Kepone-Hazard evaluation

The Science of the Total Environment, 9 (1978) 1-62 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

K E P O N E - - H A Z A R D EVALUATION*' **

SAMUEL S. EPSTEIN

School of Public Health, University o f Illinois at the Medical Center, p.o. Box 6998, Chicago, IL 60612 (U.S.A.) (Received April 17th, 1977)

ABSTRACT

Kepone is a persistent chlorinated hydrocarbon pesticide which is no longer manufactured in the U.S.A., its uses having been cancelled on April 11, 1977; previous food uses included control of the banana root borer, and non-food uses included control of tobacco wireworm, ants, and cockroaches. An adduct of Kepone, Kelevan, is now distributed by Spiess and Sohn, Chemische Fabrik, Germany, with an as yet unknown manufacturer, for control of the Colorado potato beetle in Eastern Europe and Ireland, and for control of the banana root borer in the Cameroons, Caribbean, and South America. Kepone is acutely toxic and induces cumulative and delayed toxicity, neurotoxicity, and reproductive impairment, in a wide range of species including birds, rodents and humans; it is also carcinogenic in rodents. Extensive environmental dispersion, with major evidence of aquatic toxicity has been demonstrated following its recent manufacture in Hopewell, Virginia, U.S.A. INTRODUCTION

Kepone is a highly stable chlorinated hydrocarbon pesticide, with a caged structure, closely related to Mirex and with a common approved name of Chlordecone. Kepone was introduced in 1958 by Allied Chemical as an insecticide and fungicide. Prior to its recent cancellation in the U.S., registered food uses of Kepone were primarily for control of the banana root borer; non-food uses were primarily for control of tobacco wireworm and as a bait for control of ants and cockroaches. Kepone has high acute toxicity to a wide range of avian and mammalian species, in which it induces delayed or cumulative toxic effects, including reproductive impairment and neurotoxicity. Additionally, Kepone is carcinogenic in rats and mice, in which it induces hepatocellular carcinomas. * Editor's Comments: Until now, there has been no recent systematic analysis of kepone toxicology.

In order to provide comprehensive documentation, reference in the text, is made to various memos which are now a matter of the open pubfic record. These are also available in the United States in the Environmental Protection Agency files. ** This report is based on a review of available literature and documentation prior to June 1976.

2 The toxic effects induced in humans following occupational exposure to Kepone, are similar to those observed experimentally, and include neurotoxicity, tremors, weakness, ataxia, and opsiclonus, oligospermia, disturbances in liver function, and hepatomegaly. There is evidence of substantial environmental contamination with Kepone at a distance from its site of recent manufacture in Hopewell, Virginia.

I PRODUCTION AND FORMULATION OF KEPONE AND RECOMMENDED PRECAUTIONS

1. Nomenclature and properties of Kepone a. Nomenclature The compound was originally designated decachlorotetrahydro-4-7-methanoideneone i; however, its high stability led to a subsequent correction o f this formula and the assignment a "caged" structure, in which the carbon atoms lie at the corners of a cube with a formula of decachlorooctahydro-l,3,4-metheno-2H-cyclobuta[c,d]pentalen-2-one z. The common approved name for the compound is Chlordecone. C[

C~ C ~ ."~

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O,~-'~'~C~C[ The compound was introduced as an insecticide and fungicide by Allied Chemical in 1958 with a registered trade name of Kepone and an alternative name of GCCompound 1189 (U.S. Patent Reissue, 24, 435).

b. Formulations A major U.S. registered formulation of Kepone was a 50% wettable powder, emulsifiabl~ concentrate (Kepone EM-2) containing 2 lbs/gailon, with a dosage of 0.1 oz/plant or 2 lbs/acre; this was used for control of the banana borer, constituting the only registered food or feed use, with a tolerance of 0.01 ppm. Additional registered formulations included 5 ~o and 10 Yogranules and dusts, for non-food uses, particularly wireworm control in tobacco fields, and in baits for control of ants and other insects, particularly cockroaches, in indoor and outdoor areas inaccessible to children and pets. According to pesticide petition OEO 919, occasional traces of Kepone, 5-9 ppb, were found in banana peel, but none were found in the fruit within a detection limit of 5 ppb.

