlorophenols (Table VI-6 ). When woodworkers are separated out (possible exposure to chlorophenols 1n treatment of wood) the risk ratio becomes 9.7 (Table VI-7). These data suggest the presence of some recall bias. Another focus of (Hardell and Eriksson, 1981) this study was to deter mine If observational 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 1n the frequency of reporting exposure. Stll.l a third consideration of possible bias Involves recall of exposure to phenoxy acids or chlorophenols because of subject knowledge of having cancer 1n the cases versus no knowledge of cancer 1n the referent popula tion. The study chose as a referent group for the 52 STS cases (Hardell and Sandstrom, 1979) and th? 169 malignant lymphomas (Hardell st al., 1981) a group of 15* ceaVi 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 09/18/84 TABLE VI-6 Other Occupations (Minus Forestry/Agriculture)* Group Phenoxy Ac1ds/Chlorophenols Cases Referents 11 5 ' . RR = 5.4 Source: Adapted from Hardell and Erlkkson, 1981 RR = relative risk Non-exposed 68 a 167 X2 = 11.01 (p<0.01) 01340 VI-47 09/18/84 TABLE VI-7 Other Occupations {Minus Forestry/Agrlculture/Woodworkers)* Group Phenoxy Adds/Chlorophenols Cases Referents 4 1 " RR = 9.7 Source: Adapted from Harden and Erlkkson, 1981 RR a relative risk Non-exposed 66 160 X2 = 5.98 (p<0.05) 01340 VI-48 09/18/84 Utilizing a Mantel-Haenszel rate ratio, the study found the risk of exposure to phenoxy acids 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 acids or chlorophenol exposure in 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 it does not rule out 'the possibility that recall bias can still be present in their data for other reasons. Hardell and Eriksson (1981) refer to an intense "debate about phenoxy acids and their .presumptive risk" In Sweden at the time the colon cancer study was conducted. But, there is no reason to think that colon cancer victims would assume their disease was brought about from exposure to dioxin containing chemicals if 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, 1t is 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 Hardell and Eriksson (1981) may explain any biases Introduced from secondary recall of occupation, observa tional bias Introduced from the telephone Interviewer and recall bias based on subject knowledge of cancer, 1t does not adequately answer questions of recall bias Introduced through the acquired awareness on the part of the victim of STS or non-Hodgkin's lymphoma that h1s condition may have been caused by exposure to dioxin containing herbicides. . Studies of two of the oldest; cohorts of workers knoWn to have been exposed to phenoxyacetlc acid herbicides or 2,3,7,8-TCDD or both report stomach cancer mortality rates significantly higher than expected. The results 1n each study were based 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 -1953 and 1972 were followed through October 1978. The workers were grouped' on the basis of their primary herbicide exposures: those primarily exposed to phenoxyacetlc acids (2,4-D and 2,4,5-T) only, to amltrole (anr.notrlazole) 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<0.05). None were attributable to amltrol alone, but two were assigned to phenoxy acids alone while the remaining stomach cancer death occurred In a worker exposed to both amltrol and phenoxy acids In combination. The excess was more pronounced (3 observed vs. 0.57 expected, p<0.05) among those with early exposure (1957-1961) to phenoxy acids or amltrol or both. If persons who were exposed to just amltrol alone are excluded, thus leaving Individuals exposed to phenoxy acid alone and amltrol 1n combination, the excess Is enhanced further (3 observed vs. 0.41 expected, p<0.01). 01340 VI-50 09/18/84 Axelson 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 pronounced in those exposed early to phenoxy acids alone (6 observed vs. 2:60 expected, p<0.01) and phenoxy acids in combination with amitrol (5 observed vs. 1.34 expected, p<0.05). Presumably, "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 is provided. The other study showing increased stomach cancer mortality is the follow-up of 75 workers exposed to 2,3,7,8-TCDD during nd after a 1953 run away reaction at a trichlorophenol manufacturing facility 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 Ludwigshafen is located.* .,.e The results, shown in Table VI-8 , indicate an increased rats 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 minimum period of cancer induction. All three *The report originally Included expected deaths using rates for the city of Ludwigshafen, which were later shown to be inaccurate. 01340 VI-51 09/18/84 TABLE VI-8 Analysis of Stomach Cancer Mortality 1n 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 RhlnehessenPalatlnate 1972-1975 3- 0.559 3 0.495 5.4 6.1 Source: Adapted from Thelss and Frentzol-Beyme, 1977 Significance Level 0.02 0.01 01340 VI-52 08/10/84 stomach cancer deaths In the Ludwlgshafen cohort occurred more than 10 years after Initial exposure. Employing a 10-year restriction to follow-up (as In the Swedish cohort study) would result 1n 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). Strong temporal trends 1n stomach cancer mortal ity In West Germany during the late 1950s and 1960s would make these expected figures too large. 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 11st of over 10,000 persons who had been Included 1n previous cohort studies by the same Investigators. No stomach cancer deaths occurred 1n this control group during the follow-up period. Thus, use of the Internal control groups also Indicates an excess of stomach can cers 1n the exposed workers. . In an update of this earlier study, Thelss et al. (1982) continued the follow-up of h1s cohort through 1979 by adding 2 additional years of follow up and apparently reducing the size of h1s cohort from 75 to 74. Altogether 21 deaths (4 more than from the earlier study) occurred vs. 18 and 19 deaths In the 2 matched (1 to .1) Internal comparison groups. With respect to can cer deaths, the numbers were respectively 7, 5 and 5. The first control group was manually matched from the total number of persons (5500 Included 01340 VI-53 09/18/84 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 esti mated based on 1970-1975 mortality statistics of Rhinehessin-Palatinate, 18 expected deaths based on 1970-1975 mortality statistics of Ludw1gshafen,-and 20 expected deaths based upon 1971-1974 mortality statistics of the Federal Republic of Germany. Just as in the earlier study, the three stomach car cinomas noted earlier appear to be significantly elevated regardless of which external comparison group is used (Table VI-9). On the other hand, one stomach cancer appeared in the randomized inter nal control group. None appeared in the manually 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 insensitive to the detection of a significantly elevated risk for most causes of cancer, especially STS and lymphomas. Although, stomach cancer is elevated significantly, it is based only upon three deaths and since one stomach cancer death has been noted in an internal control group in the updated version, it appears that this finding has been weakened some what.. Furthermore, as was pointed out earlier, trends in stomach cancer mortality during the 1950s, 1960s and 1970s could make the comparison of stomach cancer mortality with expected deaths less valid based upon 1970-1975 rates. 01340 VI-54 08/10/84 TABLE VI-9 Reanalysis of Stomach Cancer Mortality 1n a Group of West German Factory Workers Exposed to 2,3,7,8-TCDD* Source for Expected Deaths Stomach Cancer Deaths Observed Expected Federal Republic of Germany 1971-1974 RhlnehesslnPalatlnate 1970-1975 Ludwlgsshafen 1970-1975 3' 0.7 3 0.54 3 0.61 Source: Adapted from Thelss et al.f 1982 Relative Risk 4.3 4.7 4.9 Significance Level 0.034 0.027 0.024 01340 VI-55 09/18/84 In summary, the evidence that phenoxyacetic acids or 2,3,7,8-TCDD or both might increase the risk of stomach cancer consists of two studies, each of which reports a statistically significant excess that is based on only three stomach cancer deaths. Further follow-up of these and similar cohorts is warranted, but firm conclusions cannot yet be made. Four additional cohort studies have reported results that do not show increased stomach cancer mortality rates in groups of workers .exposed to phenoxyacetic acids or 2,3,7,8-TCDD or both. These are studies of 2,4,5-T production workers in Midland, Michigan {Ott et al.. 1980), Finnish phenoxy acetic acid herbicide applicators {Riihimakl et al., 1978), the Nitro study in which workers were exposed to 2,3,7,8-TCDD {Zack and Suskind, 1980) and trlchlorophenol manufacturing workers (Cook et al., 1980). As previously mentioned, the Nitro study included a single death from STS and a weakly suggestive increase in lymphatic and hematopoietic 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 pertinent to stomach cancer mortality in the three studies are shown in Table VI-10. Neither the Midland study nor the Nitro study contradicts the findings of the Swedish and West German investigations pre viously discussed. This can be shown in two ways. First, the upper 95% 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 ). 01340 VI-56 08/10/84 TABLE VI-10 Stomach Cancer Mortality 1n Three Studies of Workers Exposed to Phenoxyacetlc Acid Herbicides and/or 2,3,7,8-TCDD Stomach Cancer Deaths Observed Expected Relative Risk 95# Confidence Interval Reference 0 0.14a 0 0-26.3 5 6.9a *b 0.7 - 0 .2-1.7 0 0.5b 0 0-7.4 Ott et al., 1980 R11h1mak1 et al., 1978 Zack and Susklnd, 1980 aEst1mated from total cancer expected deaths (see footnote 1n text). bEntlre 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 08/10/84 This Indicates that the relative risk estimates from the Midland and N1tro studies, even though equal to zero, are nevertheless not significantly different from the estimates of 6 .1, given the sample sizes, follow-up per iods, age distribution and comparison group rates. In addition, the smallest detectable relative risk In the Midland study {a = 0.05, 9 = 0.2 one-ta1led Poisson test) was 21.4 {3 observed deaths, 0.14 expected).* Similarly, the smallest detectable relative rlsTc 1n the N1tro study {<* = 0.05, p 0.2, one-ta1led Poisson test) was 10.0 {5 observed deaths, 0.5 expected). This calculation 1s based on results for the entire follow-up period. If, as 1n the Midland study, a minimum 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.N1tro and Midland studies had very low probabilities of detecting the -6-fold In creases 1n risk suggested by the Swedish and West German Investigations. Statistically, the study of Finnish herbicide applicators 1s Incon sistent with the results of the Swedish and West German cohort studies. The smallest reasonably detectable relative risk (a = 0.05, = 0 .2 ( one- *0tt 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 tie per centage of stomach cancers among the expected deaths 1n the N U r o study {0.5/9.04 s 5.5X). The two studies used United States white ma'ia mortality rates and covered similar calendar years 1n follow-up {1949-1978 1n N1tro and 1950-1976 1n Midland), but a similarity 1n age distributions cannot be established from the published reports. 01340 VI-58 09/18/84 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 1n the Swedish and West German studies. A partial explanation for this apparent Inconsistency could lie 1n the fact that the Finnish study set the minimum period of herbicide exposure for membership 1n the cohort at 10 days (2 working weeks) and noted that the "total strength of exposure has, 1n most cases, been a few weeks only." The Swedish study of herbicide applicators set the minimum exposure at 46 days (>1 spraying season). . There are also certain Inconsistencies 1n 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 Forestry Authority workers fT of 4 groups Included 1n the cohort), even though 9.0 deaths were expected. This finding strongly suggests some defi ciency 1n follow-up or In the source records from which vital status was determined. t In summary, four cohort studies of workers exposed to phenoxyacetlc acid herbicides or 2,3,7,8-TCDD or both do not report Increased r1sk$:of stomach cancer. Only one of these, however, was statistically powerful enough to be Inconsistent with the two studies that tentatively suggest an Increase In stomach cancer risk. The available report of this study of Finnish herbl- 9. . * c1de applicators contains methodologlc questions that require clarification. *The expected stomach cancer deaths were estimated 1n the same manner as for the Midland study. A proportion of 2 0 % of all cancer deaths was applied because Finnish male mortality rates are known to be very high. 01340 VI-59 08/10/84 By adding together the number of workers exposed to phenoxy acids or chlorophenols or both from all case studies, an unusually high number of STSs 1s shown, considering the rarity of the disease. This excess 1s suggestive of an association of cancer with exposure to phenoxy acids or chlorophenols or both, and consequently, with the Impurities found 1n these herbicides, Including 2,3,7,8-TCDO. 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 tc the dioxin contaminants, however. In fact, 1n 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 dlbenzodloxlns and dlbenzofurans) are considered. The extent of possible observer bias and recall bias Intro duced Into these studies by using self-administered questionnaires Is not of sufficient magnitude to have produced the highly significant risks foun^ In the studies. Later studies did not reveal a significant excess risk of STS. However, methodology problems make these latter studies limited with respect to evaluating the risk of STSs from exposure to phenoxy acids or chloro phenols or both and, consequently, 2,3,7,8-TCDO. The Swedish case-control studies provide limited evidence for the '.ardnogenlclty of phenoxy acids or chlorophenols or both in hisu:rcs. However, with respect to the dioxin Impurities contained therein, the evidence for the human carcinogenicity for 2,3,7,8-TCBD based on the epi demiologic studies 1s only suggestive because of the difficulty or 01340 VI-60 09/18/84 evaluating the risk of 2,3,7,8-TCDD exposure in the presence of the con founding effects of phenoxy acids and/or chlorophenol. There is less evidence incriminating 2,4,5-T or 2,3,7,8-YCBO or both as the cause of malignant lymphoma and stomach cancer in humans. \ High Risk Suboopulations * Little information was found in the available 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. Tognoni and Bonaccorsi (1982) found elevated peripheral blood lymphocytes, lymphocyte blastognie response, and serum complement activity in exposed children; however, no immunological effects were detected in adults {Reggiani, 1980; May, 1982). Among adults and children exposed to contaminated soil in horse arenas in Eastern Missouri, the only adverse health effects reported were in children {Kimbrough et al., 1977). Children playing in the arena were probably in more intimate contact 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 chlorophenols from all case studies, an unusually high number of STSs is shown,, considering the rarity of the disease. This excess is suggestive of an: association of cancer with exposure to phenoxy acids and/or chloro- 01340 VI-61 08/10/84 phenols, and consequently, 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-administered questionnaires is not of sufficient magnitude to have produced 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 c'llorophenols 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, on populations previously studied will increase the pool of data on dioxin and help, evaluate its carcinogenic risk in humans. Either acute or chronic exposure to 2,3,7,8-TCDD may result in chloracne, .'iltered liver function, hematological pathologies, porphyria cutanea tard?:, hyperpigmentation, hirsutism and neural degeneration in the extremit ies. Stevens (1981) has estimated that the minimum cumulative toxic dose of 2,3,7,8-TCDD in humans is 0.1 jig/kg. 01340 VI-62 08/10/84 The toxic effects of exposure to 2,3,7,8-TCDD may persist for many years, through some effects seem to be reversible 1n some cases. Even though 2,3,7,8-TCDD has been found to be fetotoxlc and/or teratogenic 1n all animal species tested (see the Teratogenicity and Reproductive Toxicity Section 1n Chapter V), epidemiological studies have failed to demonstrate a convincing connection between 2,3,7,8-TCDD exposure and spontaneous abor tions or malformations 1ji humans. These studies are difficult to Interpret, since quantitative exposure data are not available. Some evidence of cyto genetic damage has been reported 1n 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 causa tive agents. 01340 VI-63 08/10/84 VII. MECHANISM OF TOXICITY A number of studies have attempted to determine the mechanism of toxic ity of 2,3,7,8-TCDD. The ultimate purpose Is to provide a better estimate of man's relative sensitivity to 2,3,7,8-TCDD and other compounds having a similar mode of action. Specifically, these studies may be able to explain the reason for the marked Interspecies differences 1n 2,3,7,8-TCDD 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 In 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 posslhle 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 1n mediating their toxicity and carcinogenicity (Kourl, 1976; Kourl et al., 1974; Benedict et al., 1973; Shum et al., 1979; Thomas et al., 1973; Legraverend et al., 1980; OuranReynolds et al., 1978; Robinson et al., 1975; Mattlson 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; 1n contrast, nonresponslve mice are more susceptible to the tumorlgenlc effects of PAHs at tlssue/organ sites remote from the Initial site of exposure to the PAHs. These differences 1n susceptibility are due to several factors Including AHH-med1ated toxlcation and detoxication. 2,3,7,8-TCDD: 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-TCDD. 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-TCDO and 3-MC In several genetically Inbred mice strains were similar; Like 3-MC and related PAHs, 2,3,7,8-TCDD Induced AHH 1n several responsive mouse strains (l.e., C57B1/5J). In contrast to 3-MC, 2,3,7,8-TCDO Induced microsomal AHH 1n the 0BA/2J nonresponslve mice; however, the ED5Q for this biologic response was significantly higher than values reported for the responsive mice. In genetic crosses between responsive C57B1/6 and nonresponslve D8A/2 mice 1t was also shr.wn for both 3-MC and 2,3,7,8-TCDD that the trait of responsiveness 1s Inherited 1n a simple autosomal dominant mode (Poland and 01350 VI1-2 09/18/84 Knutson, 1982). It has been suggested that the observed differences In the activities of 3-MC and 2,3,7,8-TCDO 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 trations of 3-MC caused by metabolism and excretion. Several studies with 2,3,7,8-TCDO 1n genetically Inbred mice support the receptor mediated hypothesis. The Induction of UOP-glucuranosyl transfer ase, OT dlaphorase, -aminolevulinic acid, glutath1one-S-transferase B, T-aldehyde dehydrogenase and chollneklnase by 2,3,7,8-TCOO 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; 01etr1ch et al., 1978; Ishldate et al., 1980; Poland and Slover, 1973a). Toxicology studies with genetlcally-lnbred mice confirm the role of the Ah locus 1n mediating several toxic effects Including porphyria, 1mmunotox1c1ty (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-TCOO 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 kerat1n1zat1on by 2,3,7,8-TCDO which resembles some of the characteristics of chloracne (Knutson and Poland, 1980). This cell line 1s also responsive to AHH Induction and contains a cytosolic receptor binding protein. Although the murine Ah receptor has not been characterized, several studies confirm that a protelp with high affinity for 3-MC and 2,3,7,8-TCOO 1s present 1n low concentrations 1n the hepatic (-30-50 fmolar) and extr'ahepatlc tissues of responsive C57B1/6J mice 01350 V I 1-- 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 C5781/6J mice and Sprague-Dawley rats, but not 1n nonresponslve 08A/2J mice, the Ah receptor can be Induced by pretreatment with phnobarbital which 1s 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 1n 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-TC00:nuclear protein complex 1n D8A/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 Halooenated Arvl Hydrocarbons: Structure-Actlvitv 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 1n genetically Inbred mice and other diverse animal species. A number of reviews and comparative studies (Allen et al., 1979; Allen and Norback, 1S77; Kimbrough, 1974; Kimbrough et al., 1978; McCmnell and Moore, 1979; Taylor, 1979) clearly Indicate that the toxic halogenated mixtures and Individual compounds 01350 V I 1--4 08/12/84 (Including the PCDDs, 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, 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 PCDDs, PCDFs, PCBs and PB8s and these data Illustrate the different species and organ/tlssue susceptlbl11 ties - 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-Ouke, 1979; Carlstedt-Duke et al., 1979, 1981; Okey, 1983; Okey and Vella, 1982; Mason and Okay, 1982). These observations support a common 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 1s substituted 1n 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 V I 1-5 09/18/84 Individual PCDO congeners In guinea pigs and mice (McConnell et al.f 1978b) and their AHH Induction potencies In chick embryos and rat hepatoma H-4-II-E cells In culture and their binding affinities for the C57B1/6J mouse hepatic cytosolic receptor protein (Poland et al., 1976, 1979; Bradlaw et al.t 1980; Bradlaw and Casterline, 1979). Comparable structure-activity correlations have been reported for the PCOFs In which the most active compound, 2,3,7,8TCOF, 1s an approximate Isostereomer of 2,3,7,8-TCOO (Poland et al., 1979; Poland and Knutson, 1982). Moreover, like the PCOOs, there was an excellent correlation between the toxicity of several Individual PCOFs (Yoshlhara et al., 1981), their AHH Induction potencies 1n rat H-4-II-E hepatoma cells and binding affinities to male Wlstar rat hepatic cytosolic receptor protein (8and1era 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'-hexachlorob1phenyl, 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-TCOO In their mode of Induction of the cytochrome P-450 Isozymes (34) (Parkinson et al., 1980a,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, OMAP N-demethylase and cytochromes P-450a to P-450e (Parkinson et al., 1980a,c, 1983). Quantitative structure-activity rela tionships (QSARs) within this series jf PC8s were determined by comparing their AHH Induction potencies (ECCJ) 1n rat hepatoma H-4-II-E cells and their binding affinities (ED5Q) for the 2,3,7,8-TCOO rat cytosolic recep tor protein (Sawyer and Safe, 1982; Bandlera et al., 1983). The results 01350 VII-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',4,4',5-penta- and 3,3',4,4',5,5'-hexachlorob1phenyls) > 3,4,4',5-tetrachlorob1phenyl > monoorUjo coplanar PCBs > diortho coplanar PCBs. It was also apparent that the relative tox1dt1es of this group of PCBs paralleled their biological potencies (Blocca et al., 1981; Yoshlhara et al., 1979; Harks et al., 1981; McKinney et al., 1976; Yamamoto et al., 1976; Ax and Hansen, 1975; Kurokl and Hasuda, 1977). The coplanar and monoortho coplanar PCBs also exhibit differential effects 1n the Inbred C57B1/6J and 0BA/2J mice. These compounds Induce AHH and cause thymic atrophy 1n the former "responsive" mice whereas at compar able or higher doses none of 'these effects are observed 1n the nonresponslve DBA/2J mice (Parkinson et al., 1982; Robertson et al., 1984). The results obtained for structurally diverse PCDDs, PC3s and PCDFs clearly support the role of the receptor protein 1n 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 multi parameter regres sion analysis to correlate binding avidities with the physical chemical characteristics of the critical lateral.X substituents; The equation .... log (1) = 1.53o + 1 . 4 7 1 + 1.09 HB + 4.08 01350 VII-7 08/10/84 showed that ligand binding was dependent on substituent electronegativity [ a ) , Hpoph1l1c1;y (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 v- TC00 still requires further confirmation and numerous problems must be clarified. For example: 1. Several cell culture U n e s which appear to have the Ah recep tor are highly resistant to the toxicity of 2,3,7,8-TCDD; the nonresponslve HTC and responsive H-4-II-E cell lines (1.e., for AHH 1nduc1b1l1ty by 2,3,7,8-TCDD) 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 1n rats (Wlstar and SpragueDawley), CS7B1/6J mice, hamsters and guinea pigs are compar able (Gas1ew1cz et al., 1983b); however, their susceptibility to the biologic and- toxic effects of 2,3,7,8-TCDD are highly variable: guinea pigs are highly susceptible to the lethal effects of 2,3,7,8-TCDD (LD50 = 1-2 pg/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-TCDD has been examined In the guinea pig, rat, mouse and hamster. Urine and bile from 14C-TCDD-treated animals were found to be free of unmetabollzed 2,3,7,8-TCDD, 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 In feces suggests, however, that some routes of excretion may not be dependent on pv'loi metabolism of the toxin (Olson et al., 1983). Thus, It 1s not possible to directly correlate the half-life ^or elimination of 2,3,7,8-TCDO with Its In vivo rate of metabolism 1n a given species. The relative per sistence of 2,3,7,8-TCDD 1n a given species may be related to the In. vivo 01350 V I 1--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 1n part :be related to the remarkable 1nterspec1es differences In sensitivity to 2,3,7,8-TCDD toxicity (Olson et al., 1983}. Polger et a'l. (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-TCDD metabolites. How ever, a recent study proposes that metabolites of 2,3,7,8-TCOO 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 1n vivo covalent binding of 1,6-3H-2,3,7,8-TC0D 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-TCOO derived radioactivity bound to hepatic protein of the rat. Mo data Is 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 In sensitivity to 2,3,7,8TCDD. While biliary excretion products may represent detoxified, ,poXar metabolites of 2,3,7,8-TCDD, 1t remains to be shown whether unexcreted reactive metabolites Initiate some of the toxic responses,,associated with exposure to this toxin. 01350 VI I--9 08/10/84 Vitamin A Depletion Many of the toxic effects of 2,3,7,8-TCOD 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 pg 2,3,7,8-TCDD/kg bw produces a dose-related decrease1n the hepatic storage of retinol In Sprague-Dawley rats (Thunburg et al., 1979, 1980). The authors suggested, but didnot demonstrate, that the Tow 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-TCOO. At the highest dose of 2,3,7,8-TCOD, dietary retinol supplements could not fully compensate for the 2,3,7,8-TCOD-produced decrease 1n hepatic retinol content. L1p1d Peroxidation Increased U p l d peroxidation has been suggested as a possible mechanism of 2,3,7,8-TC9D-1nduced toxicity (Sweeney and Jones, 1983). This hypothesis 1s based on th<* following limited pieces of evidence. First, Iron defi ciency Inhibits In vitro lipid peroxidation (Bus and Gibson, 1979; Sweeney et al., 1975; and reduces the hepatotoxlc effects of 2,3,7,8-TCOD (Sweeney et al., 1979). Secondly, Upofuscln pigments, by-products of lipid peroxi dation, are Increased 1n the heart muscle of rats treated with 2,3,7,8-TCOO (Albro et al., 1978). Thirdly, Sweeney and Jones (1983) reported that administration, of the antioxidant butylated hydroxyanlsde (BHA) at a level of 0 . 1 e% It the diet provided some protection from 2,3,7,8-TCDD-1nduced prophyrla and neutral I1p1d 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 (0.25%), all animals were protected from these toxic effects. No beneficial effects were observed when the antioxidant vitamin E (0.01%) was included in the diet. Recently, Stohs et al. (1983) obtained direct evidence that 2,3,7,8-TCOD acceler?tes lipid peroxidation in Sprague-Oawley rats. Groups of 4-8 female rats were created for 3 days with 2,3,7',8-TCOD at doses of 0, 10, 20 or 40 vg/kg by gavage (in a corn oil vehicle). At days 1, 6 and 11 after the last treatment the animals were sacrificed and lipid peroxidation was deter mined In isolated liver microsomes by the reaction of formed malondialdehyde with thiobarbituric acid. At all sacrifice periods, increased lipid 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 in the controls. In addition, these workers measured lipid peroxidation In. vj-VQ by the determination of conjugated dienes in rats receiving 2,3,7,8TC00 at 40 yg/kg. Using this latter method, similar Increases in lipid 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-TCOO toxicity. Endocrine Imbalance Some of the toxic response to 2,3,7,8-TCOO, Including hirsutism and diminishing libido, indicate that 2,3,7,8-TCOO may produce some of its toxicity through endocrine disturbances (Oliver, 1975). Nienstedt et al. (1979) reported that a single oral dose of 20 yg 2,3,7,8-TCDO/kg bw significantly reduced testosterone catabolism. Catabolism of exogenous estrogen in ovariectomized rats is also decreased by 2,3,7,8-TCOO pre 01350 VII-11 09/ 18/84 treatment (Shlverlck and Muther, 1982). In this study, there was a 57% Increase In serum estrone concentrations following administration of 10 mg estrone/100 g bw/day for 4 days to either control or 2,3,7,8-TCDO pretreated ovarlectoralzed rats. No differences were observed In the Increase In uterine wet weight following estrone administration In control and 2,3,7,8TCDO pretreated rats. Thus, the uterotrophlc response was not altered by any 2,3,7,8-TCDD-medlated change In estrone disposition. Shlverlck and Muther (1983) also measured estradiol metabolism In female Holtzman rats given 2,3,7,8-TCDO at a dose of 1 wg/kg bw on days 4-19 of gestation. At this fetal toxic dose, the catechol estrogen formation abil ity of Isolated liver mlcrosomes from the dams was decreased 50% when mea sured on day 20 of gestation. These mlcrosome preparations had a 4-fold Increase In the 7a-hydroxyTat1on of testosterone, while there was no change In the 16a- or 68-hydroxylase activity. Although steroid metabo lism was altered In mlcrosomes Isolated from 2,3,7,8-TCDD-treated pregnant rats, similar exposure of pregnant rats on days 4-15 of gestation did not result 1n any change 1n circulating levels of serum 178-estradiol. The authors suggested that other mechanisms besides U v e r metabolism of steroids may be Involved 1n the fetotoxlc effect of 2,3,7,8-TCDO. Gustafsson and Ingelmar-Sundberg (1979) observed that 2,3,7,8-TCDD pro duced greater change In steroid metabolism In female Sprague-Dawley rats than 1n male rats of the same strain, resulting In a U v e r enzyme pattern displaying less sex "..rferentlatlon than In uninduced rats. Pased on this result, they prooose that 'ome of the effects of 2,3,7,8-TCDD result from an Interaction with the hyoothalamo-pHuUary 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 yg/kg (the t LDj-q ), there was a slight depression 1n blood glucocorticoids during post-treatment days 1-4, followed by an -2.5-fold Increase on post-treatment days 7 and 14. While 1n competi 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 binding to the glucocorticoid receptor. It was determined, however, that the Increase 1n glucocorticoids was likely not to participate In the toxicity of 2,3,7,8-TCDD through adrenal hyperfunction, since prior adrenalectomy did not provide any protection from the lethal effects of 2,3,7,8-TCDD 1n rats. Summary .. Carcinogenic PAHs Induce cytochrome P-450-dependent monooxgenase 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 Glelen, 1972; Nebert and Jensen, 1979; Nebert et al., 1972, 1981, 1983). Since the carcinogenic and toxic effects of PAHs require oxidation to reactive electrophUs, 1t 1s likely that the Ah receptor and AHH Induction play an Important role In .mediating their toxi city and carcinogenicity. 01350 VII--13 09/18/84 Several studies with 2,3,7,8-TCOD 1n Inbred mice support the Ah receptor mediated hypothesis and have shown that effects associated with the Ah receptor (porphyria, 1mmunotox1c1ty, 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-Oawley 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 (PCODs, PCDFs, PCBs and PBBs) all elicit similar toxic responses (a wasting syndrome, skin disorder, lynphold atrophy and Immunodeficiency, porphyria, endocrine and reproductive disorders, modification of chemical carcinogene sis and hepatic enzyme ^Induction) In several different species. A common cytosolic receptor protein appears to be present 1n species that respond similarly to these compounds, Indicating a common mechanism of action (Safe, 1982; McConnell and Moore, 1979; Poland and Knutson, 1982). Structure activity studies have determined that the 2,3,7,8-(laterally substituted) member 1s the most active of the PCODs (McConnell et al., 1978b). Of the PCOFs, 2,3,7,3-TCDF 1s the most active (Poland et al., 1979). Activity of the toxic halogenated aryl hydrocarbons correlates well with their ability to Induce AHH 1n chick embryos (McConnell et al., 1578b) and rat hepatomas rt-4-n-E is (McConnell et al., 1978b; Yoshlhara et al., 1981). The activity of the various PCOO congeners has also been correlated with their binding affinity with cytosolic receptor protein 1n mouse (Poland 01350 VII-14 09/18/84 et al., 1976, 1979; Bradlow et al., 1980; Bradlow and Casterllne, 1979) or rat (Bandlera et al., 1983) hepatic cytosolic receptor protein. Activity of PCOO congeners 1s diminished by decreasing lateral substitution or Increas ing Cl substitution 1n the other positions (McConnell et al., 1978b). Slight toxicity was observed 1n guinea pigs administered extracts of the bile of dogs treated with 2,3,7,8-TCDD led Polger et al. (1982a) to conclude that metabolism represents detoxification. The structural Identification of 2,3,7,8-TCDD metabolites, however, has led Investigators (Polger 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, how 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. j `Since some of the effects of 2,3,7,8-TCOD toxicity resemble vitamin A deficiency (epithelial keratosis, Immunosuppression) Thunburg et al. (1979, 1980) Investigated the ability of single low oral doses of 2,3,7,8-TCOD to reduce hepatic storage of retinol 1n rats. A dose-related decrease 1n hepatic retinol was demonstrated. At the highest dose of 2,3,7,8-TCDD, dietary retinol supplements could not fully compensate for the 2,3,7,8-TCOD- Induced hepatic losses of retinol. . Increased 11p1d peroxidation has been suggested as a posstble mechanism for 2,3,7,8-TCDD-1nduced toxicity (Sweeney and Jones, 1983). It was demon 01350 VII-- 15 09/25/84 strated that Iron deficiency, which reduces lipid peroxidation, reduced 2.3.7.8- TCDD-1nduced hepatotoxlclty (Bus and Gibson, 1979; Sweeney et al., 1979). Llpofuscln pigments, by-products of U p l d peroxidation, accumulate 1n the cardiac muscle of 2,3,7,8-TCDD treated rats (Albro et al., 1978). Butylated hydroxyanlsole, a known antioxidant, provided some protection to mice treated with 2,3,7,8-TCDD (Sweeney and Jones, 1983). Dietary vitamin E was not protective 1n this study. Recently, a dose-related Increase 1n U v e r mlcrosomes was demonstrated 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, 197?). 2,3,7,8-TCDD has been shown to retard catabolism of testosterone (Nlenstedt et al., 1979) and estrone (Shlverlck and Huther, 1982) and estradiol (Sh1ver1ck and Huther, 1983). Since glucocorticoids are known to have a lytic effect on lymphoid tis sues and lymphoid atrophy 1s part cf the picture of 2,3,7,8-TCDD toxicity, Neal et al. (1979) Investigated the ability of the compound to stimulate cortlcold production or mimic Its effect by competitively binding to recep tor sites. Rats treated with a single dose of 2,3,7,8-TCDD responded with slightly decreased blood cortlcolds 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) pronosed > hypothalam1c-p1tu1tary axis for the action of 2,3,7,8-TCDD on steroid levels rather 'than a direct effect on steroid metabolism. C1350 VII-16 j 09/18/84 Vili. QUANTIFICATION OF TOXICOLOGICAL EFFECTS The quantification of toxicological effects of a chemical consists of an assessment of the noncardnogenlc and carcinogenic effects. In the quanti fication of noncardnogenlc effects, an acceptable dally Intake (ADI) 1s calculated. An adjusted acceptable dally Intake (AADI) and health advisory (HA) values for the chemical are then calculated to define the appropriate drinking water concentrations to limit human exposure. For Ingestion data, this approach 1s Illustrated as follows: ADI _ (NOAEL or LOAEL 1n mg/kg/dav) (body weight 1n kg) _ my/day ~ Uncert.a1nty/Safety Factor AADI = ---------------------------- --- = mg/a Drinking water volume 1n a/day where: NOAEL = no-observed-adverse-effect level LOAEL = lowest-observed-adverse-effect level Body weight = 70 kg for adult or 10 kg for child Drinking water volume = 2 a/day for adults or 1 a/day for children Uncertalnty/Safety Factor = 10, 100 or 1000 Utilizing these equations, the following drinking water concentrations are developed for noncardnogenlc effects:1 1. A 1-day HA for 10 kg child 2. A 1-day HA for 70 kg adult 3. A 10-day HA for 10 kg child 4. A 10-day HA for 70 kg adult 5. A lifetime AADI for a 70 kg adult 01360 VIII-1 10/17/85 The distinctions made between the HA calculations (Items 1-4) are asso ciated with the duration of anticipated exposure. Items 1 and 2 assume a single acute exposure to the chemical. Items 3 and 4 assume a limited period of exposure (possibly 1-2 weeks). The HA values will not be used 1n establishing a drinking water standard for the chemical. Rather, they will be used as Informal scientific guidance to municipalities and other organi zations when emergency spills or contamination situations occur. The AADI value (Item 5) Is Intended to provide the scientific basis for establishing a drinking water standard, based upon noncarclnogenlc effects. A NOAEL or LOAEL Is determined from animal toxicity data or human effects data. For animal data, this level 1s divided by an uncertainty factor because there Is no universally acceptable quantitative method to extrapolate from animals to humans. The possibility must be considered that humans are more sensitive to the toxic effects of chemicals than are ani mals. For human data, an uncertainty factor 1s also used to account for the heterogeneity of the human population 1n which persons exhibit differing sensitivities to toxic chemicals. An Office of Drinking Water (ODW) modifi cation of the guidelines set forth by the National Academy of Sciences (NAS, 1977, 1980) are typically used In establishing uncertainty factors as follows: An uncertainty factor of 10 Is used when good acute or chronic human exposure data are available and supported by acute or chronic toxicity data In other species. . An uncertainty factor of 100 Is used when good acute or chronic toxicity data Identifying N0EL/N0AEL are available for one or more species, but human data are not available. . An uncertainty factor of 1000 Is used when limited or Incom plete acute or chronic toxicity data are available or when only the acute or chronic toxicity data Identify a LOAEL (but not N0EL/N0AEL) for one or more species are available. 