c. Properties It is a highly stable odorless and colorless solid, which sublimes without melting and with some decomposition or rapid sublimation at about 350°C at atmospheric pressure a. While its vapor pressure is not accurately known, it has been estimated at 0.0004 mm at 100°C, which approximates to 0.5 ppm 4 (10 mg/m 3 or 0.01 rag/l); at 25 °C, its vapor pressure is < 3 × 10- 7 mm. It is practically insoluble in cold water and slightly soluble in boiling water, < 0.4 % at 100°C according to EPA pesticide petition OEO 919, its solubility at unspecified temperatures and pH 4-9 is 2-4 ppm. It is soluble in strongly alkaline aqueous solution, readily soluble in acetone, and less soluble in benzene or light petroleum 13. At ordinary temperature and humidity, it readily forms hydrates, and is normally used as a mono- to -tri-hydrate; the solubility of Kepone is greatly dependent on its degree of hydration. Technical grade Kepone contains unreacted hexachlorocyclopentadiene (HCP), generally in concentrations of less than 0.5 ~o. The only available data on biodegradability of Kepone is in the EPA pesticide petition No. OEO 919, relating to tolerance in bananas 4, indicating that: "... there appeared to be some disappearance of Kepone from treated soil up to 30 days after treatment, but results at 60 and 154 days after treatment indicatedlittle Joss from most soil... Also disappearance of Kepone after 5 months in soil is minimal." In studies with 3 soil types in the Nile Delta, to which Kepone had been applied at 19.6 lb/acre, fungal, actinomycete, and bacterial populations were altered to varying degrees during the course of a 45-day study period, finally returning to control levels. These findings were explained by the suggestion that microbial degradation of Kepone had occurred s' 6. However, alternative explanations include leaching or volatilization, besides Kepone-resistance. In related studies on nitrogen transformation in Kepone-treated soils, nitrification of ammonium sulfate and ammonification of peptone were initially suppressed and then returned to normal within a 60-day test period 7, 8; inhibition of nitrification was accompanied by increase in soil nitrite levels, indicating suppression of Nitrobacter, but not of Nitrosomonas bacteria. No metabolic products of Kepone have been reported, and there is no available evidence or degradation under natural conditions. Various experimental photoproducts have been identified9. Kepone hydrate has been identified as a photodegradation product of Mirex in model systems. Additionally, low concentrations of Kepone and other Mirex-related compounds have been recovered from a pond where high concentrations of Mirex bait were accidentally spilled 10; such degradation could have been induced by anaerobic bacteria, in addition to photodecomposition and photoreduction. There does not appear to be any literature on the bioaecumulation of Kepone, other than suggestive findings in fish in the James River in proximity to LSPC 11.

2. Process of manufacture Kepone manufacture involves 3 major sequential operations x' 12 as follows: Reaction. HCP and sulfur trioxide are reacted under heat and pressure, and in

4

TABLE 1 MANUFACTURERSANDPRODUCTIONOFKEPONE(Based on Allied Data)

Company

Plant location

Period

Allied

Claymont,DE Marcus Hook, PA (B & A works) Hopcwell, VA Semi-works, Hopewell,VA State College, PA State College,PA State College,PA Niagara Falls, NY. Niagara Falls, NY. Hopewell, VA

1951 3,000 1962-1963 15,395 1966--1967 183,925 1968-1974(March) 1,617,490 1959-1960 23,533 1963 39,960 1966 2,283 1965 35,880 1967 13,800• 11/15/74--7128/75 1,696,223

Nease Hooker Life Science Products Co.