01360 VIII-2 10/17/85 The uncertainty factor used for a specific risk assessment 1s Judg mental. Factors that cannot be Incorporated In the NAS/ODW guidelines (NRC, 1977) for selection of an uncertainty factor, but must be considered, Include the following: 1) the quality of the toxicology data, 2) the sig nificance of the adverse effect, and 3) the existence of counterbalancing beneficial effects. If toxicological evidence requires the chemical to be classified as a potential carcinogen mathematical models are used to calculate the estimated excess cancer risks associated with the Ingestion of the chemical by drink ing water. The bioassay data used 1n these estimates are from animal experiments. In order to predict the risk for humans, these data must be converted to an equivalent human dose. This conversion Includes correction for noncontlnuous animal feeding, nonllfetlme studies and for the difference 1n size. The factor that compensates for the size difference 1s the cube root of the ratio of the animal and human body weights. It 1s assumed that the average human body weight 1s 70 kg and that the average human consumes 2 a. of water/day. The multistage model 1s then fit to the equivalent human data to estimate the risk at low doses. The upper 95% confidence limit of this estimate Is used. Excess cancer risks can also be estimated using other models such as the one-h1t model, the Welbull model, the logit model and the probit model. There Is no basis In the current understanding of the biological mechanisms Involved 1n cancer to choose among these models. The estimates of low doses for these models can differ by several orders of magnitude. 01350 VII1-3 02/20/85 The scientific data base used to calculate and support the setting of risk rate levels has an Inherent uncertainty. This 1s because the tools of scientific measurement, by their very nature. Involve both systematic and random error. In most cases, only studies using experimental animals have been performed. There Is thus uncertainty when the data are extrapolated to humans. When developing risk rate, levels, several other areas of uncer tainty exist, such as 1) incomplete knowledge concerning the health effects of contaminants 1n drinking water, 2) the Impact of test animal age, sex and species and the nature of target organ systems examined on the toxicity study results and 3) the actual rate of exposure of Internal targets 1n test animals or humans. Dose-response data are usually only available for high levels of exposure, not for the lower levels of exposure for which a stand ard 1s being set. When there 1s exposure to more than one contaminant, additional uncertainty results from a lack of Information about possible synergistic or antagonistic effects. The U.S. EPA has established the limits of 1.3xl0"7, 1.3xl0"8 or 1.3x10" vg 2,3,7,8-TCDD/i. 1n ambient waters, based on an assumed dally consumption of 6.5 g of contaminated fish and shellfish and 2 a of drinking water (U.S. EPA, 1984a). Under these conditions, 94.2% of the total exposure would result from the consumption of aquatic organisms. The recommended levels correspond to estimated human lifetime excess cancer risks of 10"5 , 10"6 or 10"7, respectively. These values are consider ably lower than the HAs for drinking water that are recommended 1n this document (see the Summary Section of this chapter), reflecting the high b1oaccumulatlon of this compound 1n aquatic species. 01360 VIII--4 02/20/85 The FDA advises that fish containing >50 ppt of 2,3,7,8-TCDD should not be consumed and those containing >25 ppt, but <50 ppt, should not be con sumed more than twice a month (FDA, 1983). This 1s reflected In a Canadian limit of 20 ppt In the Lake Ontario commercial fish Imported Into the United States (NRCC, 1981). An ADI of I0-4 pg 2,3,7,8-TCDD/kg bw/day has previously been pro posed by the National Academy of Sciences Committee on Drinking Water and Health (NAS, 1977). This ADI was based on a 13-week rat feeding study by Koclba et al. (1976) and was proposed before convincing evidence for the carcinogenicity of 2,3,7,8-TCDD had accumulated. Noncarc1nogen1c Effects The characteristic effects of exposure to 2,3,7,8-TCDD Include thymic atrophy and weight loss (see the General Toxicity Section In Chapter V). In rats and rabbits, and to a lesser extent In guinea pigs and monkeys, U v e r damage Is a major pathological symptom. Death Is commonly preceded by a prolonged period of weight loss, during which time severe deterioration of the animals 1s 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 many short-term studies that report only minor effects at doses near, or sometimes many-fold greater that the LD5Q. 2,3,7,8-TCDD Is also an Immunosuppressant 1n mice, rats and guinea pigs. The acute toxicity of 2,3,7,8-TCDD varies among species tested. Acute oral LDj-gS ranging from 0.6 pg/kg bw for male guinea pigs to 5051 01360 VII1--5 02/07/85 pg/kg bw for hamsters have been reported (see Table V-l). The relative sensitivity of man to 2,3,7,8-TCDD toxicity, compared with other species, cannot be determined from the existing data. 1-Dav HA. The data used for the determination of a 1-day HA are sum marized 1n Table VIII--1. LD^q data, while not useful 1n the derivation of an HA, are a useful way to compare species susceptibility and are, there fore, Included 1n the table for comparison purposes. Four studies were found that Identified NOAELs or LOAELs that could be useful 1n the deriva tion of an HA (Harris et al., 1973; Madge, 1977; Smith et al., 1981; Turner and Collins, 1983). Harris et al. (1973) administered a single oral dose of 2,3,7,8-TCDD 1n acetone:corn o11 to groups of CD rats of mixed sex. Weights were determined at least once each week. Rats given 50 or 100 pg/kg bw demonstrated a decreased weight gain and Increased mortality. In the high dose group, mortality approached 50% with a mean time Interval until death of 18.3 days. A dose of 25 pg/kg bw, the LOAEL 1n this study, resulted 1n a decreased body weight In females at 1 week postdosing and a decreased rate of weight gain In males for 2 weeks postdosings. After 2 weeks, both male and female rats gained weight at the same rate as the controls. Doses of 1 or 5 pg/kg bw had no effect on body weight. Madge (1977) Investigated the effect of 2,3,7,8-TCDD on Intestinal absorption 1n CD-I mice. Mice were given single oral doses of 10, 25, 75, 150, 200 or 300 pg 2,3,7,8-TCDD/kg bw. Absorption of D-glucose, D-galactose, L-argen1ne and L-h1st1d1ne was measured 7 days later, using the 01360 VII1--6 02/07/85 TABLE V I11-1 Acute Toxicity of 2,3,7tB-TCDD 01360 VII1--7 Species Rat Rat (wle) Rat (fenale) Rat House Dniitft of Exposure oral oral oral 1 .p. l.p. House oral House House House House Guinea pig (feaale) oral oral oral oral oral Guinea pig (Bale) Guinea pig (female) Guinea pig (male) Rabbit Rabbit Hamster Hamster Hamster Honkey oral oral oral oral dermal oral oral 1 .p. oral l.p. - Intraperitoneal Dose vg/kg/day 25 2 2 .0 45.0 2.5 1 10 50 15 2B4 114 0 .1 0 .6 2 .1 2.0 115 275 5051 1157 >3000 <70 Duration of Exposure 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 Duration of Experiment Effect Level Endpoints 8-9 weeks 2 -8 weeks 2 -8 weeks 8 weeks NA ,1 7 days 1 2 weeks 12 weeks 30 days 2 months 42 days 2 -8 weeks 2 -8 weeks 30 days 2 -8 weeks . 3 weeks 55-71 days 50 days 50 days >35 days L0AEL ~ Decreased body weight L P 50 L D 50 L0EL L0AEL L0AEL L0AEL NOAEL Increased serum triglycerides Decreased macrophage and natural killer cell number Decreased Intestinal absorption of d-glucose Porphyria Porphyria LD50 LD50 LOAEL HIId histopathologic effects on liver LD50 LD50 LO50 L D 50 LO5O LD50 L D 50 L D 50 L D 50 Reference Harris et al., 1973 Schwetz et al., 1973 Schweiz et al., 1973 Poli et al., 1980 Hantovanl 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 Schweiz et al., 1973 HcConnell et al., 1978a Schwetz et al., 1973 Schwetz et al., 1973 Henck et al., 1981 Olson et al., 19B0b Olson et al., 19B0b HcConnell et al., 1978b 08/11/84 everted Intestinal sac technique. Absorption of D-glucose was decreased at all dose levels; however, absorption of the other compounds was not affected by any of the treatment. The decrease 1n 0-glucose absorption was doserelated over the range of 0-75 yg/kg bw. In this study, 10 yg/kg bw constituted a LOAEL. Smith et al. (1981) Investigated the effect of 2,3,7,8-TCDD on hepatic porphyrin levels 1n C57B1/10 and DBA/2 mice. A single oral dose was admin istered 1n arachls oil (.0, 5, 15, 50, 75, 150, 300, 600 or 1200 yg/kg bw) and hepatic porphyrin levels were determined at Intervals for up to 12 weeks. There were large strain differences 1n susceptibility to porphyria Induction, with the C57B1/10 strain being -20 times as sensitive as the DBA/2 strain. In this study, the lowest dose which Induced porphyria was 50 vg/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 yg/kg bw of 2,3,7,8-TCDD 1n aqueous methyl cellulose to groups of 4-6 female guinea pigs. Survivors were killed 42 days after dos ing and examined for histopathologic changes 1n the U v e r . Four of the 6 animals 1n the highest dose group and 1 of 5 In the 12.5 yg/kg group died before the end of the observation period. HI Id histopathologic changes Including steatosles (fatty change), focal necrosis and cytoplasmic degener ation were noted 1n animals from all treated groups, but not 1n controls. The authors Indicated that qualitative differences among the dosage groups were not detectable by light microscopy. 01360 V I I 1--8 02/07/85 All of the LOAELs and NOAELs determined for rats and mice are above the LDorUn for guinea pigs (0.6-2.1 yg/kg). Although no NOEL or NOAEL 1s available for guinea pigs, a LOAEL of 0.1 yg/kg can be derived from the study of Turner and Collins (1983). 10-Dav H A . Studies which can possibly be used for calculation of a 10-day HA are Identified 1n Table VIII-2. However, the study by Turner and Collins (1983) used for the calculation of the 1-day HA 1s proposed as the basis for calculation of a 10-day HA. By dividing the 1-day HA by 10 to convert the 1-day HA to 10-day HA, a 10,000-fold uncertainty factor 1s, 1n effect, applied to the 0.1 yg/kg LOAEL 1n the Turner and Collins (1983) study. The calculation of a 10-day HA on this basis 1s concluded to be consistent with 10-day HA calculations which could be considered using the data In Table VIII-2 and applying uncertainty factors of 1000 and 100 to LOAEL and NOAEL data as appropriate. Long-Term Exposure. The health effects of long-term exposure to 2,3,7,8-TCDD are summarized In Table VIII-3. Host of the long-term studies have been performed using rats. There 1s, therefore, only limited Informa tion on the chronic toxicity of 2,3,7,8-TCDD 1n other species. Of the studies Included 1n Table VIII-3, only those by Koclba et al. (1978a,b, 1979) and Murray et al. (1979) were done with administration of 2,3,7,8-TCDD 1n the diet on a dally basis. ' 01360 VII1--9 10/17/85 TABLE VIII-2 Effects of 4-13 Weeks Exposure to 2,3,7,8-TCDD 01360 VII1-10 Species Rout of Exposure Rat oral Rat oral Rat oral Rat oral Rat oral Rat oral Rat oral Rat oral Rat oral Rat oral House House House House oral oral oral oral House oral House House House oral oral oral Dose yg/kg 0 .0 1 0 .1 0.5 1.0 0 .1 1.0 0 .1 5.0 1.0 0.001 No. of Treatments/ Week 5 5 2 2 7 1 7 1 1 7 1.0 5.0 25.0 5.0 2 1 1 1 1.0 1 1.0 1.5 10 ppb 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 LOAEL N0AEL LOAEL LOAEL N0AEL LOAEL LOAEL N0AEL LOAEL 13 weeks 4 weeks 4 weeks 4 weeks LOAEL N0AEL LOAEL LOAEL 4 weeks NOAEL 4 weeks 4 weeks 5 weeks LOAEL LOAEL LOAEL 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. colli susceptibility Decreased tetanus response, antigenic RBC response, s e n s 1 M 7 a t 1 n n to DNFB. resistance to Salmonella infection, resistance to Listeria Infection Koclba et al., 1976 Koclba 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., 19 Goldstein et a l ., 19 Vos et al., 1973 Vos et al., 1973 Thigpen et al., 197 Vos et al., 1978a Hlnsdlll et a l ., 19( 01360 TABLE VI 11-2 (coni.) Species Route of Exposure Dose yg/kg House Guinea p1g Guinea p1g 1 .p. oral oral 0.4 0.00B 0.04 1.p. Intraperitoneal No. of Treatments/ Week 1 1 1 Duration of Exposure 4 weeks 8 weeks 8 weeks Effect Level LOAEL N0AEL LOAEL 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 V I 1 1-11 CO TABLE VI11-3 Effects of Long-Term Exposure to 2,3,7,0-TCQD 21-IIIA Species Rat Rat Rat ^ Rat pat Rat Rat Rat Rat Rat House House Route of Exposure oral oral oral oral oral oral oral oral oral oral oral oral Dose vg/kg 0.001 0 .0 1 0.1 0.1 0.001 0 .0 1 0 .0 1 0.05 1.0 0 .0 1 0 .0 1 0.007 No. of Treatments/ Week 7 1 1 2 7 7 2 2 1 1 2 1 Duration of Exposure 3 generation 16 weeks 16 weeks 28 weeks 104 weeks 104 weeks 104 weeks 104 weeks 45 weeks 45 weeks 104 weeks 52 weeks exposure, 104 weeks study duration Effect Level Endpoints LOAEL NOAEL LOAEL LOAEL NOAEL Decreased body weight, decreased fertility, ; decreased fetal survival Elevated porphyrin levels Elevated porphyrin levels Fatty changes 1n U v e r , decreased body weight Degenerative and necrotic changes 1n the liver LOAEL Degenerative and necrotic changes 1n the U v e r NOAEL LOAEL LOAEL LOEL LOAEL LOAEL Toxic hepatitis Toxic hepatitis Porphyria ' Hepatic enzyme Induction, liver weight Toxic hepatitis Dermatitis and amyloidosis Reference Hurray et al ., 1979; Goldstein et al., 1982b Goldstein et al., 1982b King and Roesler, 1974 Koclba et al., 1978a,b, 1979 Koclba et al., 1978a,b, 1979 NTP, 1980a HTP, 1980a Cantonl et al., 1981 Cantonl et al., 1981 NTP, 1980a Toth et al., 1978, 1979 The EPA has developed an ADI based on noncarclnogenlc effects as Indicated 1n U.S. EPA (1984a). For consistency, the rationale used by EPA for the ADI calculation 1n U.S. EPA (1984a) 1s used for the ADI calculation herein. The rationale as presented 1n U.S. EPA (1984a) 1s as follows: 2,3,7,8-TCDD displays an unusually high degree of reproductive toxicity. It 1s teratogenic, fetotoxlc and reduces fertility. In a 3-generat1on reproductive study, Murray et al. (1979) reported a reduction 1n fertility after dally dosing at 0.1 or 0.01 pg 2.3.7.8- TCDD/kg 1n the F-j and ?2 generations of Sprague-Dawley rats. Although Murray et al. (1979) considered the lowest dose tested, 0.001 pg/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 1n the gestation Index, decreased fetal weight. Increased liver to body weight ratio, and Increased Incidence of dilated renal pelvis at the 0.001 pg/kg dose. The re-evaluated data would suggest that equivocal adverse effects were seen at the lowest dose (0.001 pg/kg/day) and that this dose should, therefore, represent a lowest-observed-adverse-effect level (L0AEL). Schantz et al. (1979) found reductions 1n fertility and various other toxic effects 1n rhesus monkeys fed a 50 ppt 2,3,7,8-TCDD diet for 20 months. This corresponds to a calculated dally dose of 0.0015 pg 2.3.7.8- TCDD/kg/day. These results suggest that monkeys may be somewhat more sensitive than rats, since the effects 1n monkeys were more severe and not equivocal. Since the data from the limit ed study by Schantz et al. (1979) are supportive of the findings by Murray et al. (1979), 1t seems reasonable to determine an ADI based on the L0AEL. Quantification of Noncarclnogenlc Effects Calculation of 1-Day HA. Th^ data on very short-term exposures, 1-14 days, are sufficient for the derivation of an HA. As previously discussed 1n the 1-Day HA Section, the available studies establish LOAELs for rats and mice which are greater than the LD,.g for guinea pigs. A N0AEL 1s not available for guinea pigs, but a L0AEL of 0.1 pg/kg can be derived from t e study of Turner and Collins (1983). This L0AEL can be used to calculate a 1-day HA, using an uncertainty factor (UF) of 1000 for an animal L0AEL. 01360 VIII-13 10/17/85 This UF consists of two 10-fold factors to account for both 1ntra and Inter species variability to the toxicity of this chemical 1n lieu of chemicalspecific data, and an additional 10-fold factor because the HA 1s based on a LOAEL and not a NOAEL. The UFs are based on previous guidelines (NAS, 1977; Federal Register, 1980). = where HA = dose x bw * (UF x wc) bw = body weight, 70 kg for adult 10 kg for child wc = water consumption, 2 i/day for adult 1 /day for child Thus, for a 70 kg adult the 10-day HA 1s as follows: 1-day HA = (0.1 pg/kg bw/day x 70 kg bw) * (1000 x 2 9,/day) = 0.0035 pg/!, This HA 1s equivalent to 0.0070 pg/day or 0.00010 pg/kg bw/day. For a 10 kg child the 10-day HA 1s as follows: 1-day HA = (0.1 pg/kg bw/day x 10 kg bw) = (1000 x 1 l/day) = 0.0010 pg/8. This HA 1s equivalent to 0.0010 pg/day or 0.00010 pg/kg bw/day. Calculation of 10-Dav H A . A 10-day HA 1s calculated by dividing 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 LOAEL (1.e., 10-fold for Intra- and 10-fold for Interspecies variability to the toxicity of a chemical 1n H e u of specific data, and an additional 10-fold because the estimate 1s based on a LOAEL rather than a NOAEL), a 10-day HA can be calcu lated from the LOAEL of 0.1 pg/kg/day reported by Turner and Collins (1983). 