Production (lbs)

a Residualinventoryof off-specificationmaterial. the presence of antimony pentachloride as a catalyst. Quenching. The reaction mass is hydrolyzed with aqueous alkali and then neutralized with acid, forming a gelatinous slurry of Kepone. Drying. The Kepone is recovered by centrifugafion or filtration and then hotair dried in a dryer house. The dried material is then moved by conveyor to the bag house where it is packaged and prepared for shipment.

3. Manufacturers of Kepone Based on receipts at the Allied Formulating Plant in Baltimore, MD xa, the manufacturers of Kepone and their production volumes are listed in Table 1. As can be seen (Table 1), the major manufacturers of Kepone were Allied and Life Science Products (LSPC). It may be noted that LSPC manufactured more Kepone in an approximate period of 8 months, than did Allied over approximately 6 years in its Semiworks. LSPC Was formed in November, 1973 by two previous principal Allied executives, and commenced production of Kepone, its only product, in March, 1974 and ceased operations on July 24, 1975.

4. Formulation of Kepone Kepone manufactured at Allied plants and by other companies from 1951 to 1975 was shipped to the Allied Formulating and Shipping Plant at Baltimore, MD (Table 2). The major formulation of Kepone was the 90 Yotechnical grade for export to Europe. The 90 ~o grade is also milled and diluted to 80 ~o for worldwide export to formulators o f 5 ~o dusts used for control of the banana root borer; it is also processed for marketing by Velsicol in the U.S. and Canada for ant and roach baits, and for control of the grass mole cricket in Florida. Over 90 ~o of the total Kepone produced was exported, largely to Europe, and

TABLE 2 MAJOR PRODUCT5 FORMULATED AT THE ALLIED, BALTIMORE PLANT

% Kepone in product

Major u~age

0.125 5 25 50 90

Ant and roach traps, U.S.A. Exported for banana and potato dusting. Ant and roach bait, U.S.A. Control grass mole cricket, Florida. Exported to Europe for conversionto Kelevan.

80 % of this was sent to Spiess and Sohn, Germany, for further conversion into an adduct, Kelevan or Despirol, used for control of the Colorado potato beetle primarily in eastern European countries, although not registered in the U.S.A. The remainder was largely sold to the Cameroons and Jamaica for control of the banana root borer. During 1974 and the first 10 months of 1975, when over 1.5 million pounds of Kepone were manufactured, only 12,000 pounds were sold domestically to formulators of various Kepone-containing insecticides, of which 26 were federally registered in 197514; this figure is likely to be an underestimate, excluding small formulators unaware of EPA requirements. There are approximately 55 different commercial formulations of Kepone ~~ On March 24, 1976, EPA issued a "Notice of Presumption against Continued Registration" for Kepone (41 F.R. 12333) allowing its cancellation 260 days later. Allied Chemical Co. subsequently requested cancellation of its registrations in June, 1976. It is believed that Kepone is now being produced in Europe by Sandoz, AG, Switzerland, under license to Allied Chemicals, largely for sale to Spiess and Sohn. 5. Safety precautions recommended by Allied Chemical There is extensive documentation by Allied on necessary precautions recommended during the manufacture and handling of Kepone, although the extent of the general availability of these documents is not apparent in all cases. In addition to the Allied documentation, summarized chronologically below, use precautions have been published in a variety of pesticide manuals, handbooks and compendia, such as the Pesticide Official Compendium s . a. Allied Chemical t s, 1958 One of the earliest available references to precautions recommended by Allied was apparently issued in 1958. This presented LD50 values in rats and rabbits and described "typical DDT-like tremors---even as long as 4 weeks after the exposure"; this reference appeared in a standard toxicological text ~s published in 1959. b. Information provided to Nease Chemical Co. 16, 1958 The letter provides detailed information on a wide range of areas relating to the proposed manufacture o f Compound 1189 by Nease Chemical for Allied. Topics covered include the results of batch analyses, chemical and physical properties,

packaging, toxicological data which stress the cumulative effects and tremors induced by Kepone, and also needed safety precautions. The letter further states: "Working areas should not be allowed to become contaminated with compound. To assist in avoiding skin contact, clean work clothing should be provided and changed daily. During handling of the compound, exposed skin areas should be washed frequently with soap and water." Attached to this letter was an undated Allied document entitled D e v e l o p m e n t Information Bulletin 6-58 Kepone. This summarized the chemistry o f Kepone, pest control results, recommended dosage, and cautions in handling formulations. T h e document states: "IMPORTANT: The chemical physical and toxicological properties of this product have not been fully investigated and its handling or use may present additional hazards. CAUTION "May cause irritation of eyes, nose, throat and skin. "May be harmful ff inhaled or swallowed. "Avoid breathing spray mist or fumes. "Avoid contact with skin, eyes, or clothing."

c. Application f o r Kepone labelling, 1961-196217

An undated application for a label for 50 Yo K e p o n e wettable powder, was submitted by Allied as an F D A petition. The application is based on a p r o p o s e d label which clearly recognized the toxic effects o f K e p o n e and states: "Warning. Hazardous by skin contact inhalation or swallowing. Do not get in eyes, on skin, or on clothing. Wear heavy synthetic rubber gloves. Wash thoroughly with soap and water after handling and before eating or smoking. During commercial operations, also wear clean waterproof or fresh laundered clothing (coveralls, caps, etc.) and use clean clothing daily. In case of contact, immediately remove all contaminated clothing and wash skin thoroughly with soap and water. For eyes flush with water for at least 15 minutes and get medical attention. Wash clothing before re-use. "Do not breathe vapor or spray mist. During commercial operations on prolonged or repeated exposure, wear a respirator of a type passed by the Department of Agriculture for Kepone protection. Do not handle indoors unless adequate mechanical exhaust ventilation is provided. Do not contaminate food or foodstuffs." d. L e t t e r to U S D A 1 s, 1963

This letter discussed possible hazards to applicators o f 10 Yo K e p o n e dust without p r o p e r protective equipment and also makes an attempt to estimate such hazards. Simulating conditions of dusting around b a n a n a plants, with a perforated can spreading 100 lbs dust and a dusting time of a b o u t 2 h daily, model experiments f o r 8-rain dusting periods demonstrated average yields o f 0.375 mg on fresh falter p a d s o f MSA respirators, 80~o effective for 0.3 p m particles. Based on "light w o r k " respiration of 30 l/min, 8 min dusting corresponds to 2401 respiratory volume, which contained approximately 0.375 mg o f Kepone, equivalent to 0.0016 mg/l. This is stated to correspond to 4.7 mg/day for a 2-h dusting day; this presumably is in error, as it appears to correspond to 5.7 rag/day (30 × 120 x 0.0016 -----5.7). Using a rat oral LDS0 of 130 mg/kg, the equivalent LD50 for a 70 kg m a n is 9.1 g, and an a n n u a l estimated dusting period of 10 days/worker, the annual intake for a 70 kg m a n w o u l d

be 47 rag, equivalent to a p p r o x i m a t e l y 1/200 o f the o r a l LD50. A suggested f u r t h e r safety factor c o u l d be t h a t d u s t ingested in a q u e o u s f o r m w o u l d have a higher LD50. I t should be n o t e d t h a t these c a l c u l a t i o n s are b a s e d o n 2-h e x p o s u r e for 10 d a y s annually. It was c o n c l u d e d t h a t o r a l t o x i c i t y m a y n o t be c o m p a r a b l e to i n h a l a t i o n toxicity, b u t " . . . r a t h e r s u b s t a n t i a l q u a n t i t i e s o f p o w d e r e d K e p o n e a n d 50 70 K e p o n e wettable p o w d e r have been h a n d l e d . . , w i t h o u t evidence o f lung i r r i t a t i o n o r o t h e r adverse effects." T h e letter then discusses t h e d i r e c t i o n for use o f l 0 7o d u s t as follows: "Actually our directions for use of this product prescribe use of a respirator and if directions axe followed no inhalation would result . . . . We do know from animal experimentation that rats fed 0.1 LDS0 daily develop DDT-like tremors chaxactefistic of poisoning from chlorinated hydrocarbon pesticides. After 4 doses, i.e., 0.4 LD50. This effect would also be expected if absorption occurred via the respiratory tree . . . . Our argument here is that symptoms, tremors or lung irritation would be evident from ovvr-exposure and would serve as a warning that exposure should cease immediately. This can be added to the label, if it appears desirable or ner~ssary."