01360 HA = (dose x bw) * (UF x wc x 10) V I 1 1 -- 14 10/17/85 where bw = body weight, 70 kg for adult 10 kg for child wc = water consumption, 2 i./day for adult 1 l/day for child 10 = division by 10 to convert 1-day HA to 10-day HA Thus, for a 70 kg adult the 10-day HA 1s as follows: ' 10-day HA = (0.001 pg/kg bw/day x 70 kg bw) * (1000 x 2 k/day) = 0.00035 vg/k This HA 1s equivalent to 0.0007 pg/day or 0.0000099 pg/kg bw/day. For a 10 kg child the 10-day HA 1s as follows: 10-day HA = (0.001 pg/kg bw/day x 10 kg bw) 4- (1000 x 1 l/day x 10) = 0.0001 pg/i. This HA 1s equivalent to 0.0001 pg/day or 0.00001 pg/kg bw/day. Quantification of Lifetime AADI. The studies Included In Table VII1-3 are considered for calculation of an AADI. The AADI 1s based on a L0AEL of 0.001 pg/kg for reproductive effects In the 3-generat1on reproductive study 1n rats by Murray et al. (1979) along the rationale discussed 1n the Long-Term Exposure Section. Using the L0AEL of 0.001 pg/kg bw/day, because the Murray et al. (1979) study suggested that dose of 0.001 pg/kg bw/day may be a L0AEL for reproductive effects, and dividing this by an uncertainty factor of 1000 for an animal L0AEL, a lifetime AADI can be calculated. This uncertainty factor 1s based on previous guidelines (NAS, 1977; Federal Register, 1980). Lifetime AADI = (0.001 pg/kg bw/day x 70 kg bw) 4- (1000 x 2 i./day) = 0.000035 pg/fi. 01360 VIII-15 10/17/85 This AADI 1s Identical to the 10-day HA for adults and Is equivalent to an ADI of 70 pg/day or 1.0 pg/kg bw/day. This ADI 1s the same as that esti mated 1n the AWQC document for TCDD for comparison purposes to the criteria derived from carcinogenicity data (U.S. EPA, 1984a). Carcinogenic Effects 5 A number of epidemiological studies have attempted to relate 2,3,7,8TCDD 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-TCDD and the development of tumors 1n humans, though an association has been suggested with soft-tissue sarcomas (Hardell and Sandstrom, 1979; Eriksson et al., 1979, 1981), lymphomas (Hardell et al., 1980, 1981) and stomach cancer (Axelson et al., 1980; Thelss and Frentzel-Beyme, 1977). In comparison, a number of cancer bioassays have clearly demonstrated the carcinogenic potential of 2,3,7,8-TCDD 1n animals (Koclba et al., 1978a,b; Van Miller et al., 1977a,b; Toth et al., 1979; NTP, 1980a,b). These studies are summarized 1n Table VIII-4. Oral administration of 2,3,7,8-TCDD, either 1n the diet or by gavage, results 1n the production of hepatocellular carcinomas 1n female rats and both sexes of mice (Koclba et al., 1978a,b; NTP, 1980a; Toth et al., 1979). Foll1cular-cell adenomas of the thyroid have been observed 1n both male rats and female mice (NTP, 1980a). Various squamous cell carcinomas have also been reported 1n both sexes of rats (Koclba et al., 1978a,b). 01360 V j.II-16 10/17/85 TABLE VII1-4 Carcinogenicity Bloassays of 2,3,7,8-TCDO by Oral and Dermal Exposure. 01360 Species Rat (male) Route Dose Range Duration of Treatment gavage 0-0.5 pg/kg bw/week 104 weeks Rat (female) gavage 0-0.5 pg/kg bw/week 104 weeks Rat (male) Rat (male) dtet dtet 0 -10 0 0 ppb 0 -0 .1 pg/kg bv/day 78 weeks 105 weeks Rat (female) dlet 0 -0 .1 pg/kg bw/day 05 weeks House (male) House (female) gavage gavage 0-0.5 pg/kg bw/week 0-0 .2 pg/kg bw/week 104 weeks 104 weeks House (male) House (male) gavage dermal 0-7.0 pg/kg bw/week 0-0.03 pg/week House (female) dermal 00.015 pg/week 365 days 104 weeks 104 weeks Duration of Study Animals/ Group 105-107 weeks 50 105-107 weeks 50 95 weeks 105 weeks 10 50 105 weeks * 105-107 weeks 105-107 weeks 424-649 days 104 weeks 104 weeks 50 50 50 45 30 30 Tumor Types Reported Reference folllcular-cell adenoma or carcinoma of the thyroid ^ Neoplastic nodule or hepatocellular carcinoma of the liver All tumors Squamous cell carcinoma of the hard palate and the tongue, adenoma of the adrenal cortex Hepatocellular carcinoma, squamous cell carcinoma of the tongue and lung Hepatocellular carcinoma Hepatocellular carcinoma, fo1 )1 cular-cell adenomas of the thyroid Liver tumors fibrosarcoma of the Integumentary system Fibrosarcoma of the Integumentary system HTP, 1980a NTP, 1980a Van Hiller et al., 1977a,b Koctba et al., 1978a,b Koctba et al.. 1978a,b NTP, 1980a NTP, 1980a Toth et al., 1979 NTP, 1980b NTP, 1980b Toth et al. (1979) administered weekly gavage doses of 0.0, 0.007, 0.7 and 7.0 pg/kg bw to groups of 45 male Swiss mice for 1 year. The rela tively short duration of exposure limits the sensitivity of this assay for determining the carcinogenic potency of 2,3,7,8-TCOD. Van Miller et al. (1977a,b) maintained groups of 10 male Sprague-Oawley Tats on diets containing 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 1n doses of 0.0, 0.003, 0.001, 0.01, 0.1, 0.4, 2.0, 24, 240 and 500 pg/kg bw/week, respectively. The small group sizes (10 rats/group) and relatively short exposure times (78 weeks) limit the usefulness of this study 1n quantitative risk assess ment. Both the NTP (1980a) study and the Koclba et al. (1978a,b) study contain sufficient anlmals/group and Involved sufficiently long dosing schedules to be used for quantitative risk assessment. Koclba et al. (1978a,b) main tained groups of 50 Sprague-Dawley rats on diets providing doses of 0.0, 0.001, 0.01 or 0.1 pg 2,3,7,8-TCDD/kg bw/day for 2 years. The high dose resulted 1n reduced lifespans for the female rats, reduced body weight gain 1n both sexes and signs of tissue toxicity. Statistically significant Increases 1n hepatocellular neoplastic nodules were observed 1n females at doses of 0.1 and 0.01 pg/kg bw/day. At the high-dose, Increases were observed 1n stratified squamous cell carcinomas of the hard palate and/or nasal turbinates 1n both sexes, 1n keratinizing squamous cell carcinomas of the lungs 1n females, 1n squamous cell carcinomas of the tongue 1n males and 1n hepatocellular carcinomas 1n females. 01360 VIII-18 10/17/85 In the NCI bioassay (NTP, 1980a), groups of 50 Osborne-Mendel rats or Swiss mice were dose twice weekly by gavage with 2,3,7,8-TCDD 1n a 9:1 corn o1l:acetone solution. The rats and male mice received TWA doses of 0.0, 0.0014, 0.0071 and 0.071 yg/kg bw/day. The corresponding doses for female mice were 0.0, 0.0057, 0.029 and 0.29 yg/kg bw/day. Dose-related depres sions 1n mean body weight were reported for both sexes of rats. In male *rats, a dose-dependent Increase 1n the Incidence of folUcular-cell adenomas or carcinomas of the thyroid was observed. The Incidence of subcutaneous tissue fibromas was significantly Increased 1n the high-dose group. In female rats, observed Increases 1n the Incidence of subcutaneous tissue fibrosarcomas, adrenal cortical adenomas and hepatocellular carcinomas and neoplastic nodules were statistically significant 1n the h1gn-dose group. In mice, statistically significant Increases 1n tumor Incidences were ob served only 1n the high-dose group. AnN Increase 1n hepatocellular carci nomas and neoplastic nodules was noted 1n the males. In females. Increases were observed 1n hepatocellular carcinomas and adenomas, fibrosarcoma, histiocytic lymphoma, thyroid follicular-cell adenoma and cortical adenoma or carcinoma. Quantification of Carcinogenic Risk A summary of 9554 upper-11m1t human carcinogenic potency estimates for 2,3,7,8-TCDD derived from the K o d b a et al. (1978a,b) and NCI (NTP, 1980a) studies In rats and mice, with two pathologists' findings for the K o d b a study, are given 1n Table VII1-5. These potency estimates have been calcu lated using the linearized multistage model by a previously described methodology (Federal Register, 1980). The largest of these potency factors { * ) comes from data 1n an Independent pathologist's (Dr. R. Squire) review of the K o d b a feeding study of female Sprague-Dawley rats. An 01360 VIII--19 10/17/85 TABLE VIII--5 Summary of Human Potency Estimates for 2,3,7,8-TCDD3 Species Study Sex Pathologist Human Potency Estimate q-|* In (mg/kg/day)-1 Rat Rat Rat Mouse Mouse K o d b a et al.b K o d b a et al .b NCId NCId NCId male female female male female Kodba Squire Kodba Unadjusted Adjusted for early mortality Squire Unadjusted Adjusted for early mortality NCI-reviewed NCI-reviewed NCI-reviewed 1.47 x 104 1.73 x 104 2.52 x 10s 1.51 x 105C 4.25 x 10s 1.61 x 10sC 3.28 x 104 7.52 x 104 4.56 x 104 aSource: U.S. EPA, 1984b bK o d b a et al., 1978a,b cValues used to determine the geometric mean of 1.56x10s (mg/kg/day) 1 dNTP, 1980a 01360 V I I I-- 20 10/17/85 adjustment for high early mortality In the high dose groups led to a slightly lower estimate. The mean of the two pathologists' estimates after mortality adjustment 1s as follows: qi* = [(1.51 x 10s ) x (1.61 x 10s )]l/2 _ n .56 x kjs (mg/kg/day)_1 These potency estimates were derived from the Koclba feeding study. The `responses and parameters of the Koclba feeding study 1n female rats are given 1n Table VII1--6. The number with tumors refers to the number of ani mals with at least one of Uver, lung, hard palate or nasal turbinate tumors. Adjustment for early mortality refers to eliminating those which animals died during the first year of study. The first tumor appeared 1n the high-dose group during the thirteenth month. With these parameters, the mean 95% upper-limit carcinogenic potency factor for humans, q ^ , 1s 1.56x10s (mg/kg/day)"1 . For a 70 kg human drinking 2 8, water/day, the water concentration should be <2.2xl0"6 vg/8. 1n order to keep the upper-limit Individual lifetime cancer risk <10_ s . Water concentration corresponding to excess cancer risk of 10-4 and 10~* are, therefore, <2.2xl0~s and <2.2xl0-7, respectively. Special Considerations Synergistic Effects. Enzyme Induction -- 2,3,7,8-TCDD has been demonstrated to signifi cantly alter the toxicity of other toxicants, primarily as a result of enzyme Induction (see the Interactive Effects Section 1n Chapter VI). These changes may either Increase toxicity, 1f metabolism 1s predominantly an activation pathway, or decrease toxicity 1f metabolism 1s predominantly a detoxification mechanism. Thus, Grieg (1972) has demonstrated a 54% 01360 V I I I -21 10/17/85 TABLE Vili--6 Responses and Parameters of the K o d b a Feeding Study* Dose (mg/kg/day) 0 0.001 x 10"9 0.01 x 10"3 0.1 x 10"9 le = 720 days Le = 720 days L = 720 days No. with Tumors/No. Examined Adjusted for Early Mortality Saulre 16/85 8/48 27/48 24/40 Kodba 9/85 3/48 18/48 34/40 Wh = 70 kg W0 = 0.450 kg R = 5000 i./kg Source: K o d b a et al., 1978a,b 01360 V I I I -- 22 10/17/85 decrease 1n the duration of zoxazolamlne-lnduced paralysis and a 2-fold Increase 1n hexabarbltone sleeping time. Cocardnoqenesls__ and Promotion -- In addition to being a complete carcinogen, 2,3,7,8-TCDD has been demonstrated to function as a promoter of DEN-1n1 tlated hepatocardnogenesls (Pitot et al., 1980). Positive results 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 al., 1982). Another attempt to demonstrate the tumor promoting ability of 2,3,7,8-TCDD on mouse skin ("wild" type) have produced negative results (NTP, 1980b; Berry et al., 1978, 1979). 2,3,7,8-TCDD has also been demon strated to be cocardnogenlc with 3-methylcholanthrene (Kourl et al., 1978). His!!-- Risk-- Subpopulatlons. The data from human studies are Insuffi cient to establish the existence of sensitive subpopulatlons, though there 1s suggestive evidence that children may be more sensitive than are adults (see the High Risk Subpopulatlons Section 1n Chapter VI). Summary The recommended HAs developed 1n this document are summarized 1n Table VIII-7. The 1-day HA 1s based on a single-dose LOAEL 1n the most sensitive species, the guinea pig (Turner and Collins, 1983). The 10-day HA 1s calcu lated by dividing the 1-day HA by 10. An AADI for noncarclnogenlc effects from lifetime exposure 1s derived from the LOAEL 1n the 3-generat1on repro ductive study by Hurray et al (1979) along the rationale developed by EPA; however, the carcinogenicity risk assessment based on the linearized multi stage model and the carcinogenicity data 1n the Koclba et al. (1978a,b, 1979) study Indicates lower HAs for lifetime exposure. 01360 VIII--23 10/17/85 TABLE VII1--7 Health Advisories for 2,3,7,8-TCDD 1-day (adult) 1-day (child) 10-day (adult) 10-day (child) Lifetime 10" Excess Cancer Risk 10"5 Excess Cancer Risk 10"6 Excess Cancer Risk v q/i 3.5xl0~3 1 .OxlO"3 3.5xl0"5 1 .OxlO"5 3.5xlO"5 2.2xlO"5 2.2xlO"6 2.2x10"' 01360 VII1--24 10/17/85 IX. REFERENCES A1t1of A. and M.G. Parkkl. 1978. Organ specific Induction of drug metabo lizing enzymes by 2,3,7,8-tetrachlorodlbenzo-p-d1ox1n 1n the rat. Toxicol. Appl. Pharmacol. 44(1): 107-114. Albro, P.W. 1979. Problems 1n analytical methodology: Sample handling, extraction, and clean up. Ann. NY Acad. Sc1. 320: 19-27. Albro, P.W., J.T. Corbett, M. HarMss and L.O. Lawson. 1978. Effects of 2,3,7,8-tetrachlorodibenzo-p-dloxln on- I1p1d profiles 1n tissue of the Fischer rat. Chem. Biol. Interact. 23(3): 315-330. Aldred, J.E. 1978. Report of-the Consultative Council on Congenital Abnor malities In the Yarrom District. Minister of Health, Melbourne, Victoria, Aust. (Cited In M1lby Pt al., 1980) Allen, J.R. and J.J. Lallch. 1962. Response of chickens to prolonged feed ing of crude "toxic fat." Proc. Soc. Exp. Biol. Med. 109: 48-51. Allen, J.R. and D.H. Norback. 1977. Carcinogenic potential of the poly chlorinated biphenyls. In: Origins of Human Cancer, H.H. Hiatt, J.D. Watson and J.A. Winston, Ed. 173-186. Cold Spring Harbor Conf., Cell Proliferation. ' 4: Allen, J.R., J.P. Van Miller and D.H. Norback. 1975. Tissue distribution, excretion, and biological effects of (14--C) tetrachlorodlbenzo-p-dloxin in rats. Food Cosmet. Toxicol. 13(5): 501-505. 01370 IX-1 09/18/84 Allen, J.R., D.A. Barsottl, J.P. Van Miller, L.3. Abrahamson and J.J. Lallch. 1977. Morphological changes 1n monkeys consuming a diet containing low levels of TCOO. Food Cosmet. Toxicol. 15: 401-410. Allen, 3.R., D.A. Barsottl, L.K. Lambrecht and J.P. Van Miller. 1979. Reproductive effects of halogenated aromatic hydrocarbons on nonhuman pri mates. Ann. NY Acad. Sc1. 320: 419-425. Appelgren, L.E., I. Brandt, E.B. Brlttelos, M. Glllner and S.A. Gustafsson. 1983. Autoradiography of 2,3,7,8-tetrachloro-iCl-d1benzo-p-d1ox1n TCOO: Accumulation 1n the nasal mucosa. Chemosphere. 12(4/5): 545-548. Ax, R.L. and L.G. Hansen. 1975. Effects of purified PCB analogs on chicken reproduction. Poult. Sc1.- 54:. 895-900. .. Axelson, 0. 1980. A note on observational bias case-referent studies. Scand. J. Work Environ. Health. 6(1): 80-82. Axelson, 0., L. Sundell, K. Anderson, C. EdUng, C. Hogstedt and H. KUng. 1980. Herbicide exposure and tumor mortality: An updated epidemiologic Investigation on Swedish railroad workers. Scand. J. Work. Environ. Health. 6: 73-79. Baars, A.J., M. Jansen and D.D. Brelmer. 1978. The Influence of phno barbital, 3-methylcholanthrene, and 2,3,7,8-tetrachlorodlbenzo-p-dloxln on glutathione S-transferase activity of rat liver cytosol. Blochem. Phar macol. 27(21): 2487-2494. 01370 IX--2 09/18/84 Ball, l.M. and R.S. Chhabra. 1981. Intestinal absorption of nutrients In rats treated with 2,3 ,7 ,8-tetiachlorod1benzo-p-d1ox1n (TCDD). J. Toxicol. Environ. Health. 8/4: 629-638. 8and1era, S., T.W. Sawyer, M.A. Campbell, P. Fujita and S. Safe. 1983. Comparative binding to the cytosolic 2,3,7,8-tetrachlorod1benzo-p-d1ox1n receptor: Effects of structure on the affinities' of substituted halogenated biphenyls -- a QSAR analysis. 81ochem. Pharmacol. 32: 3803-3813. Bartsch, H., L. Tomatls and C. Malavellle. 1982. Qualitative and quantita tive comparisons between mutagenic activities of chemicals. In: Mutagenic ity: New Horizons 1n Genetic Toxicology. J.A. Heddle, Ed. Academic Press, NY. p. 36-72. Beatty, P. and R.A. Neal. 1976a. Induction of 0T-d1aphorase by 2,3,7,8tetrachlorod1benzo-p-dlox1n (TCDO). Blochem. Blophys. Res. Commun. 12. 68(1): 197-204. Beatty, P.W. and R.A. Neal. 1976b. Induction of DT-d1aphorase activity of rat liver by 2,3,7,8-tetrachlorod1benzo-p-d1ox1n. Toxicol. Appl. Pharmacol. 37: 189. Beatty, P. and R.A. Neal. 1978. Factors affecting the Induction of OT-dlaphorase by 2,3,7,8-tetrachlorod1benzo-p-d1ox1n. Blochem. Pharmacol. 27(4): 505-510. 01370 IX--3 09/18/84 Beatty, P.W. and R.A. Neal. 1975. Effect of alteration of hepatic mixed function oxidase activity on the toxicity of 2 ,3,7,8-tetrachlorodlbenzodloxln (TCOO) In rats. Toxicol. Appl. Pharmacol. 33: 151. Beatty, P.W., W.K. Vaughn and R.A. Neal. 1978. Effect of alteration of rat hepatic mixed-function oxidase (MFO) activity on the toxicity of 2k3,7,8tetrachlorod1benzo-p-d1ox1n (TCDD). Toxicol. Appl. Pharmacol. 45(2): 513-519. Benedict, W.F., N. Consldlne and O.W. Nebert. 1973. Genetic differences 1n aryl hydrocarbon hydroxylase Induction and benzo[a]pyrene-produced tumorlgenesls 1n the mouse. Hoi. Pharmacol. 9: 266-277. Berry, O.L., P.K. Zachar-lah, -M.J. Namkung, H.R. Juchau. 1976. Trans placental Induction of cardnogen-hydroxylatlng systems with 2,3,7,8-tetrachlorodlbenzo-p-d1oxln. Toxicol. Appl. Pharmacol. 36(3): 569-584. Berry, O.L., T.J. Slaga, N.H. Wilson et al. 1977. Transplacental Induction of mixed-function oxygenases In extra-hepatic tissues by 2,3,7,8-tetrach1orod1benzo-p-d1ox1n. Blochem. Pharmacol. 26(15): 1383-1388. Berry, D.L., J. 01G1ovann1, M.R. Juchau, W.M. Bracken, G.L. Gleason and T.J. Slaga. 1978. Lack of tumor-promoting ability of certain environmental chemicals 1n a two-stage mouse skin tumodgenesls assay. 20(1): 101-108. 01370 IX--4 09/18/84 Berry, O.L., T.J. Slaga, J. DIGlovannl and M.R. Juchau. 1979. Studies with chlorinated d1benzo-p-d1ox1ns, polybromated biphenyls, and polychlorinated biphenyls 1n a two-stage system of mouse skin tumorlgenesls: Potent ant1carclnogenlc effects. Ann. NY Acad. Scl. 320: 405-414. Blocca, M., B.W. Gupta, K. Chae, J.D. McKinney and J.A.Moore. 1981. Toxicity bf selected symmetrical hexachloroblphenyl Isomers 1n the mouse. Toxicol. Appl. Pharmacol. 58: 461-474. B1sant1, L., F. Bonettl, F. Caramaschl et al. 1980. Experiences from the accident of Seveso. Acta. Morphol. Acad. Sc1. Hung. 28(1-2): 139-157. Bishop, C.M. and A.H. Jones. 1981. Non-Hodgkin's lymphoma of the scalp 1n workers exposed to dioxins-. L-ancet. 2(8242): 369. Boerl, E. 1978.Report to Lombardy Regional Authority. Unpublished. (Cited 1n Pocchlarl et al., 1979) * Bogen, G. 1979. Symptoms of Vietnam veterans exposed to Agent Orange. J. Am. Med. Assoc. 242(22): 2391. Bonaccorsl, A., R. Fanelll and G. Tognonl. 1978. In the Wake of Seveso. Amblo. 7(5-6): 234-239. 8otre, C., A. Memoll and F. Alhalque. 1978. TCOO solubilization and photo decomposition 1n aqueous solutions. Environ. Scl. Technol. 12(3): 335-336. 01370 IX-5 09/18/84 Bradlaw, J.A. and J.L. Casterline, Jr. 1979. Induction of enzyme activity 1n cell culture: A rapid screen for detection of planar polychlorinated organic compounds. J. Assoc. Off. Anal. Chem, 62: 904-906. Bradlaw, J.A., L.H. Garthoff, 0. Graff and N.E. Hurley. 1975. Detection of chlorinated dioxins Induction of aryl hydrocarbon hydroxylase activity 1n rat hepatoma cell culture. Toxicol. Appl. Pharmacol. 33(1): 166. Bradlaw, J.A.; L.H. Garthoff, N.E. Hurley and 0. Firestone. 1976. Aryl hydrocarbon hydroxylase activity of twenty-three halogenated d1benzo-pdloxins (Meet. Abstr.). Toxicol. Appl. Pharmacol. 37(1): 119. Bradiaw, J.A., L.H. Garthoff, N.E. Hurley and 0. Firestone. 1980. Compara tive Induction of aryl hydrocarbon hydroxylase activity In. vitro by ana logues of d1benzo-p-d1ox1n. Food Cosmet. Toxicol. 18: 627-635. Brewster, D.W., 8.V. Madhukar and F. Matsumura. 1982. Influence of 2,3,7,8-TCOD on the protein composition of the plasma membrane of hepatic cells from the rat. Blochem. Btophys. Res. Commun. 107(1): 68-74. Bronzettl, G., E. Zelger, I. Lee, K. Suzuki and H.V. Hailing. 1983. Muta genicity study of TCDO and ashes from urban Incinerator "In, vitro" and "Tn vivo" using yeast 07 strain. Chemosphere. 12: 549-553. 8urns, L.H., D.M. C U n e and R.R. Lassiter. 1981. Exposure analysis model ing system (EXAMS). Prepared by Environmental Research Laboratory, 0R0, U.S. EPA, Athens, GA. 01370 IX-6 09/18/84 Bus, J.S. and J.E. Gibson. 1979. L1p1d peroxidation and U s role 1n toxi cology. In: Reviews In Biochemical Toxicology. E. Hodgson. J.R. Bend and R.M. Phllpot, Ed.- New York, Elsev1or/North Holland. 1: 125-149. Buu-Ho1, N.P., Pham-Huu-Chanh, G. Sesque, M.C. Azum-Gelade and G. Sa1nt-Ruf. 1972. Organs as targets of dioxin {2,3,7,8-tetrachlorod1benzo-p-d1ox1n) Intoxication. Naturwlssenschaften. 59(4): 174-175. Cantonl, L., M. R.1zzard1ni, g. Belvedere, R. Cantonl, R. Fanelll and H. Salmona. 1981. Induction of mixed-function oxidase by chronic treatment with 2,3,7,8-teirachlorod1benzo-p-d1oxin 1n female rats. 21(2): 159-167. Toxicology. Caramaschl. F., G. del Crono,-C. Favarett, S.E. Glambelluca, E. Moniesarehlo and G.H. Fara. 1981. Chloracne following environmental contamination by TCOD 1n Seveso, Italy. Int. J. Epidemiol. 10(2): 135-143. Carlstedt-Ouke, J.H. 1979. Tissue distribution of the receptor for 2,3,7,8-tetrachlorod1benzo-p-d1ox1n 1n the rat. 3172-3176. Cancer Res. 39(8): Carlstedt-Ouke, J.M., G. Elfstrom, B. Hogberg and J.A. Gustafsson. 1979. Ontogeny of the rat hepatic receptor for 2,3,7,8-tetrachIorod1benzo-p-d1ox1n and Its endodne Independence. Cancer Res. 39(11): 4653-4656. Carlstedt-Ouke, J.M.B., U.B. Harnemo, B. Hogberg and J.A. Gustafsson. 1981. Interaction of the hepatic receptor protein for 2,3,7,8-tetric;v ..benzop-d1ox1n with ONA. J. Blochim. Blophys. Acta. 672(2): 131-141. 01370 IX--7 09/18/84 / Chhabra, R.S., J.M. Tredger, R.M. Phllpot and J.R. Fouts. 1976. Relation between Induction of aryl hydrocarbon hydroxylase and de novo synthesis of cytochrome P-448 (Pl-450) In mice. Chem.-B1ol. Interact. 15: 21-31. Chlapplno, G., R. Gllloll and V. Foa. 1378. Report of Lombardy Regional Authority. Unpublished. (Cited 1n Pocchlarl et al., 1979) Clark, D.A., J. Gauldle, M.R. Szewczuk and G. Sweeney. 1981. Enhanced sup pressor cell activity as a mechanism of Immunosuppression by 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (41275). Proc. Soc. Exp. Biol. Med. 168(2): 290-299. Clark, D.A., G. Sweeney, S. Safe, E. Hancock, O.G. Kllburn and J. Gauldle. 1983. Cellular and genetic basis for suppression of cytotoxic T cell gen eration by haloaromatlc hydrocarbons. Immunopharmacology. 6: 143-153. Cockerham, L.G., A.L. Young and C.E. Thalken. 1980. Hlstopathologlcal and ultrastructural studies of liver tissue from TCOO-exposed beach mice (Peromyscus pollonotus). J. A0-A083 323/6 PC A04/MF A01. 61 p. Cohen, G.M., W.M. Bracken, R.P. Iyer, D.L. Berry, J.K. Selkirk and T.J. Slaga. 1979. Ant1carc1nogen1c effects of 2,3,7,8-tetrachlorod1benzo-pdloxln on benzo(a)pyrene and 7,l2-d1methylbenz(a)anthracene tumor Initiation and U s relationship to DNA binding. Cancer Res. 39(10): 4027-4033. Cole, P. 1979. The evolving case-control study J. Chron. 01s. 32: 15-27. 01370 IX-8 09/18/84 Col 11ns, T.F.S., C.H. Williams and G.C. Gray. 1971. Teratogenic studies with 2,4,5-T and 2..4-0 1n the hamster. Bull. Environ. Contain. Toxicol. 6(6): 559-567. Cook R.R. 1981. Dioxin, chloracne and soft-tissue sarcoma. Lancet. J. 618-619. Cook, R.R., J.C. Townsend, M.G. Ott and L.6..S1Iversteln. 1980. Mortality experience of employees exposed to 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCOD). J. Occup. Med. 22: 530-532. Conway, C.C. and F. Matsumura. 1975. Alteration of cellular utilization of thymidine by TCOO (2,3,7,8-tetrachlorod1benzo-p-d1ox1n). Bull. Environ. Contam. Toxicol. 13(1): 52-56-.- Coulston, F. and E.J. Olajos. 1980. Panel report: Panel to Discuss the Epidemiology of 2,4,5-T. Mew York City, July 10-11, 1979. Ecotoxlcol. Environ. Safety. 4: 96-102. Courtney, K.O. 1976. Mouse teratology studies with chlorodlbenzo-pdloxlns. Bull. Environ. Contam. Toxicol. 16(6): 674-681. Courtney, K.O. 1977. Prenatal effects of herbicides evaluation by the pre natal development Index. Arch. Environ. Contam. Toxicol. 6(1): 33-46. Courtney, K.O. and J.A. Moore. 1971. Teratology studies with 2,4,5-T and 2,3,7,8-TCQQ. Toxicol. Appl. Pharmacol. 20: 396-403. 01370 IX--9 09/18/84 Courtney, K.D., D.W. Gaylor, M.D. Hogan and H.L. Falk. 1970a. Teratogenic evaluation of pesticides: Large-scale screening study. Teratology. 3: 199. Courtney, K.D., D.W. Gaylcr, M.D. Hogan, H.L. Falk, R.R. Bates and I. Mitchell. 1970b. Teratogenic evaluation of 2,4,5-T. Science. 168: 864-866. Courtney, K.D., J.P. Putnam and J.E. Andrews. 1978. Metabolic studies with TC00 (dioxin) treated rats. Arch. Environ. Contain. Toxicol. 7(4): 385-396. Crosby, D.G., A.S. Wong, J.R. PUmmer and E.A. Woolson. 1971. Photodecom position of chlorinated d1benzo-p-d1ox1ns. Science. 173(3998): 746-749. Crow, K. 1978. Chloracne-: The-chemical disease. . New Sc1. 78(1098): 78-80. Crow, K.D. 1981. Chloracne and Its potential clinical Implications. CUn. Exp. Dermatol. 6/3: 243-257. Crummett, W.B. and. R.H. Stehl. 1973. Determination of chlorinated dlbenzop-d1ox1ns and dlbenzofurans 1n various materials. Environ. Health Perspect. 5: 15-25. Cunningham, H.M. and O.T. Williams. 1972. Effect of 2,3,7,8-tetrachlorodlbenzo-p-d1ox1n on growth rate and the synthesis of 11plds and proteins 1n rats. Bull. Environ. Contam. Toxicol. 7(1): 45-51. 01370 IX-10 09/18/84 Czelzel, E. and J. Klraly. 1976. Chromosome examinations 1n workers pro ducing Klorlnol and Bumlnol. In: The Development of a Pesticide as a Com plex Scientific Task, L. Bankl, Ed. (Cited 1n NRCC, 1981) Medldna: Budapest. p. 239-256. Dees, J.H., B.S. Masters, U. Muller-Eberhard and E.F. Johnson. 1982. Effect of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n and phnobarbital on the occurrence and distribution of four cytochrome P-45G Isozymes 1n rabbit kidney, lung, and liver. Cancer Res. 42(4): 1423-1432. 0e1tr1ch, R.A., P. Bludeau, T. Stock and M. Roper. 1977. Induction of different rat liver supernatant aldehyde dehydrogenases by phnobarbital and tetrachlorod1benzo-p-d1ox1n. J. B1ol. Chem. 252(17): 6169-6176. Department of Health, New Zealand. 1980. Report to the Minister of Health of an Investigation Into allegations of an association between human con genital defects and 2,4,5-T spraying In and around teKultl. Med. J. p. 314-315. New Zealand De Verneull, H., S. Sassa and A. Kappas. 1983. Effects of polychlorinated biphenyl compounds, 2,3,7,8-tetrachlorod1benzo-p-d1ox1n, phnobarbital and Iron on hepatic uroporphyrinogen decarboxylase. Blochem. J. 214; 145-151. Dickens, M., H.D. Seefeld and R.E. Peterson. 1981. Enhanced U v e r ONA synthesis 1n partially hepatectomlzed rats pretreated with 2,3,7,8-tetrachlorod1benzo-p-d1ox1n. Toxicol. Appl. Pharmacol. 58(3): 389-398. 01370 IX-11 09/18/84 Dietrich, R.A., R. Bludeau, M. Roger and J. Schmuck. 1978. Induction of aldehyde dehydrogenases. 81ochem. Pharmacol. 27: 2343-2347. D1G1ovann1, J., A. Viaje, O.L. Berry, T.J. Slaga and H.R. Juchau. 1977. Tumor-1n1t1at1ng ability of 2,3,7,8-telrachlorod1benzo-p-d1ox1n (TCDD) and arochlor 125*, In the two-stage system of mouse skin carcinogenesis. Bull. Environ. Contam. Toxicol. 18(5): 552-557. . D1G1ovann1, J., D.L. Berry, T.J- Slaga, A.H. Jones and M.R. Juchau. 1979a. Effects of pretreatment with 2,3,7,8-tetrachlorod1benzo-p-d1ox1n on the capacity of hepatic and extrahepatlc mouse tissues to convert procarcinogens to mutagens for Salmonella tvphlmurlum auxotrophs. Toxicol. Appl. Phar macol. 50(2): 229-239. . D1G1ovann1, J., D.L. Berry, H.R. Juchau and T.J. Slaga. 1979b. 2,3,7,8tetrachlorod1benzo-p-d1ox1n: Potent ant1carc1nogen1c activity 1n CO-1 mice. Blochem. Blophys. Res. Commun. 86(3): 577-584. 0161ovann1, J., O.L. Berry, G.L. Gleason, G.S. Klshore and T.J. Slaga. 1980. Time-dependent Inhibition by 2,3,7,8-tetrachlorod1benzo-p-d1oxln of skin tumorlgenesls with polycyclic hydrocarbons. Cancer Res. 40(5): 1580-1587. D1Lern1a, R., C. Crlmaudo and G. Pacchectl. 1982. The study of X-rays and TCDD effects on satellite associations may suggest a simple model for appli cation 1n environmental mutagenesis. Hum. Genet. 61(1): 42-47. 01370 IX--12 09/18/84 Duran-Reynolds, H., F. Ullie, H. Bartsch and K.J. Blank. 1978. The genetic basis of susceptibility to leukemia Induction 1n mice by 3-methylcholanthrene applied subcutaneously. J. Exp. Med. 147: 459-469. Edling, C. and S. Granstam. 1979. Summaries transaction of the Swedish Medical Society. XXXVI. Natl. Conf. Med. Soc. sponsored by the Swedish Med. Soc., Stockholmsmasan, Alv1f1, Stockholm, Oecember 5-8. 88(3): 73-74 (Translation) Elovaara, E., H. Savolalnen, M.G. Parkkl, A. A1t1o and H. Valnic. 1977. Neurochemical effects of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n 1n WVstar and . Gunn rats. Res. Commun. Chem. Pathol. Pharmacol. 18: 487-494. Emerson, J.L., O.J. Thompson, C.G . Gerblg and V.B. Robinson. 1970. Terato genic study of 2,4,5-tr1chlorophenoxyacet1c .acid 1n the rat. Toxicol. Appl. Pharmacol. 17: 317. .' Emerson, J.L., O.J. Thompson, R.J. Streblng, C.G. Gerblg and B. Robinson. 1971. Teratogenic studies of 2,4,5-T In the rat and rabbit. Food Cosmet. Toxicol. 9: 395-404. Eriksson, M., L. Hardell, N.O. Berg, T. Mller and 0. Axelson. 1979. Casecontrol study on malignant mesenchymal tumors of the soft-tissue and expo sure to chemical substances. Lakart1dn1ngen. 76: 3872-3875. Eriksson, M., L. Hardell, N. O 'Berg, T. Mller and 0. Axelson. 1981. Softtissue sarcomas and exposure to chemical substances: A case-referent study. Br. J. Ind. Med. 38: 27-33. 01370 IX-- 13 09/18/84 Facchetti, S., A. Fornari and M. Montagna. 1980. Distribution of 2,3,7,8tetrachlorodlbenzo-p-dloxln In the tissues of a person exposed to the toxic cloud at Seveso (Italy). Adv. Mass Spectrom. SB: 1405-1414. Faith, R.E. and H.I. Luster. 1979. Investigations of the effects of 2,3,7,8-tetrachlorodlbenzo-p-dioxin (TCDD) on parameters of various Immune functions. Ann. NY Acad. Sc1. 320: 564-571. Panel11, R., C. Chiabrando, M. Salmona, S. Grattlnl and P.G. Caldera. 1978. Degradation of 2,3,7,8-tetrachlorod1benzo-p-diox1n In organic solvents by gamma ray Irradiation. Experlentla. 34: 1126-1127. . FDA (Food and Qrug Administration). 1983. Statement by S.A. Miller, Director, Bureau of Foods,... FDA, before The Subcommittee on Natural Resources, Agriculture Research and Environment, U.S. House of Representa tives, June 30. Federal Register. .1980. U.S. EPA. Water Quality Criteria Documents: Availability. 45(251): 79318. Field, B. and C. Kerr. 1979. Herbicide use and Incidence of neural-tube defects. Lancet. 1(8130): 1341-1342. Fowler, 8.A., G.W. Lucler, H.W. Brown and O.S. McDaniel. 1973. Ultra structural changes 1n rat Uver cells following a single oral dose of TCDD. Environ. Health Perspect. 5: 141-148. 01370 IXrl4 09/18/84 Fries, G.F. and G.S. Harrow. 1975. Retention and excretion of 2,3,7,8tetrachlorod1benzo-p-d1ox1n by rats. J. Agrlc. Food Chem. 23(2): 265-269. Garattlnl, S. 1982. TCOD toxicology with particular reference to Seveso: Introductory remarks. Druf; Metab. Rev. 13(3): 345-353. Gaslewlcz, T.A. and R.A. Neal. 1979. 2,3,7,8-Tetrachlorod1benzo-p-d1ox1n tissue distribution, excretion, and effects on clinical chemical parameters 1n guinea pigs. Toxicol. Appl. Pharmacol. 51(2): 329-340. Gaslewlcz, T.A. and R.A. Neal. 1982. The examination and quantitation of tissue cytosolic receptors for 2,3,7,8-tetrachlorod1benzo-p-d1ox1n using hydroxyapatite. Anal. Blochem. 124: 1-11. Gaslewlcz, T.A., H.A. Holscher and R.A. Neal. 1980. The effect of total parenteral nutrition on the toxicity of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n 1n the rat. Toxicol. Appl. Pharmacol'. 54: 469-488. Gaslewlcz, T.A., J.R. Olson, L.E. Geiger and R.A. Neal. 1983a. Absorption, distribution and metabolism of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCOO) 1n experimental animals. In: Human and Environmental Risks of Chlorinated Dioxins and Related Compounds, R.E. Tucker, A.L. Young and A.P. Gray, Ed. Plenum Press, NY. p. 495-525. Gaslewlcz, T.A., L.E. Geiger, G. Ru e d and R.A. Neal. 1983b. Distribution, excretion and metabolism of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n 1n C57B1/6J, D8A/2J and B602F1/J mice. Drug Metab. Dlspos. (In press) 01370 IX--15 09/18/84 Geiger, L.E. and ft.A. Neal. 1981. Mutagenicity testing of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n In histidine auxotrophs of Salmonella typhlmurlum. Toxicol. Appl. Pharmacol. 59(1): 125-129. Glanottl, F. 1977. Chloracne due to tetrachloro-2,3,7,8-d1benzo-p-d1ox1n 1n children. Ann. Dermatol. Venereol. 104(12): 825-829. Glavini, E., M. Pratl and C. Vlsmara. * 1982a. Effects of 2,3,7,8-tetrachlorod ibenzo-p-d1ox1n administered to pregnant rats during the prelraplantat1on period. Environ. Res. 29(1): 185-189. Glavini, E., M. Pratl and C. Vlsmara. 1982b. Rabbit teratology study with 2.3.7.8- tetrachlarod1benzo-p~d1ox1n. Environ. Res. 27(1): 74-78. Glavini, E., M. Pratl and C. Vlsmara. 1983.Embryotoxlc effects of 2.3.7.8- tetrachlorod1benzo-p-d1ox1n administered to female rats before mating. Environ. Res. 31: 105-110. Gilbert, P., G. Sa1nt-Ruf, F. Poncelet and H. Mercler. 1980. Genetic effects of chlorinated anilines and azobenzenes on Salmonella typhlmurlum. Arch. Environ. Contam. Toxicol. 9(5): 533-541. Goldstein, J.A., P. Hickman, H. Bergman, J.O. McKinney and M.P. Walker. 1977. Separation of pure polychlorinated biphenyl Isomers Into two types of Inducers on the basis of Induction of cytochrome P-450 or P-448. Chem.81ol. Interact. 17: 69-87. 01370 IX-- 16 09/18/84 Goldstein, J.A., M. Frlesen, T.M. Scottl, P. Hickman, J.R. Hass and H. 8ergman. 1978. Assessment of the contribution of chlorinated d1benzo-pdloxlns and dlbenzofurans to hexachlorobenzene-lnduced toxicity, porphyria, changes In mixed function oxygenases, and hlstopathologlcal changes. Toxicol. Appl, Pharmacol. 46(3): 633-649. Goldstein, J.A., P. Unko, J.N. Hucklns and O.L. Stalling. 1982a. Structure-activity relationships of chlorinated benzenes as Inducers of different formsof cytochrome P-450 1n rat liver. Chem. B1ol. Interact. 41(2): 131-139. Goldstein, J.A., P. L1nko and H. Bergman. 1982b. Induction of porphyria 1n the rat by chronic versus acute exposure to 2,3,7,8-tetrachlorod1benzo~p- dloxln. 81ochem. Pharmacol. .3.1(8): 1607-1613. .. Green, S. and F.S. Moreland. 1975. Cytogenetic evaluation of several dioxins 1n the rat. Toxicol. Appl. Pharmacol. 33: 161. Green, S., F. Moreland and_C. Sheu.1977. Cytognie effect of 2,3,7,8tetrachlorod1benzo-p-d1ox1n on rat bone marrow cells. FDA, Washington, DC. FDA By-Lines. 6: 292. Greenlee, W.F. and A. Poland. 1978. An Improved assay of 7-ethoxycoumarln 0-deethylase activity: Induction of hepatic enzyme activity 1n C57B1/6J and D8A/2J mice by phnobarbital, 3-methylcholanthrene and 2,3,7,8-tetrachlorod1benzo-p-d1ox1n. J. Pharmacol. Exp. Ther. 205(3): 596-605. 01370 IX-17 09/18/84 Greenlee, W.F. and A. Poland. 1979. Nuclear uptake of 2,3,7,8-tetrachlor"d1benzo-p-d1ox1n In C57B1/6J and 08A/2J mice. Role of the hepatic cytosol receptor protein. J. 81ol. Chera. 254(19): 9814-9821. Grelg, J.B. 1972. Effect of 2,3,7,8-tetrachlorod1benzo-l,4-d1ox1n on drug metabolism 1n the rat. Blochem. Pharmacol. 21(23): 3196-3198. Grelg, J.B., G. Jones, W.H. 8utler and J.H. Barnes. 1973. Toxic effects of 2,3,7 ,8-tetrachlorod1benzo-p-d1ox1n. Food Cosmet. Toxicol. 11: 585-595. Grieg, J.B., D.M.- Taylor and J.D. Jones. 1974. Effects of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n on stimulated DNA synthesis 1n the Uv e r and kidney of the rat. Chem. B1ol. Interact. 8(1): 31-39. . * Guenthner,. T.M., O.W. Nebert and R.H. Menard. 1979. Hlcrosomal aryl hydro carbon hydroxylase In rat adrenal: Regulation by ACTH but not by polycyclic hydrocarbons. Mol. Pharmacol. 15(3): 719-728. Gupta, B.N., J.G. Vos, J.A. Moore, J.G. Z1nkl and B.C. Bullock. 1973. Pathologic effects of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n in laboratory animals. Environ. Health Perspect. 5: 125-140. Gustafsson, J.A. and M. Ingelman-Sundberg. 1979. Changes In steroid hormone metabolism 1n rat Uv e r mlcrosomes following administration of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCOD). Blochem. Pharmacol. 28(4): 497-499. 01370 IX--18 09/18/84 Hajdu, S.I. 1983. Classification of soft tissue tumors and tumor-Ulce lesions. Presented at Symp. on Public Health Risks of the Dioxins, Rockefeller Univ., October 19-20. Hanlfy, J.A., P. Metcalf, C.L. Nobbs and R.J. Worsley. 1981. Aerial spray ing of 2,4,5-T and human birth malformations: An epidemiological Investiga tion. Science. 212: 349-351. Harden, ,L. 1977. Soft-tissue sarcomas and exposure to phenoxy acids: A clinical observation. Lakartldnlngen. 74(33): 2753-5754. Harden, L. 1979. Malignant lymphoma of histiocytic type and exposure to phenoxyacetlc acids or chlorophenols. Lancet. 1: 55-56. Harden, L. 1981. Relation of soft-tissue sarcoma, malignant lymphoma and colon cancer to phenoxy acids, chlorophenols and other agents. Scand. J. Work Environ. Health. 7: 119-130. ; Harden, L. 1983. Letter to D. 8ayl1ss, Carcinogen Assessment Group, U.S. EPA, Washington, DC, August 5. Hardell, L. and M. Eriksson. 1981. Soft-tissue sarcomas, phenoxy herbi cides and chlorinated phenols. Lancet. 2(8240): 250. Hardell, L. and A. Sandstrom. 1979. Case-control study: Soft-tissue sar comas and exposure to phenoxyacetlc acids or chlorophenols. Br. J. Cancer. 39: 711-717. 01370 IX--19 09/18/84 Hardell, L.f M. Eriksson and P. Lenner. 1980. Malignant lymphoma and expo sure to chemical substances, especially organic solvents, chlorophenols and phenoxy-aclds. Lakart1dn1ngen. 77: 208-210. (Trans.) Hardell, L., M. Eriksson, P. Lenner and E. Lundgren. 1981. Malignant lym phoma and exposure to chemicals, especially organic solvents, chlorophenols and phenoxy acids: A case-control study. 8r. J. Cancer. 43: 169-176. Harris, H.W., J.A. Moore, J.G. Vos and 8..N. Gupta. 1973. General biologi cal effects of TCDD 1n laboratory animals. Environ. Health. Perspect. 5: 101-109. Hay, A. 1982. Toxicology of dioxins. In: The Chemical Scythe: Lessons of 2,4,5-T and D1ox1n. Plenum Pr-ess, NY and London, p. 41-47. . Henck, J.W., H.A. New, R.J. Kodba.and K.S. Rao. 1981.. 