e. Research Memorandum 884, 1966 This m e m o r a n d u m describes the p r o d u c t i o n o f K e p o n e at the A l l i e d S e m i w o r k s in H o p e w e l l a n d s u m m a r i z e s processes, costs a n d safety precautions19: "The raw materials and desired products of this process are very toxic or reactive materials and must be handled carefully and kept from skin, eyes, respiratory system and mouth. Neoprene gloves, rubber shoes, and face shield must be worn when handling these chemicals and adequate ventilation maintained to remove vapors and dust. Matedai contacting the skin must be immediately washed off with soap and water . . . . operators should shower thoroughly after work before dressing in street clothes." I t m a y be n o t e d t h a t a c o p y o f this d o c u m e n t was sent o n J u n e 27, 1966 to Mr. W . P. M o o r e 2°, t h e n an A l l i e d e m p l o y e e , a n d s u b s e q u e n t l y p r e s i d e n t o f LSPC. I t s h o u l d also be n o t e d t h a t this m e m o r a n d u m was s u b s e q u e n t l y i n c l u d e d in the d o c u m e n t a t i o n 21 s u b m i t t e d b y A l l i e d to M o o r e on A u g u s t 5, 1975. These safety i n s t r u c t i o n s were s u b s e q u e n t l y c o m m e n t e d on, b y an A l l i e d consultant 22, as follows: " . . . These are basically sound with the exception that a precaution to avoid inhalation of dust is not included. Since the operating instructions did not cover milling of the dried filter cake, this may well explain why dust inhalation was not covered. For the similar reason that solutions were not handled accelerated absorption through skin from solutions was not mentioned."

f. Draft material safety and data sheet 2 a This was i n c l u d e d in d o c u m e n t a t i o n 21 s u b m i t t e d b y A l l i e d to L S P C A u g u s t 5, 1975. This d r a f t is u n d a t e d a n d uses s t a n d a r d 1971 D e p a r t m e n t o f L a b o r F o r m s . T h e d r a f t states: "Section V. Health hazard data TLV: Non-established, suggest 0.35 mg/m a for dust. Effect of overexposure: DDT-like tremors and convulsions.

"Section viii. Specialprotection information Respiratory protection: Dust mask with etfacient filter. Local exhaust ventilation: Yes. Protective gloves: Heavy neoprene, particularly when handling solution. Eye protection: Yes---goggles. Other protective equipment: use fresh work clothing daily.

"Section IX. Specialprecautions Precautions to be taken in handling and storing. Kepone is a cumulative toxic compound and could produce chronic effects. Major route of ingestion is through skin from non-aqueons solutions but inhalation of dust should also be strictly avoided" (12).