2,3,7,8-Tetrachlorod1benzo-p-d1ox1n: Acute oral toxicity In hamsters. Toxicol. Appl. Pharmacol. 59: 405-407. H1les, R.A. and R.O. Bruce. 1976. 2,3,7,8-Tetrachlorod1benzo-p-d1ox1n elimination 1n the rat: First order or zero order. Food Cosmet. Toxicol. 14: 597-600. . H1nsd1ll, R.D., O.L. Couch and R.S. Spelrs. 1980. Immunosuppression 1n mice Induced by dioxin (TCOO) 1n feed. J. Environ. Pathol. Toxicol. 4(2-3): 401-425. .. 01370 / IX-20 09/18/84 Holden, C. 1979. Agent Orange furor continues to build. Science. 205: 770-772. Holmstedt, B. 1980. Prolegomena to Seveso Ecclesiastes I 18. Toxicol. 44(4): 211-230. Arch. Honchar, P.A. and W.E. Halperln. 1981. 2,4,5-T, trlchlorophenol and softtissue sarcoma. Lancet. 1(8214): 268-269. Hook, G.E.R., J.K. Haseman and G.W. Lucler. 1975a. Induction and suppres sion of hepatic and extrahepatlc microsomal foreign-compound-metabolizing systems by 2,3,7,8-tetrachlorod1benzo-p-d1ox1n. Chem.-81ol. Interact. 10(3): 199-214. Hook, G.E.R., T.C. Orton, J.A. Moore and G.W. Lucler. 1975b. Tetrachlorod1benzo-p-d1ox1n-1nduced changes In the hydroxylatlon of biphenyl by rat liver mlcrosomes. Blochem. Pharmacol. 24(3): 335-340. Hook, J.B., K.M. McCormack and W.M. Kluwe. 1977. Renal effects of 2,3,7,8tetrachlorod1benzo-p-d1ox1n. In: Pentachlorophenol, K.R. Rao, Ed. Plenum Press, NY. p. 381-387. Houk, V.N. 1983. Testimony by Dr. Vernon W. Houk, Olrector, Center for Environmental Health, CDC, PHS, DHHS before the United States House of Representatives Science and Technology Committee, Subcommittee on Natural Resources, Agricultural Research and Environment, March 23. 01370 IX--21 09/18/84 Hueper, W.C. and W.O. Conway. 1964. Chemical carcinogenesis and cancers. Springfield, IL. Thomas. Huetter, R. and M, Philippi. 1982. Studies on microbial metabolism of TCDD under laboratory conditions. Pergamon Ser. Env'iron. Sc1. 5: \87-93. Hussain, S., L. Ehrenberg, G. Lofroth and T. Gejvall. effects of TCDO on bacterial systems. Amblo. 1: 32-33. 1972. Mutagenic * Hwang, S.W. 1973. Effect of 2,3,7,8-tstrachlorod1benzo-p-d1ox1n on the biliary excretion of Indocyanine green 1n rat. Environ. Health Perspect. 5: 227-231. IARC (International Agency for. Research on Cancer). 1977. LARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Han. Some Fumi gants, the Herbicides 2,4-D and 2,4,5-T, Chlorinated 01benzod1ox1ns and Mis cellaneous Industrial Chemicals. Vol. IS, IARC, Lyon, France, p. 41-102. ICO (International Classification of Diseases). 1975. 9th revision. WHO, Geneva. Ideo, G., G. Bellatl, A. Bellobuono, P. MocarelH, A. Marocchl and P. Brambllla. 1982. Increased urinary D-glucar1c acid excretion by children living In an area polluted with tetrachlorod1benzoparad1ox1n (TCDO). Cl 1n . Chlm. Acta. 120(3): 273-283. 01370 IX-22 09/18/84 Isensee, A.R. and G.E. Jones. 1975. Distribution of 2,3,7,8-tetrachlorodibenzo-p-d1ox1n (TCDD) 1n aquatic model ecosystem. Environ. Sc1. Technol. 9: 668-672. Ishldate, K., H. Tsuruoka and Y. Nakazawa. 1980. Induction of choline kinase by polycyclic aromatic hydrocarbon carcinogens 1n rat liver. Blo chern. Blophys. Res. Coiwnun. 96: 946-952. Johnson, E.F. and U. Mller-Eberhard. 1977a. Resolution of two forms of cytochrome P-450 from liver mlcrosomes of rabbits treated with 2,3,7,8tetrachlorod1benzo-p-d1ox1n. J. Biol. Chem. 252(9): 2839-2845. Johnson, E.F. and U. Muller-Eberhard. 1977b. Multiple forms of cytochrome P-450 from liver mlcrosomes of rabbits treated with 2,3,7,8-tetrachlorodlbenzo-p-d1ox1n (Meet. Abstr.). Fed. Proc. 36(3): 833. Johnson, E.F. and U. Muller-Eberhard. 1977c. Purification of the major cytochrome P-450 of liver mlcrosomes from rabbits treated with 2,3,7,8tetrachlorodlbenzo-p-dloxln (TCOD). 81ophys. Res. Commun. 76(3): 652-659. Johnson, E.F. and U. Muller-Eberhard. 1977d. Multiple forms of cytochrome P-450 resolution and purification of rabbit liver aryl hydrocarbon hydroxy lase. Blochern. Blophys. Res. Commun. 76(3): 644-G51. Johnson, E.F., G.E. Schwab and U. Muller-Eberhard. 1979. Multiple forms of cytochrome P-450: Catalytic differences exhibited by two homogeneous forms of rabbit cytochrome P-450. Mol. Pharmacol. 15(3): 708-718. 01370 IX-23 09/18/84 Johnson, F.E., M.A. Kugler and S.M. Brown. 1981. Soft-tissue sarcomas and chlorinated phenols. Lancet. 2(8236): 40. . Jones, G. 1975. A hlstochemlcal study of the Uver lesion Induced by 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (dioxin) In rats. J. Pathol. 116(2): 101-105. Jones, G. and W.H. Butler. 1974. A morphological study of the Uver lesion Induced by 2,3,7,8-tetrach1orod1benzo-p-d1ox1n 1n rats. J. Pathol. 112: 93-97. Jones;, G. and J.8. Grelg. 1975. Pathological changes In the liver of mice given 2,3,7,8-tetrachlcrod1benzo-p-d1ox1n. Experlentla. 31(11): 1315-1317. Jones,, K.G. and G.O. Sweeney. 1977. Association between Induction of aryl hydrocarbon hydroxylase and depression of uroporphyrinogen decarboxylase activity. Res. Comrnun. Chem. Pathol. Pharmacol. 17(4): 631-638. Jones, K.G. and G.O. Sweeney. 1980. Dependence of the porphyrogenlc effect of 2,3,7,B-tetrachlorod1benzo-p-d1ox1n upon Inheritance of aryl hydrocarbon hydroxylase responsiveness. Toxicol. Appl. Pharmacol. 53(1): 42-49. Kapltulnlk, J. and J.D. Ostrow. 1978. Stimulation of bilirubin catabolism 1n jaundiced Gunn rats by an Inducer of mlcrosmal mixed-function monooxy genases. Proc. Natl. Acad. Sc1. 75(2): 682-685. 01370 IX-24 09/18/84 Karickhoff, S.W., D.S. Brown and T.A. Scott. 1979. Sorption of hydr-pholic pollutants on natural sediments. Water Res. 13: 241-248. Khera, K.S. and W.P. HcKinley. 1972. Pre- and postnatal studies on 2,4,5-T and 2,4-0 and their derivatives in rats. Toxicol. Appl. Pharmacol. 22: 14-28. Khera, X.S. and J.A. Ruddick. 1973. Polychlorod1benzo-p-dioxins: Perinatal effects and the dominant lethal test in Wlstar rats. Adv. Chem. Ser. 12C: 70-84. Khera, K.S., 8.L. Huston and W.P. McKinley. 1971. Pre- and postnatal studies on 2,4,5-T, 2,4-0, and derivatives in Wistar rats. Toxicol. Appl. Pharmacol. 19: 369-370......... .. Kimbrough, R.D. 1974. The toxicity of polychlorinated polycyclic compounds and related chemicals. CRC Crit. Rev. Toxicol. 2: 445-489. Kimbrough, R.O., C.D. Carter, J.A. Liddle and R.E. Cline. 1977. Epidemi ology and pathology of a tetrachlorodlbenzodioxin poisoning episode. Arch. Environ. Health. 32(2): 77-86. Kimbrough, R.O., J. Buckley, L.'Fishbein, et al. 1978. Animal toxicology. Environ. Health Perspect. 24: 173-184. . Kimmig, J. and K.H. Schulz. 1957. Berufliche akna (sog. chlorakne) durch chloriette aromatische zylkische ather. Dermatulogla. 115: 540-546. (Ger.) 01370 IX--25 / 09/18/84 King, M.E. and A.ft. ftoesler. 1974. Subacute Intubation study on rats with the compound 2,3,7,8-tetrachlorldloxln. U.S. EPA. NTIS PB 257 677. p. 27. Klrsch, et al. 1975. Structural and functional studies of Ugandln, a major renal organic an1on-b1nd1ng protein. J. Clin. Invest. 55: 1009. Kltchln, K.T. and J.S. Woods. 1977. 2,3,7,8-Tetrachlorod1benzo-p-d1ox1n (TCOD) Induced synthesis of cytochrome P-448 and aryl hydrocarbons hydroxy lase (AHH) activity In female rat liver (Meet. Abstr.). Pharmacol. 19(2): 232. Kltchln, K.T. and J.S. Woods. 1878a. 2,3,7,8-Tetrachlorod1benzo-p-d1ox1n Induction of aryl hydrocarbon hydroxylase (AHH) In hepatic mlcrosomes from female rats. Toxicol. Appl. Pharmacol. 45(1 ): 297. . Kltchln, K.T. and J.S. Woods. 1978b. 2,3,7,8-Tetrachlorod1benzo-p-d1ox1n Induction of aryl hydrocarbon hydroxylase In female rat Uver, evidence for de novo synthesis of cytochrome P-448. Mol. Pharmacol. 14: 890-899. Knutson, J.C. and A. Poland. 1989. 2,3,7,8-Tetrachlorod1benzo-p-d1ox1n: Failure to demonstrate toxicity In twenty-three cell types. Toxicol. Appl. Pharmacol. 54: 377-383. Koclba, R.J., P.A. Keeler, C.N. Park and P.J. Gehrlng. 1976. 2,3,7,8Tetrachlorod1benzo-p-dTox1n results of a 13-week oral toxicity study 1n rats. Toxicol. Appl. Pharmacol. 35: 553-574. 01370 IX--26 09/18/84 Kociba, R.J., o.fi. Keyes, J.E. Beyer et al. 1978a. Results of a two-year chronic toxicity and oncogenicity study of 2.3.7,8-tetrachlorod1benzo-pdloxin 1n rats. Toxicol. Appl. Pharmacol. 46(2): 279-303. Kociba, R.J., O.G. Keyes, J.E. Beyer and R.M. Carreon. 1978b. Toxicologic studies of 2,3,7,8-tetrachlorod1benro-p-d1ox1n (TCOO) 1n rats. Occup. Med. 4: 281-287. TOX1col. Kociba, R.J., O.G. Keyes, J.E. Beyer, R.M. Carreon and P.J. Gehrlng. 1979. Long-term toxicologic studies of 2,3,7,8-tecrachlorod1benzo-p-d1oxln (TCOO) 1n laboratory animals. Ann. NY'Acad. Sci. 320: 397-404. Kohll, K.K. and J.A. Goldstein. 1981. Effects of 2,3,7,8-tetrachlorodl- benzo-p-d1ox1n on hepatic and- renal prostaglandin synthetase. 29(3): 299-305. Life Sc1. Kondorosi, A.-, I. Fedorcsak, F. Solymosy, L. Ehrenberg and S. OstermanGolkar. i973. Inactivation of QBRNA by electrophiles. Mutt. Res. 17: 149-161. KouM, R. 1976. Relationship between levels of aryl hydrocarbon hydroxy lase activity and susceptibility to 3-methylcholanthrene and benzo(a)pyreneInduced cancers 1n Inbred strains of mice. In: Polynuclear Aromatic Hydro carbons: Chemistry, Metabolism and Carcinogenesis, Vol. 1, R.j. Freudenthal and P.W. Jones, Ed. Raven Press, NY. p. 139. 01370 09/18/84 KouM, R.E., H. Ratrie, III, S.A. Atlas, A. N1wa and O.W. Nebert. 1974. Aryl hydrocarbon hydroxylase Induction in human lymphocyte cultures by 2,3,7,8-tetrachlorod1benzo-p-d1o*1n. Life Sc1. 15(9): 1585-1595, Kouri, R.E., T.H. Rude, R. Joylekar, et al. 1978. 2,3,7,8-Tetrachlorodlbenzo-p-diox1n as cocardnogen causing 3-methylcholanthrene-1n1 Mated sub cutaneous tumors 1n mice genetically "nonresponslve" at Ah locus. Cancer Res. 38(9): 2777-2783. Kurl, R.N., 2. Lund, L. PoelUnger and J.A. Gustafsson. 1982. Differential effect of 2,3,.7,8-tetrachlorod1benzo-p-d1ox1n on nuclear RNA polymerase activity In the rat liver and thymus. Blochem. Pharmacol. 31(15): 2459-2462. . Kurokl, H. and Y. Hasuda. 1977. Structures and concentrations of. the main components of polychlorinated biphenyls retained In patients with Yusho. Chemosphere. 6: 465-469. Lamb, J.C., J.A. Moore and T.A. Markes. 1980. Evaluation of 2,4-0, 2,4,5-T, and TC00 tcxlclty 1n C57B1/6 mice: Reproduction and fertility 1n treated male mice and evaluation of congenital malformation 1n their off spring. NTP, Research Triangle Park, NC. Lee, I.P and K. Suzuki. 1980. Induction of aryl hydrocarbon hydroxylase activity 1n the rat prostate glands by 2,3,7,8-tetrchlorod1benzo-p-d1ox1ns. J. Pharmacol. Exp. Ther. 215(3): 601-605. 01370 IX-28 09/18/84 Legraverend, C., B. Mansour, D.W. Nebert and J.M. Holland. 1980. Genetic differences 1n benzo[a]pyrene-1n1 Mated tumorlgenesls 1n mouse skin. Phar macology. 20: 242-255. Levin, J.O., C.F. Rappe and C.A. Nllssen. 1976. Use of chlorqphenols or fungicides 1n sawmills. Scand. 3. Work Environ. Health. 2: 71-81. L1bert1, A., 0. Brocco, I. Allegrlnl and G. 8erton1. 1978. Field photo degradation of TCDO by ultraviolet radiations. Toxicol. Chem. Aspects. 1: 195-200. L1em, H.H., U. Muller-Eberhard and E.F. Johnson. 1980. Differential Induc tion by 2,3,7,8-tetrachlorod1benzo-p-d1ox1n of multiple forms of rabbit microsomal cytochrome P-450: Evidence for tissue specificity. J. Hoi. Phar macol. 18(3): 565-570. . Lindahl, R., M. Roper and R.A. 01etr1ch. 1978. Rat liver aldehyde dehydro genase -- Immunochemical Identity of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n Inducible normal Uver and 2-acetylam1nofluorene Inducible hepatoma iso zymes. Blochem. Pharmacol. 27(20): 2463-2465. Llss, Q.S. and P.G. Slater. 1974. Flux of gases across the a1r-sez Inter face. Nature. 247: 181-184. ; 01370 IX-29 09/18/84 Loprieno, N., I. Sbrana, D. Rusdano, D. Lasdalfarl and T. Lari. 1982. In vivo cytognie studies on mice and rats exposed to 2,3,7,8-tetrachlorodl- benzo-jj-d1ox1n (TCDO). In.: Chlorinated Dioxins and Related Compounds: Impact on the Environment, 0. Hutzlnger, et al., Ed. Pergamon Press, NY. p. 419-428. : Lucler, G.W. and O.S. McDaniel. 1979. Developmental toxicology of the halogenated aromatics: Effects on enzyme development. Ann. NY Acad. S d . 320: 449-457. - Luder, G.W., 8.R. Sonawane, O.S. McDaniel and G.E.R. Hook. 1975. Post natal stimulation of hepatic microsomal enzymes following administration of TCDD to pregnant rats. Chem. 81ol. Interactions. 11: 15-26. Luster, M.I., R.E. Faith and G. Clark. 1979a. Laboratorystudies on the Immune effects of halogenated aromatics. Ann. NY Acad. Scl. 320: 473-484. Luster, M.I., G. Clark, L.O. Lawson and R.E. Faith. 1979b. Effects of a -brief 1n vitro exposure to 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCDD) on mouse lymphocytes. J. Environ. Pathol. Toxicol. 2(4): 965-977. Luster, M.I., G.A. 8oorman, J.H. Oean et al.1980. Examination of bone marrow, Immunologic parameters and host susceptibility following pre- and postnatal exposure to 2.3,7,8-tetrachlorod1benzo-p-d1ox1n (TCOO). Immunopharmacol. 2(4): 301-310. Int. J. 01370 IX-30 09/18/84 Mabey, W.R., J.H. Smith, R.T. Podoll, et al. 1981. Aquatic fate processes data for organic priority pollutants. Monit. Data Support Div., OWRS, Wash ington, DC. EPA 440/4-81-014. p. 107-108. Madge, D.S. 1977. Effects of trichlorophenoxyacetic acid and chlorodioxins on small intestinal function. Gen. Pharmacol. 8: 319-324. Malik, N. and I.S. Owens. 1977. Studies on the requirements for induction of UDP glucuronosyl-transferase (T'ASE) activity in the Reuber hepatoma H-4It-E established cell line (Meeting Abstract). Pharmacology. 19(2): 150. Malik, N., G.M. Koteen and I.S. Owens. 1979. Induction of UDP-glucuronosyl-transferase activity in the Reuber H-4-II-E hepatoma cell, culture. Mol. Pharmacol. 16(3): 950-960-. ' Manara, L.. P. Coccia and T. Croci. 1982. Persistent tissue levels of TCDD in the mouse and their reduction as related to prevention of toxicity. Drug Metab. Rev. 13(3): 423-446. Manis, J. and R. Apap. 1979. Intestinal organic anion transport, gluta thione transferase and aryl hydrocarbon hydroxylase activity: Effect of dioxin. Life Sci. 24(15): 1373-1380. Manis, J. and G. Kim. 1979a. Stimulation of Iron absorption by polychlori nated aromatic hydrocarbons. Am. J. Physiol. 236(6): E763-E768. 01370 IX--31 09/18/84 Manls, J. and 6. K1m. 1979b. Introduction of Iron transport by a potent Inducer of aryl hydrocarbon hydroxylase, 2,3,7,8-tetrachlorodlbenzo-pdloxln. Arch, Environ. Health. 34(3): 141-145. Mantovanl, A., A. Vecchl, W. Lu1n1 et al. 1980. Effect of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n on macrophase and natural killer cell-mediated cyto toxicity 1n mice. B1pmed1c1ne. 32(4): 200-204. Harks, T.A., G.A. Klmmel and R.E. Staples. 1981. Influence of symmetrical polychlorinated biphenyl Isomers on embryo and fetal development 1n mice. Toxicol. Appl. Pharmacol. 61: 269-276. Harselos, M., R. Torronen and A. A1t1o. 1978. Responses of the 0-glucuronlc acid pathway 1n rat -tissues to treatment with tetrachlorodlbenzo dioxin. Xenoblotlcs. 8(7): 397-402. Mason, M.E. and A.8. Okey. 1982. Cytosolic and nuclear binding of 2,3,7,8tetrachlorod1benzo-p-d1ox1n to the AH receptor 1n extra-hepatic tissues of rats and mice. Eur. J. Blochem. 123(1): 209-215. Matsumura, F., J. Quensen and G. Tsushlmoto. 1983. Microbial degradation of TCD0 In a model ecosystem. In: Human and Environmental Risks of Chlori nated Q1ox1ns and Related Compounds, R.E. Tucker, A.L. Young and A.P. Gray, Ed. Plenum Press, NY. p. 191-192. Mattlson, O.R. and S.S. Thorgelrsson. 1978. Gonadal aryl hydrocarbon hydroxylase 1n rats and mice. Cancer Res. 38(5): 1368-1373. 01370 IX-32 09/18/84 Mattlson, O.R. and S.S. Thorgelrsson. 1979. Ovarian aryl hydrocarbon hydroxylase activity and primordial oocyte toxicity of polycyclic aromatic hydrocarbons 1n mice. Cancer Res. 39: 3471-3475. Hay, G. 1973. Chloracne from the accidental production of tetrachlorodlbenzodloxln. 8r. J. Ind. Hed. 30(3): 276-283. Hay, G. 1982. . Tetrachlorod1benzod1ox1n: A survey of subjects ten years after exposure. 8r. J. Ind. Hed. 39(2): 128-135. HcCann, J. 1978,. Unpublished study. (Cited 1n Wassom et al., 1978) HcConnel, E.E. 1980. Acute and chronic toxicity, carcinogenesis, reproduc tion, teratogenesls and mutagenesis In animals. In: Halogenated Biphenyls, Terphenyls, Naphthalenes, 01benzod1ox1ns and Related Products, R.O. Kimbrough, Ed. Elsevier/North-Hoi land Biomedical Press, NY. p. 109-150. HcConnel1, E.E. and J.A. Hoore. 1979. Toxlcopathology characteristics of the halogenated aromatics. Ann. NY Acad. Sc1. 320: 138-150. HcConnel1, E.E., J.A. Hoore, J.K. Haseman and H.W. Harris. 1978a. The com parative toxicity of chlorinated d1benzo-p-d1oxlns 1n mice and guinea pigs. Toxicol. Appl. Pharmacol. 44(2): 335-356. HcConnel1, E.E., J.*. Hoore and O.W. Oalgard. 1978b. Toxicity of 2,3,7,8tetrachlorod1benzo-p-d1ox1n 1n rhesus monkeys (Hacacas mulattaV following a single oral dose. Toxicol. Appl. Pharmacol. 43: 175-187. r 01370 IX--33 09/18/84 McKinney, J.D., K. Chae, B.N. Gupta, J.A. Moore and J.A. Goldstein. 1976. Toxicological assessment of hexachlorobiphenyl isomers and 2,3,7,8-tetrachlorodibenzofuran in chicks. I. Relationship of chemical parameters. Toxicol. Appl. Pharmacol. 36: 65-80. McNulty, W.P. 1978. Direct testimony before the administrator, U.S. EPA, FIFRA Oocket No. 415, EPA Exhibit No. 106. . McNulty, W.P., K.A. N1elsen-Smith, J.O. Lay, Jr. et al. 1982. Persistence of TCDO in monkey adipose tissue. Food Cosmet. Toxic. 20: 985-987. McQueen, E.G., A.M.O. Veale, W.S. Alexander and M.N. Bates. 1977. 2,4,5-T and human birth defects. Report of the Division of Public Health, New Zealand Dept. Health. (Cited -In Mllby et al.,' 1980) Michigan Department of Public Health. 1983a. Evaluation of congenital mal formation rates for Midland and other selected Michigan Counties compared nationally and statewide 1970-1981. Michigan Dept. Public Health, May 4. Michigan Department of Public Health. 1983b. Evaluation of soft and con nective tissue cancer mortality rates for Midland and other selected Michigan counties compared nationally and statewide. Michigan Dept. Public Health, May 4. Miettinen, 0. 1976. Estlmability and estimation in case referent studies. Am. J. Epidemiol. 103: 226-235. 01370 IX-34 09/18/84 HI 1by, T.H., E.L. Hustlng, H.D. Whorton and S. Larson. 1980. Potential health effects associated with the use of phenoxy herbicides: A summary of recent scientific literature. Report for the National Forest Products Assoc, from Environ. Health Assod?ted, Inc., Berkeley, CA. Moore, J.A. and R.E. Faith. 1976. Immunologic response and factors affect ing Its assessment. Environ. Health Perspect. 18: 125-131. Moore, J.A. and J.G. Vos. 1974. Evaluating the rodent Immune system as a focus for teratogenic effects. Teratology. 9(3): A-29. Moore, J.A., 8.N. Gupta,'J.G. Z1nkl and J.G. Vos. 1973. Postnatal effects of maternal exposure to 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCDD). Environ. Health Perspect. - 5: -8-1-85. - Moore, J.A., M.W. Harris and P.W. Albro. 1976. Tissue distribution of [14C] tetrachlorodlbenzo-p-dloxln 1n pregnant and neonatal rats. Toxicol. Appl. Pharmacol. 37(1): 146-147. Moore, J.A., E.E. McConnell, D.W. Dalgard and M.W. Harris. 1979. Compara tive toxicity of three halogenated dlbenzofurans 1n guinea pigs, mice and rhesus monkeys. Ann. NY Acad. Sc1. 320: 151-163. Moses, M. and I. Sellkoff. 1981. Soft tissue sarcomas, phenoxy herbicides and chlorinated phenols. Lancet. 1(8234): 1370. 01370 IX-35 09/18/84 Mottura, A., G. Ze1, F. Nuzzo et al. 1981. Evaluation of results of chromosome analyses on lymphocytes of TCOO exposed subjects after the Seveso accident. Mutat. Res. 85(4): 238-239. . .J Mulcahy, M.T. 1980. Chromsome aberrations and "Agent Orange". Med. J. Aust. 2(10): 573-574. .. Murray, F.J., F.A. Smith, K.D. Nltschke, C.G. Humlston, R.J. Koclba and 8.A. Schwetz. 1979. Three-generation reproduction study of rats given 2,3,7,8- tetrachlorod1benzo-p-d1ox1n (TCOO) 1n the diet. Toxicol. Appl. Pharmacol. 50: 241-251. . Nagarkattl, P.S., G.O. Sweeney, J. Gauldle and O.A. Clark. 1984. Sensitiv ity to suppression of cytotoxic T cell generation by 2,3,7,8-tetrachlorodlbenzo-p-dloxin (TCOO) 1s dependent on the AH genotype of the murine host. Toxicol. Appl. Pharmacol. 72: 169-176. NAS (National Academy of Sciences). 1977. Drinking Hater and Health: Part II. NAS, Washington, DC. Nau, H. and R. Bass. 1981. Transfer of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCOO) to the mouse embryo and fetus. Toxicology. 20(4): 299-308. Nau, H., R. Bass and 0. Neubert. 1982. Transfer of 2,3.7.8-tetrachlorod1benzo-p-d1ox1n (TCOO) to the mouse embryo, fetus and neonate. In: Chlo rinated Dioxins and Related Compounds. Impact on the Environment, 0. Huntzlnger, R.W. Fre1, E. Merlan and F. Pocchlarl, Ed. Pergamon Press, NY. p. 325-337. 01370 IX-36 09/18/84 Neal, R.A., P.W. Beatty and T.A. Gas1ew1cz. 1979. Studies of the mecha nisms of toxicity of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCDD). Ann. NY Acad. Sc1. 320: 204-213. Meal, R.A., J.R. Olsen, T.A. Gas1ew1cz and L.E. Geiger. 1982. The toxico kinetics of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n 1n mammalian systems. Drug Metab. Rev. 13: 355-385. Nebert, D.W. 1979. Multiple forms of Inducible drug-metabol1z1ng enzymes: A reasonable mechanism by which any organism can cope with adversity. Mol. Cell. 81ochem. 27: 27-45. ' Nebert, O.W. 1982. Pharmacogenetics and human cancer. Host factors 1n human carcinogenesis. IARC Scientific Pub. No. 39. Lyon, France. Nebert, D.W. and J.E. Glelen. 1972. Genetic regulation of aryl hydrocarbon hydroxylase Induction 1n the mouse. Fed. Proc. 31: 1315-1327. Nebert, O.W. and N.M. Jensen. 1979. The Ah locus: Genetic regulation of the metabolism of carcinogens, drugs, and other environmental chemicals by cytochrome P-450 mediated monooxygenases. CRC Cr1t. Rev. Blochem. 6: 401-437. Nebert, D.W., F.W. Goujon and J.E. Glelen. 1972. Aryl hydrocarbon hydroxylase Induction by polycyclic hydrocarbons: Simple autosomal dominant trait 1n the mouse. Natl. New. Biol. 236: 107-110. 01370 IX--37 09/18/84 Nebert, D.W., et al. 1975. Genetic expression of aryl hydrocarbon hydroxy lase activity 1n the mouse. J. Cell. Physiol. 85: 393-414. _ Nebert, 0., S. Thorglersson and J. Felton. 1976. Genetic differences 1n mutagenesis, carcinogenesis, and drug toxicity. In: In v1tro Metabolic Activation 1n Mutagenesis Testing, F. de Serros, J. Folets, J. Bend and R. Philpot, Ed. Elsevier/North Holland Biomedical Press, Amsterdam, p. 105-124. ' * Nebert, D.W., H.J. E1sen, M. Neglshl, M.A. Lang, L.M. Hjelmeland and A.B. Qkey. 1981. Genetic mechanisms controlling the Induction- of polysubstrate monooxygenase (P-450) activities. Ann. Rev. Pharmacol. Toxicol. 21: 431-462. Nebert, D.W., H. Neglshl and H.J: E1sen. 1983. Genetic differences 1n enzymes which metabolize drugs, chemical carcinogens and other environmental pollutants. In.: Human and Environmental Risks of Chlorinated Dioxins and Related Compounds, R.E. Rucker, A.L. Young and A.P. Gray, Ed. Plenum Press, NY. p. 441-462. Nelson, C.J., J.F. Holson, H.G. Green and D.W. Gaylor. 1979. Retrospective study of the relationship between agricultural use of 2,4,5-T and cleft palate occurrence 1n Arkansas. Teratology. 19: 377-384. Neubert, D. and I. Dlllmann. 1972. Embryotoxlc effects 1n mice treated with 2,4,5-tr1chlorophenoxyacet1c acid and 2,3,7,8-tetrachlorod1benzo-pd1ox1n. Arch. Pharmacol. 272(3): 243-264. 01370 IX-38 09/18/84 Neubert, 0., P. Zens, A. Rothenwallner and H.J. Merker. 1973. A survey of the embryotoxlc effects of TCOD 1n mammalian species. Environ. Health Perspect. 5: 67-79. Nlenstedt, W.f M. Parkkl, P. Uotlla and A. A1t1o. 1979. Effects of 2,3,7,8-tetrachlorodlbenzo-p-dloxln on hepatic metabolism of testosterone 1n the rat. Toxicology. ~ 13{3): 233-236. Nlsbet, I.C.T. and M.8. Paxton. 1982. Statistical aspects of three- generation studies of the reproductive toxicity of TCDO and 2,4,5-T. Am. Stat. Vol. 36(37: 290-298. N1wa, A., K. Kumakl, D.W. Nebert and A.P. Poland. 1975. Genetic expression of aryl hydrocarbon hydroxylase .activity 1n the mouse. Arch. Blochem. Blophys. 166: 559-564. _ Nolan, R.J., F.A. Smith and J.G. Hefner. 1979. Elimination and tissue distribution of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCOD) 1n female guinea pigs following a single oral dose. Toxicol. Appl. Pharmacol. 48(1): A162. Norback, D.H. and J.R. Allen. 1973. Biological responses of the nonhuman primate, chicken and rat to chlorinated d1benzo-p-d1ox1n Ingestion. Environ. Health Perspect. 5: 233-240. Norman, R.L., E.F. Johnson and U. Muller-Eberhard. 1978. Identification of the major cytochrome P-450 form transplacentally Induced 1n neonatal rabbits by 2,3,7,8-tetrachlorod1benzo-p-d1ox1n. J. 81ol. Chem. 253(23): 8640-8647. 01370 IX-39 09/18/84 NRC {National Research Council). 1977. Drinking Water and Health. NRC Safe Drinking Water Committee. National Academy Press, Washington, DC. Volume 1. NRCC (National Research Council of Canada). 1981. Polychlorinated t)1benzo-p-D1ox1ns: Criteria for their Effects on Han and his Environment. NRCC/CNRC Associate Committee on Scientific Criteria for Environmental Quality, Ottawa, Canada. Publ. No. NRCC 18574, ISSN 0316-0114. 251 p. NTP (National Toxicology Program). 1980a. Bioassay of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n for possible carcinogenicity (gavage study). Carcino genesis Testing Program, NCI, NIH, Bethesda, HO; NTP, Research Triangle Park, NC. DHHS Publ. No. (NIH) 62-1765. NTP (National Toxicology Program). 1980b. Bioassay of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n for possible carcinogenicity (dermal study). Carcino genesis Testing Program, NCI, NIH, Bethesda, HO; NTP, Research Triangle Park, NC.' DHHS Publ. No. (NIH) 80-1757. NTP (National Toxicology Program). 1980c. Bioassay of 1,2,3,7,8- and 1.2.3.7.8.9- hexachlorod1benzo-p-d1ox1n for possible carcinogenicity (dermal study). OHHS Publ. No. (NIH) 80-1758. Carcinogenesis Testing Program, NCI, NIH, Bethesda, HD, and National Toxicology Program, NTP, Box 12233, NC. NTP (National Toxicology Program). 1980d. Bioassay of 1,2,3,7,8- and 1.2.3.7.8.9- hexachlorod1benzo-rp-d1ox1n (gavage). for possible carcinogenic ity. DHHS Publ. No. (NIH) 80-1754. Carcinogenesis Testing Program, NCI, NIH, Bethesda, HD, and National Toxicology Program, RTP, Box 12233, NC. 01370 IX--40 09/18/84 Okey, A.G. 1983. The Ah receptor: A specific site for action of chlori nated dioxins. .In: Human and Environmental Risks of Chlorinated Dioxins and Related Compounds, R.E. Tucker, A.L. Young and A.P. Gray, Ed. Plenum Press, NY. p. 423-440. Okey, A.B. and L.H. Vella. 1982. Binding of 3-methylcholanthrene and 2,3,7,8-tetrachlorod1benzo-p-d1ox1n to a common Ah receptor site 1n mouse and rat hepatic cytosls. Eur. J. Blochem. 127(1): 39-47. Okey, A.B. and L.H. Vella. 1984. Elevated binding of 2,3,7,8-tetrachlorod1benzo-p-d1oxin and 3-methylcholanthrene to the Ah receptor 1n hepatic cytosols from phenobarbltal-treated rats and mice. Blochem. Pharmacol. (In press) Okey, A.B., G.P. Bondy, H.E. Mason, et al. 1979. Regulatory gene product of the Ah locus. J. B1ol. Chem. 254: 11636-11648. Okey, A.B., G.P. Bondy, H.E. Mason et al. 1980. Temperature-dependent cytosol-to-nucleus translocation of the Ah receptor for 2,3,7,8-tetrachlorod1benzo-p-d1ox1n in continuous cell culture lines. J. 81ol. Chem. 255(23): 11415-11422. Oliver, R.K. 1975. Toxic effects of 2,3,7,8-tetrach1orod1benzo-l,4-d1ox1n 1n laboratory workers. Br. J. Ind. Med. 32(1): 49-53. Olson, J.R. and W.E. Bittner. 1983. Comparative metabolism and elimination of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCDD). Toxicologist. 3: 103. 01370 IX--41 / 09/18/84 Olson, J.R., T.A. Gas1ew1cz and R.A. Neal. 1980a. Tissue distribution, excretion, and metabolism of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCDO) 1n the Golden Syrian hamster. Toxicol. Appl. Pharmacol. 56: 78-85. Olson, J.R., M.A. Holscher and R.A. Neal. 1980b. Toxicity of 2,3,7,8- tetrachlorod1benzo-p-d1ox1n 1n the Golden Syrian hamster. Pharmacol. 55: 67-78. Toxicol. Appl. Olson, J.R., M. Gudz1now1cz and R.A. Neal. 1981. The 1n vitro and In vivo metabolism of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCDD) In the rat. Toxicologist. 1: '69-70. Olson, J.R., T.A. Gas1ew1cz, L.E. Geiger and R.A. Neal. 1983. The metabo lism of 2,3,7,8-tetrachlorod-1benzo-p-d1ox1n In mammalian systems. In: Accldeatal Exposure to Oloxlns: Human Health Aspects, R. Coulston and F. Pocchlarl, Ed. Academic Press, NY. p. 81-100. Olsson, H. and L. Brandt. 1981. Non-Hodgkin's lymphoma of the skin and occupational exposure to herbicides. Lancet. 2(8246): 579. Orris, P. 1981. Unjustified conclusion? J. Occup. Med. 23(1): 7-8. Ott, H.G., B.8. Holder and R.0. Olson. 1980. A mortality analysis of employees engaged 1n the manufacture of 2,4,5-tr1chlorophenoxyacet1c acid. J. Occup. Med. 22(1): 47-50. 01370 IX-42 09/18/84 Owens, I.S. 1977. Genetic regulation of UDP-glucuronosyltranferase induc tion by polycyclic aromatic compounds in mice. J. Biol. Chem. 252: 2827-2833. Parkinson, A., R. Cockerline and S. Safe. 1980a. Polychlorinated biphenyl Isomers and congeners as Inducers of both 3-methylcholanthrene- arid pheno- barb1tone-type microsomal- enzyme activity. 277-289. ! Chem.-B1ol. INteract.l 29: Parkinson, A., L. Robertson, L. Safe and S. Safe. 1980b. Polychlorinated biphenyls as Inducers of hepatic microsomal .enzymes: Structure-activity rules. Chem.-B1ol. Interact. 30: 271-285. Parkinson, A., R. Cockerline- nd S. Safe. 1980c. Induction of both 3-methylcholanthrene- and phenobarbltone-type microsomal enzyme activity by a single polychlorinated biphenyl Isomer. Blochem. Pharmacol. 29: 259-262. Parkinson, A., L. Robertson, L. Uhllg, M.A. Campbell and S. Safe. 1982. 2,3,4,5',5-Pentachlorob1phenyl: Differential effects on CS6BL/6J and DBA/2J Inbred mice. Blochem. Pharmacol. 31: 2830-2833. Parkinson, A., S: Safe, L. Robertson, et al. 1983. Immunochemical quanti tation of cytochrome P-450 Isozymes and epoxide hydrolase 1n liver mlcrosomes from polychlorinated and polybromlnated biphenyls: A study of struc ture activity relationships. J. B1ol. Chem. 258: 5967-5976. 01370 IX--43 0 9 / i8/84 Pass 1v S., H. Nazzaro-Porro, L. Boniforti and F. Gianottl. 1981. Analysis of lipids and dioxin 1n chloracne due to tetrachlcro-2,3,7,8-p-d1benzodloxln. Br. J. Dermatol. 105(2): 137-143. Pazderova-Vejlupkova, J., M. Nemcova, J. Plckova, L. Jlrasek and E. Lukas. 1981. The development and prognosis of chronic Intoxication by tetrachlorod1benzo-p-d1ox1n 1n men. Arch. Environ. Health. 36(1): 5-11. Pegg, D.G., W.R. Hewitt, K.M. McCormack and J.B. Hook. 1976. Effect of 2,3,7,8-tetrachlorod1benzo-rho-d1ox1n on renal function 1n the rat. J. Toxicol. Environ. Health. 2(1): 55-65. ' Peterson, R.E., N. Hamada, K.H. Yang, B.V. Hadhukar and F. Matsumura. 1979a. Depression of adenosine triphosphatase activities In Isolated liver surface membranes of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n-treated rats: Cor relation with effects on ouabain biliary excretion and bile flow. J. Phar macol. Exp. Ther. 210(2): 275-282. Peterson, R.E., N. Hamada, K.H. Yang, B.V. Kaahukar and F. Matsumura. 1979b. Reversal of 2,3,7,8-tetrchlorod1benzo-p-d1ox1n-1nduced depression of ouabain biliary rr.cretlon by pregnenolone-16-alpha-carbonltrlle and spi ronolactone 1n Isolated perfused rat livers. Toxicol. Appl. Pharmacol. 50(3): 407-416. Philippe, H., V. Krasnobagew, J. Zeyer and R. Huetter. 1981. Fate of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TC00) 1n microbial cultures and soil under laboratory conditions. FEHS Symp. 12: 2210-2233. 01370 IX-44 09/18/84 Piper, W.N., R.Q. Rose and P.J. Gehring. 1973. Excretion and tissue dis tribution of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n 1n the rat. Environ. Health Perspect. 5: 241-244. Pitot, H.C., T. Goldsworthy and H. Poland. 1980. Promotion by 2,3,7,8tetrachlorod1benzo-p-d1oxin of hepatocarclnogenesls from dlethylnltrosamlne. Cancer Res. 40: 3616-3620. 0 PUmmer, J.R., U.I. Khingeblel, D.G. Crosby and A.S. Wong. and fate. In: Chlorod1ox1ns. Adv. Chem. Ser. 120: 44. 1973. Origin Pocc:n1ar1, F., V. Sllano and A. Zamplerl. 1979. Human health effects from accidental release of tetrachlorod1benzo-p-d1ox1n (TCOD) at Seveso, Italy. Ann.: NY-Acad. Sc1. 320: 311-320. Poiger, H. and C. Schlatter. 1979. Biological degradation of TCOD 1n rats. Nature. 231(5733): 706-707. Poiger, H. and C. Schlatter. 1980. Influence of solvents and adsorbents on dermal and Intestinal absorption of TCOD. Food Cosmet. Toxicol. 18(5): 477^481. Poiger, H., H. Weber and Ch. Schlatter. 1982a. Special aspects of metabo lism and kinetics of TCOD In dogs and rats. Assessment of toxicity of TCDQ-metabollte(s) 1n guinea pigs. In: Chlorinated Dioxins and Related Com pounds. Impact on the Environment, 0. Hutzlnger, R.W. Fre1, E. Merlan and F. Pocchlarl, Ed. Pergamon Press, NY. p. 317-325. 01370 IX--45 * 09/18/84 Poiger, H., H.R. Buser, H. Weber, U. Zwelfel and C. Schlatter. 1982b. Structure elucidation of mammalian TCDD-metabol1tes. Experlentla. 38(4): 484-486. Poland, A. and E. Glover. 1973a. Chlorinates c;<benzo-p-d1ox1ns: Potent Inducers of s-am1nolevul1n1c a d d synthetase and aryl hydrocarbon hydroxy lase. II. A study of the structure activity relationship. Hoi. Pharmacol. 9: 736-747. ' Poland, A. and E. Glover. 1973b. 2,3,7,8-Tetrachlorod1benzo-p-d1ox1n. Potent Inducer of delta-am1nolevu!1n1c acid synthetase. Science. 179(4072): 476-477. Poland, A. and E. Glover.- 19-74. Comparison of 2,3,7,8-tetrachlorodlbenzop-d1ox1n, a potent Inducer of aryl hydrocarbon hydroxylase, with Inducer 3-methylcholanthrene. Hoi. Pharmacol. 10(2): 349-359. Poland, A. and E. Glover. 1975. Genetic expression of aryl hydrocarbon hydroxylase by 2,3,7,8-tetrachlorod1benzo-p-d1ox1n: Evidence for a receptor mutation In genetically non-respons1ve mice. Hoi. Pharmacol. 11(4): 389-398. Poland, A. and E. Glover. 1977. Chlorinated biphenyl Induction of aryl hydrocarbon hydroxylase activity: A study of the structure activity rela tionship. Holec. Pharmacol. 13: 924-938. 01370 IX-46 09/18/84 Poland, A. and E. Glover. 1979. An estimate of the maximum in. vivo covalent binding of 2 ,3 ,7 ,8-tetrachlorodibenzo-p-d1ox1n to rat liver pro tein, ribosomal RNA and DNA. Cancer Res. 39(9): 3341-3344. Poland, A. and E. Glover. 1980. 2,3,7,8-Tetrachlorod1benzo-p-dioxin: Segregation of toxicity with the Ah locus. Mol. Pharmacol. 17(1): 86-94. Poland, A. and J.C. Knutson. 1982. 2,3,7,8-Tetrachlorod1benzo-p-d1ox1n and related halogenated aromatic hydrocarbons: Examination of the mechanism of toxicity. Ann. Rev. Pharmacol. Toxicol. 22: 517-554. Poland, A.P., D. Smith, G. Hetter and P. Posslck. 1971. A health survey of workers ih a 2,4-D and 2,4,5-T plant with special attention to chloracne, porphyria cutanea tarda, and psychologic parameters. Arch. Environ. Health. . 22: 316-327. Poland, A.P., E. Glover, J.R. Robinson and D.W. Nebert. 1974. Genetic expression of aryl hydrocarbon hydroxylase activity. Induction of mono oxygenase activities and cytochrome PI--450 formation by 2,3,7,8-tetrachlorod1benzo-p-d1ox1n 1n mice genetically "nonresponsive* to other aromatic hydrocarbons. J. 81ol. Chem. 249(17): 5599-5606. Poland, A., E. Glover and A.S. Kende. 1976. Stereospeclf1c, high affinity binding of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n by hepatic cytosol. Evidence that the binding species 1s a receptor for Induction of aryl hydrocarbon hydroxylase. J. B1ol. Chem. 251(16): 4936-4946. 01370 IX--47 09/18/34 Poland, A., W.F. Greenlee and A.S. Kende. 1979., Studies on the mechanisms of action of the chlorinated d1benzo-p-d1ox1ns and related compounds. Ann. NY Acad. Sc1. 320: 214-230. Poland, A., 0. Palen and E. Glover. 1982. Tumour promotion by TCDO 1n skin of HRS/J mice. Nature. 300(5889): 271-273. Poland, A., J. Knutson and E. Glover. 1983. A consideration of the mechan ism of action of 2,3,,7,8-tetrachlorod1benzo-p-d1ox1n and related halogenated aromatic hydrocarbons. In.: Human and Environmental Risks of Chlorinated Dioxins and Related Compounds, R.E. Rucker, A.L. Young and A.P. Gray, Ed. Plenum Press, NY. p. 539-559. Poll, A., G. Grancesclnl,- L.--Pugl1s1 and C.R. Slrtorl. 1980. Increased total and high density lipoprotein cholesterol with apoprotein changes resembling streptozotocln diabetes In tetrachlorodlbenzodloxln (TCDD) treated rats. Blochem. Pharmacol. 28(5): 835-838. Poole, C. 1983. Statement of Charles Poole before the United States House of Representatives Science and Technology Committee, Subcommittee on Natural Resources, Agricultural Research and Environment. March 23. Potter, C.L., I.G. Sipes and D.H. Russell. 1982. Inhibition of ornithine decarboxylase activity by 2,3,7,8-tetrachlorod1benzo-p-d1ox1n. Blochem. Pharmacol. 31(21): 3367-3371. 01370 IX-48 09/18/84 Ramsey, J.C., J.G. Hefner, R.J. Karbowskl, W.H. Braun and P.J. Gehrlng. 1979. The Ijn vivo biotransformation of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCOD) 1n the rat. Toxicol. Appl. Pharmacol. 48: A162. Ramsey, J.C., J.G. Hefner, R.J. Karbowskl, W.H. Braun and P.J. Gehrlng. 1932. The In. v1tro b1otransforr?t1on of 2,3,7,8-tetrachlorod1behzo-p-d1ox1n {TCOO) tn the rat. Toxicol. Appl. Kharmacol. 65: 180-184. Regg1an1, G. 1980. Acute human exposureto TCDO 1n Toxicol. Environ. Health. 6(1): 27-43. Seveso, Italy. J. R11h1mak1, V., S. Asp, A.M. Seppalalnen and S. Herberg. 1978. Symptomatol ogy, Morbidity, and Mortality Experience of Chlorinated Phenoxy Acid Herbi cide (2,4-0 and 2,4,5-T) Sprayers 1n Finland: A Clinical and Epidemiological Study. Working paper for an IARC working group meeting on coordination of epidemiological studies on the long-term hazards of chlorinated dlbenzodloxlns and chlorinated dibenzofurans. Lyon, France. Jan. 10-11. R11h1mak1, V., A. S1sko and S. Hernberg. 1982. Mortality of 2,4-d1chlorophenoxyacetlc acid and 2,4,5-trlchlorophenoxyacetlc acid herbicide appli cators 1n Finland. Scand. J. Work Environ. Health. 8: 37-42. R11h1mak1, V., S. Asp, E. Pukkala and S. Hernberg. 1983. Mortality and cancer morbidity among chlorinated phenoxy acid applicators 1n Finland. Chemosphere. 12(4/5): 779-784. 01370 IX-49 09/18/84 R1kansf L.E., 0.0. Gibson and P.B. McCay. 1979. Evidence for the presence of cytochrome P-450 in rat mammary gland. 3039-3042. Biochem. Pharmacol. 28(19): Robertson, L,,, A. Parkinson, S. Bandiera, et al. 1984. Polychlorinated and polybrominated biphenyls: Differential effects on genetically inbred C57B1/6J and DBA/2J mice. (In preparation) Robinson, J.R., J.S. Felton, R.C. Levitt, S.S. Thorgelrsson and D.W. Nebert. 1975. Relationship between aromatic hydrocarbon responsiveness and the sur vival times 1n mice treated with various drugs anti environmental compounds. Hoi. Pharmacol. 11: 850-865. Rogers, A.M., H.E. Anderson and K.C. Back. 1982. Mutagenicity of 2,3,7,8tetrachlorod1benzo-p-d1ox1n and perfluoro-n-decano1c acid 1n LSI78y mouse lymphoma cells. Mutat. Res. 105: 445-449. Roll, R. 1971. Studies of the teratogenic effect of 2,4,5-T 1n mice. Food Cosmet. Toxicol. 9(5): 671-676. Rose, J.Q., J.C. Ramsey, T.H. Wentzler, R.A. Hummel and P.J. Gehrlng. 1976. The fate of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n following single and repeated oral doses to the rat. Toxicol. Appl. Pharmacol. 36(2): 209-226. Safe, S., L.W. Robertson, L. Safe, et al. 1982. Halogenated biphenyls: Molecular toxicology. Can. J. Physiol. Pharmacol. 60: 1057-1064. 01370 IX-50 09/18/84 Sarnia, P.R. and J. Jacops. 1981. Thoracic soft-tissue sarcoma in Vietnam veterans exposed to Agent Orange. Lancet. 302(18): 1109. Sawahata, T.t J.R. Olson and R.A. Neal. 1982. Identification of metabo lites 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCOO) formed on Incubation with isolated rat hepatocytes. Blochem. Blophys. Res. Commun. 105(1): 341-346. * Sawyer, T*. and S. Safe. 1S82. PCB Isomers and congeners: Induction of aryl hydrocarbon hydroxylase and ethoxyresorufin O-deethylaesenzyme activities 1n rat hepatoma cells. Toxicol. Lett. 13: 87-94. Schwetz, B.A., J.M. Norris, G.L. Sparschu et al. 1973. Toxicology of chlorinated d1benzo-p-d1ox1ns. Environ. Health Perspect. 5: 87-99. Seefeld, M.O. and R.E. Peterson. 1983. 2,3,7,8-Tetrachlorodibenzo-pd1ox1n-1nduced weight loss: A proposed mechanism. In: Human and Environ mental Risks of Chlorinated Dioxins and Related Compounds, R.E. Tucker, A.L. Young and A.P. Gray, Ed. Plenum Press, NY. p. 405-413.' Seefeld, M.O., R.M. Albrecht and R.E. Peterson. 1979. Effects of 2,3,7,8tetrachlorod1benzo-p-d1ox1n on Indocyanine green blood clearance 1n rhesus monkeys. Toxicology. 14(3): 263-272. Seefeld, M.D., R.M. Albrecht, K.W. Gilchrist and R.E. Peterson. 1,980. Blood clearance tests for detecting 2,3,7,8-tetrachlorod1benzo-p-d1ox1n hepatotoxlclty 1n rats and rabbits. Arch. Environ. Contam. Toxicol. 9(3).: 317-327. 01370 IX--51 09/18/84 Seller, J.P. 1973. A survey on the mutagenicity of various pesticides. Experlentla. 29: 622-623. Sharma, R.P. and P.J. GehMng. 1979. Effects of 2,3,7,8-tetrachlorodlbenzo-p-d1oxin (TCOD) on splenic lymphocyte transformation In mice after single and repeated exposures. Ann. NY Acad. Scl. 320: 487-497. Shlverick, K.T. and T.F. Muther. 1982. Effects of 2,3,7,8-tetrachlorodlbenzo-p-d1ox1n on serum concentrations and the uterotrophlc action of exogenous estrone 1n rats. Toxicol. Appl. Pharmacol. 65(1): 170-176. Shlverick, K.T. and T.F. Muther. 1983. 2,3,7,8-Tetrachlorod1benzo-p-d1ox1n (TCOD) effect on hepatic microsomal steroid metabolism and serum estradiol of pregnant rats. Blochem-. Pharmacol. 32: 991-995. t .. Shum, $., N.M. Jensen and D.W. Nebert. 1979. The Ah Locus. In utero tox icity and teratogenesls associated with genetic differences 1n benzo[a]pyrene metabolism. Teratology. 20: 365-375. Sllkworth, J., D. McMartln, A. DeCaprio, R. Rej, P. O'Keefe and L. Kaminsky. 1982. Acute toxicity 1n guinea pigs and rabbits of soot from a polychlori nated biphenyl-containing transformer fire. Toxicol. Appl. Pharmacol. 65: 425-439. Singer, R., M. Moses, J. Valclukas, R. LI 11s and I.J. Sellkoff. 1982. Nerve conduction velocity studies of workers employed In the manufacture of phenoxy herbicides. Environ. Res. 29: 297-311. 01370 IX-- 52 09/18/84 S1rch1af G. 1978. Report to Lombardy Regional Authority. (Cited 1n Pocchlarl et al., 1979) Unpublished. Smith, A.H. 1983. School of Public Health, University of Letter to Carcinogen Assessment Group, U.S. EPA, Oecember 13. California. Smith, A.H., J.E. Francis, S.J. Kay and J.B. Grelg.. 1981. Hepatic toxicity and uroporphyrinogen decarboxylase activity following a single dose of 2.3.7.8- tetrachlorod1benzo-p-d1ox1n to mice. Blochem. Pharmacol. 30(20): 2825-2830. Smith, A.H., 0.0. Fisher, N.P. Dip and C.J. Chapman. 1982a. Congenital defects and miscarriages among New Zealand 2,4,5-T sprayers. Arch. Environ. Health. 37: 197-200. - ... * .- Smith, A.H., 0.0. Fisher, N. Pearce and C.A. Teague. 1982b. Do agricul tural chemicals cause soft-tissue sarcoma? Initial findings of a casecontrol study In New Zealand. Community Health Studies. 6(2): 114-119. Smith, A.H., 0.0. Fisher, H.J. Giles and N. Pearce. 1983. The New Zealand soft tissue sarcoma case-control study: Interview findings concerning phenoxyacetlc acid exposure. Chemosphere. 12(4/5): 565-571. Smith, F.A., 8.A. Schwetz and K.D. Nltschke. 1976. Teratogenicity of 2.3.7.8- tetrachlorodlbenzo-p-dloxln In CF-1 mice. Toxicol. Appl. Pharmacol. 38(3): 517-523. 01370 IX-- 53 09/18/84 Smith, J. 1979. EPA halts most uses of herbicide 2,4,5-T. 203(4385): 1090-1091. Science. Sparschu, G.L., Jr., F.L. Dunn, Jr., V.K. Rowe, Jr. 1971a. Study of the teratogenicity of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n in the rat. Cosmet. Toxicol. 9: 405-412. Food Sparschu, G.L., F.L. Dunn, R.W. Lisowe and V.K. Rowe. 1971b. Effects of high levels of 2,4.,5-tr1chlorophenoxyacet1c acid on fetal development In the rat. Food Cosmet, Toxicol. 9(4): 527-530. Stehl, R.H., R..R. Pap.erfuss, R.A. Bredeweg and R.W. Robers. 1973. The stability of pentachlorophenol and chlorinated dioxins to sunlight, heat and combustion. Adv. Chem. Ser. T20, Am. Chem. Soc., Washington, DC. Stevens, K.M. 1981. Agent Orange toxicity: A quantitative perspective. Human Toxicol. 1: 31-39. . Stohs, S.J., M.Q. Hassas and W.J. Murray. 1983. Lipid peroxidation as a possible cause of TCDO toxicity. Blochem. Blophys. Res. Comm. Ill: 854-859. Strlk, J.J. and J.M. de W1t. 1980. Health aspects of rabbits In a low-TCDD contaminated area. Int. J. Blochem. 12(5-6): 999-1001. Sweeney, G.D. arid K.G. Jones. 1978. On the mechanism of porphyria due to chlorinated hydrocarbons. Int. Congr. Ser. Excerpta Med. 440: 229-231. 01370 IX--54 09/18/84 Sweeney, G.G. and K.G. Jones. 1983. Studies of the mechanism of action of hepatotoxlclty of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCOO) and related compounds. In: Human and Environmental Risks of Chlorinated Dioxins and Related Compounds, R.E. Tucker, A.L. Young and A.P. Gray, Ed. Plenum Press, NY. p. 415-422. Sweeney, G.O., K.G. Jones, F.M. Cole, 0. Basford and f. Krestynskl. 1979. Iron deficiency prevents liver toxicity of 2,3,7,8-tetrachlorod1benzo-pdloxin. Science. 204(4390): 332-335. Swift, L.L., T.A. Gas1ew1cz, G.D. Dunn, P.0. Soul'e and R.A. Neal. 1981. Characterization of the hyperlipidemia 1n guinea pigs Induced by 2,3,7,8tetrachlorod1benzo-p-d1ox1n. Toxicol. Appl. Pharmacol. 59(3): 489-499. Taylor, J.S. 1979. Environmental chloracne: Update and overview. Ann. NY Acad. Sci. 320: 295-307. Thibodeaux, L.J. 1979. Chemodynamlcs - Environmental Movement of Chemicals 1n A1r, Water and Soil. John Wiley and Sons, NY. p. 1976-1977. Thibodeaux, L. 1983. 0ff-s1te transport of 2,3,7,8-tetrachlorodlbenzo-p<11ox1n from a production disposal facility. In: Chlorinated Dioxins and Dibenzofurans 1n the Total Environment, Vol. I, L.H. Keith et al., Ed. 8utterworth Publishers, Woburn, MA. 01370 IX--55 1 . 09/18/84 / Thiess, A.M. and R. Frentzel-Beyme. 1977. Mortality study of persons exposed to dioxin following an accident which occurred 1n the 8ASF on 13, November, 1953. Working Papers, Joint NIEHS/IARC Working Group Report, Lyon, France, June. Thiess, A.M., R. Frentzel-Beyme and R. Link. 1982. Mortality study of per sons exposed to dioxin 1n a trlchlorophenol-process accident that occurred 1n the BASF AG on November 17, 1953. Am. J. Ind. Med. 3: 179-189. Thigpen, J.E., R.E. Faith, E.E. McConnell and J.A. Moore. 1975. Increased susceptibility to bacterial Infection as a sequela of exposure- to 2,3,7,8tetrachlorod1benzo-p-d1ox1n. Infect. Immun. 12(6): 1319-1324. Thomas, H.F. 1980. Internal- memo to P. Cohn, Office of Toxic Substances, U.S. EPA, Washington, 0C. Thomas, P.E., J.J. Hutton and B.A. Taylor. 1973. Genetic relationship between aryl hydrocarbon hydroxylase 1nduc1b1l1ty and, chemical carcinogen Induced skin ulceration In mice. Genetics. 74: 655-659. Thomas, P.T. and R.D. H1nsd1ll. 1979. The effect of perinatal exposure to tetrachlorod1benzo-p-d1ox1n on the Immune response of young mice. Drug Chem. Toxicol. 2(1,2): 77-98. Thompson, D.J., J.L. Emerson and G.L. Sparschu. 1971. Study of the effects of 2,4,5-trlchlorophenoxyacetlc acid (2,4,5-T) on rat and rabbit fetal development. Teratology. 4: 243. 01370 IX--56 09/18/84 Thunberg, T.t U.G. Ahlborg and H. Johnsson. 1979. Vitamin A (retinol) status 1n the rat after a single oral dose of 2,3,7,8-tetrachlorod1bsnzo-pdloxln. Arch.-Toxicol. 42(4): 265-274. Thunberg, T., U.G. Ahlborg, H. Hakansson, C. Krantz and M. Honler. 1980. Effect of 2,3,7,8-tetrachlorod1benzo-p-d1oxin on the hepatic storage of Retinol 1n rats with different dietary supplies of vitamin A (Retinol). Arch. Toxicol. (Berl). 45(4): 273-285. . Tofllon, P.J., P.G. Peters, R.P. Clement, D.M. Hardwlcke and W.N. Piper. 1980. Depressed guinea pig testicular microsomal cytochrome P-45G content by 2,3,7,8-tetrachlorod1benzo-p-d1ox1n. L1fe:Sc1. 27(10): 871-876. Tognonl, G. and A. 8onaccors1. 1982. Epidemiological problems with 2.3.7.8- tetrachlorod1benzo-p-d1ox1n (TCDD). Drug Hetab. Rev. 13: 447-469. Toth, K., J. Sugar, S. Somfal-Relle and J. Bence. 1978. Carcinogenic bio assay of the herbicide, 2,4,5-trlchlorophenoxyethanol (TCPE) with different 2.3.7.8- tetrachlorod1benzo-p-d1ox1n (dioxin) content 1n Swiss mice. Prog. Blocher. Pharmacol. 14: 82-93. Toth, K., S. Somfal-Relle, J. Sugar and J. Bence. 1979. Carcinogenicity testing of herbicide 2,4,5-trlchlorophenoxyethanol containing dioxin and of pure dioxin 1n Swiss mice. Nature. 278(5704): 548-549. Townsend, J.C., K.M. Bodner, P.F. Van Peenen, R.D. Olsen and R.R. Cook. 1982. Survey of reproductive events of wives of employees exposed to chlorinated dioxins. Am. J. Epidemiol. 115(5): 695-713. 01370 IX-57 09/18/84 Tsushlmoto, G., F. Matsumura and R. Sago. 1982. Fate of 2,3,7,8-tetrachlorod1benzo-p dioxin (TCOO) 1n an outdoor pond and 1n model aquatic eco system. Environ. Toxicol. Chem. 1: 61-68. Turner, J.N. and D.N. Collins. 1983. Liver morphology In guinea pigs administered either pyrolysis products of a polychlorinated biphenyl trans former fluid or 2,3,7,8-tetrachlorod1benzo-p-d1ox1ns. Toxicol. Appl. Pharmacol. 67: 417-429. United Kingdom Ministry of Agriculture, Fisheries and Food. 1983. Advisory coirmltteee on pesticides: Report on phenoxy acid herbicides. Whitehall Place, London, February 7. ' U.S. EPA. 1979a. Report of assessment of a field Investigation of six-year spontaneous abortion rates 1n three Oregon areas 1n relation to forest 2,4,5-T spray practice. Off. Toxic Subst., U.S. EPA. U.S. EPA. 1979b. Report of the FIFRA Scientific Advisory Panel on proposed Section 6(b)(2) notices for 2,4,5-T and Sllvex. Memorandum from Or. H. Wade Fowler, Jr., Executive Secretary H F R A Scientific Advisory Panel to the Deputy Administrator for Pesticide Programs, September 27. U.S. EPA. 1980. Dioxins. Industrial Environmental Research Lab., U.S. EPA, Cincinnati, OH. EPA 600/2-80-197. U.S. EPA. 1984a. Ambient Water Quality Criteria for 2,3,7,8-Tetrachlorodlbenzo-p-d1ox1n. Environmental Criteria Assessment Office, Cincinnati, OH. EPA 440/5-84-007. 01370 IX--58 09/18/84 U.S. EPA. 1984b. Health Assessment Document for Polychlorinated Dlbenzo-jjDloxlns. Environmental Criteria and Assessment Office, Cincinnati, OH. EPA 600/8-84-014A. van den Berg, M., K. 011e and 0. Hutzlnger. 1983; Uptake and selective retention 1n rats of orally administered chlorinated dioxins and dlbenzo. forans from fly ash and fly ash extract. Chemosphere. 12(4/5): 537-544. van Logten, M.J., B.N. Gupta, E.E. McConnell and J.A. Moore. 1980. Role of the endocrine system 1n the action of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCOD) on the thymus. Toxicology. 15{2): 135-144. van Logten, M.J., B.N. Gupta, E.E. McConnell and J.A. Moore. 1981. The Influence of malnutrition- on-the toxicity of 2,3,7,8-tetrachlorod1benzo-pdloxln (TCDO) 1n rats. Toxicology. 21(1): 77-88. Van Miller, J.P., R.J. Marlar and J.R. Allen. 1976. Tissue distribution and excretion of trltlated tetrachlorod1benzo-p-d1ox1n 1n non-human primates and rats. Food Cosmet. Toxicol. 14(1): 31-34. Van Miller, J.P., J.J. Lallch and J.R. Allen. 1977a. Increased Incidence of neoplasms 1n rats exposed to low levels of 2,3,7,8-tetrachlorodlbenzo- rho-d1ox1n. Chemosphere. 6(10): 625-632. : Van Miller, J.P., J.J. Lallch and J.R. Allen. 1977b. Increased Incidence of neoplasms In rats exposed to' low levels of 2,3,7,8-tetrachlorod1benzo-pdloxln. Chemosphere. 6(9): 537-544. 01370 IX-59 09/18/84 Vecchl, A., A. Mantovanl, H. Sironl, W. Lulnl, F. Spreaflco and S. Garattlni. 1980. The effect of acute administration of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n (TCOO) on humoral antibody production and cellmediated activities In mice. Arch. Toxicol. (Suppl.) 4: 163-165. Vecchl, A., M. Sironl, M.A. Canegratl, M. Recchla and S. Garattlnl. 1983. Immunosuppressive effects of 2,3,7,8-tetrachiorod1benzo-p-d1ox1n 1n strains of mice with different susceptibility to Induction of aryl hydrocarbon hydroxylase. Toxicol. Appl. Pharmacol. 68: 434-441. ' Vlnopal, J.H. and J.E. Caslda. 1973. Metabolic stability of 2,3,7,8-tetrachlorod1benzo-p-diox1n In mammalian Uver microsomal systems and 1n living mice. Arch. Environ. Contain. Toxicol. 1(2): 122-132. Vos, J.G. 1977. Immune suppression as related to toxicology. Crlt. Rev. Toxicol. 5(1): 67-101. Vos, J.G. and R.8. Beems. 1971. Oermal toxicity studies of technical poly chlorinated biphenyls and fractions thereof 1n rabbits. Toxicol. Appl. Pharmacol. 19: 617-633. Vos, J.G. and J.H. Koeman. 1970. Comparative toxicologic study with poly chlorinated biphenyls In chickens with special reference to prophyrln, edema formation, Uver necrosis and tissue residues. Toxicol. Appl. Pharmacol. 17: 656-668. . 01370 IX-60 09/18/84 Vos, J.G. and J.A. Moore. 1974. Suppression of cellular immunity in rats and mice by maternal treatment with 2,3,7,8-tetrachlorod1benzo-p-d1ox1n. Int. Arch. Allergy Appl. Immunol. 47(5): 777-794. Vos, J.G., J.A. Moore and J.G. Zinkl. 1973. Effect of 2,3,7,8-tetrachloro- dibenzo-p-dioxin on the immune system of laboratory animals. Environ. Health Perspect. 5: 149-162. Vos, J.G.., J.A. Moore and J.G. Zinkl. 1974. Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in C57B1/6 mice. Toxicol. Appl. Pharmacol. 29: 229-241. Vos, J.G., J.G. Kreeftenberg and L. Kater. 1978a. Immune suppression by TCDO. In: Dioxin: Toxicological and Chemical Aspects. SO Medical and Scientific Books, NY. p. 163-175. ' Vos, J.G., J.G. Kreeftenberg, H.W. Engel, A. Minderhoud and J.L.M. Van Noorle. 1978b. Studies on 2,3,7,8-tetrachlorodibenzo-p-dioxin induced Immune suppression and decreased resistance to infection: Endotoxin hyper sensitivity, serum zinc concentrations and effect of thymus in treatment. Toxicology. 9(1-2): 75-86. Walker, A.E. and J.V. Martin. 1979. workers. Lancet. 1: 446-447. Lipid profiles in dioxin-exposed 01370 IX--61 09/18/84 Ward, A.M. 1982. Investigation of the Immune capability of workers pre viously exposed to2,3,7,8-tetrachlorod1benzo-para-d1ox1n (TCDO). In: The Chemical Scythe: Lessons of 2,4,5-T and Dioxin, A. Hay, Ed. Plenum Press, NY. p. 117-118. Ward, C . a n d F. Hatsumura. 1977. Fate of 2,4,5-T contaminant, 2,3,7,8- tetrachloroaibenzo-p-d1ox1n (TCDD) In aquatic environments. NTIS PB 264187. 28 p. ^ Ward, C.T. and F. Matsumura. 1978. Fate of ?,3,7,8-tetrachlorod1benzo-p- dloxln (TCDD) -1n a model aquatic environment. Arch. Environ. Contam. Toxicol. 7:349-357. : Wassom, J.S., J.E. Huff and-N. Loprleno. 1978. A review of the genetic toxicology of chlorinated d1benzo-p-d1ox1ns. Mutat. Res. 47(3,4): 141-160. Weber, G., P. Luz1, L. Res1, P. TanganelH, M.R. Lovatl and A. Poll. 1983. Natural history of TCDD-1nduced U v e r lesions 1n rats as observed by trans mission electron microscopy during a32-week period after a single 1ntraperltoneal Injection. J. Toxicol. Environ. Health. 12: 533-540. Welssberg, J.8. and J.G. Z1nkl. 1973- Effects, of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n upon hemostasis and hematologic function 1n the rat. Environ. Health Perspect. 5: 119-123. Woods, J.S. 1973. Studies of the effects of 2,3,7,8-tetrachlorod1benzo-p- dloxln on mammalian hepatic -aminolevulinic acid synthetase. Environ. Health Perspect. 5: 221. . 01370 IX-62 09/18/84 Yamamoto, H., H. Yoshlmura, H. Fujlta and T. Yamamoto. 1976. Metabolic and toxicologic evaluation of 2,3,4,4' ,4'-pentachlorobiphenyl 1n rats and mice. Chem. Pharm. Bull. 24: 2168-2174. Yang, K.H. and R.E. Peterson.1977. Differential effects of halogenated aromatic hydrocarbons on pancreatic excretory functions 1n rats. Fed. Proc., Fed. Am. Soc. Exp. 81ol. 36(3): 356. Yang, K.H., W.A. Croft and R.E. Peterson. 1977. Effects of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n on plasma disappearance and biliary excretion of foreign compounds In rats. Toxicol. Appl. Pharmacol. 40: 485-496. Yang, K.H., E.J. Choi and S.Y. Choe. 1983. Cytotoxicity of 2,3,7,8-tetrachlorod1benzo-p-d1ox1n on-primary cultures of adult rat hepatocytes. Arch. Environ. Contain. Toxicol. 12: 183-188. Yoshlhara, S., K. Kawano, H. Yoshlmura, H. Kurokl and Y. Masuda. 1979. Toxicological assessment of highly chlorinated biphenyl congeners retained In the Yusho patients. Chemosphere. 8: 531-538. Yoshlhara, S., K. Nagata, H. Yoshlmura, H. Kurokl and Y. Masuda. 1981. / Inductive effect on hepatic enzymes and acute toxicity of Individual poly chlorinated dlbenzofuran congeners 1n rats. Toxicol. Appl. Pharmacol. 59: 580-588, Young, A.L., H.K. Kang and B.M. Shepard. 1983. Chlorinated dioxins as herbicide contaminants. Environ. Sc1. Technol. 17: 530A-540A. 01370 IX-63 09/18/84 Zack, J.A. and R.R. Susklnd. 1980. The morality experience of workers exposed to tetrachlorod1benzod1ox1n In a trlchlorophenol process accident. J. Occup. Med. 22(1): 11-14. Zelger, E. 1983. Memorandum from Dr. Zelger of Or. E.E. McConnell on the results of test performed for the Environmental Mutagenesis Development Program. NTP, HIEHS. Z1nkl, J.G., J.G. Vos, J.A. Moore and B.N. Gupta. 1973. Hematologic and clinical chemistry effects of. 2,3,7,8-tetrachlorod1benzo-p-d1ox1n In labora tory animals.- Environ. Health Perspect. 5: 111-118. 01370 IX-64 09/18/84