g. Draft Kepone Safety and Handling Precautions and First Aid Procedures 24 This u n d a t e d d r a f t was described as " b a s i c a l l y a n e n l a r g e m e n t o f t h e l a b e l p r e c a u t i o n s " , a n d was i n c l u d e d in the d o c u m e n t a t i o n 21 s u b m i t t e d b y A l l i e d t o L S P C o n A u g u s t 5, 1975. T h e d r a f t states: "It is toxic if taken by mouth, if inhaled or absorbed through the skin, particularly from solutions. Prolonged or repeated exposure by any route of entry increases the danger of toxic effect. Accordingly, strict hygiene procedures must be followed when working with this material. "To avoid contamination, food and/or smoking materials must not be stored o r consumed in or near areas where Kepone is manufactured or processed. "Do not get dust or solutions in eyes, on skin or on clothing. In case of contact, immediately remove all contaminated clothing and wash thoroughly with soap and plenty of water. "Operators manufacturing or processing Kepone should wear heavy synthetic rubber gloves and wear freshly laundered clothing dally. . . . Consequently, if clothing becomes seriously contaminated with dust or with solution as by spillage, change clothing immediately. Shoes contaminated with solution cannot be adequately cleaned and should be discarded. "Do not breathe dust. Any dust operation should be provided with mechanical ventilation to remove and collect dust and where this is not possible the worker should wear approved dust mask. I n this event, clothing contaminated with dust should be changed frequently. "Good personal hygiene is important. Wash thoroughly with soap and water after handling and before eating or smoking. Employees should be provided with double lockers to keep street clothes and work clothes separate and they should not be permitted to wear contaminated work clothes to their homes. Skin absorption from contaminated clothing is substantially in proportion to degree o f contamination and time of contact with skin. "Symptoms of overexposure to Kepone are similar to those of DDT and other highly chlorinated pesticides, namely fatigue, salivation, nausea, vomiting, etc., progressing to tremors a n d convulsions. "Good housekeeping is essential to safe practice in chemical operations. Spilled solids should be vacuumed up or carefully swept up and disposed of in a safe place. Spilled solutions should be absorbed on an inert carrier and similarly disposed of in a safe ~4ace. Although vapor pressure of Kepone is extremely low, any heated operations (dryers) should be mechanically vented outside work area."

h. Estimate of inhalation hazard of Kepone vapor and dust 2s This u n d a t e d a n d u n s i g n e d d o c u m e n t was i n c l u d e d in d o c u m e n t a t i o n 2 o s u b m i t t e d b y A l l i e d to L S P C o n A u g u s t 5, 1975. B a s e d o n a claim o f a 1 p p m " n o effect" level in rats, a n d a n a v e r a g e f o o d c o n s u m p t i o n o f 50 g / k g / d a y , the " n o effect" d i e t a r y c o n c e n t r a t i o n o f K e p o n e is thus, 5 0 / z g / k g / d a y . A s s u m i n g all i n h a l e d d u s t is a b s o r b e d , the " n o effect" h u m a n level w o u l d then be 3.5 m g ] d a y . A s s u m i n g a h e a v y w o r k i n h a l a t i o n rate o f 10 m 3 o f a i r f o r a n 8-h w o r k i n g day, the " c a l c u l a t e d p e r m i s s i b l e c o n c e n t r a t i o n " w o u l d b e 0.35 m g / m 3.

These calculations exclude a contribution f r o m Kepone vapor, which it was claimed not to be a significant factor. While the 0.35 m g / m 3 figure excludes a safety factor, some probable margin o f safety was claimed as the inhalation and deposition of dust particles is not complete and as absorption f r o m the lungs is comparable to that from the alimentary tract. Based on the above assumptions, a TLV of 0.25-0.5 m g / m 3 was suggested. i. Recent Allied labels 2 6 Recent registered Allied labels on various K e p o n e formulations contain statements such as: "Wamingl May be fatal if swallowed, inhaled or absorbed through skin . . . . Do not breathe vapor or dust. During commercial operations or prolonged or repeated exposures, wear a respirator of a type passed by the USDA for Kepone protection. Do not handle indoors unless adequate mechanical exhaust ventilation is provided. During commercial operations also wear clean waterproof or freshly laundered clothing and use clean clothing daily." j. Allied Memorandum on "Kepone Vapor ''27, 1975 The m e m o r a n d u m states that the vapor pressure of Kepone is not accurately known, but that literature indicates sublimation at 350°C at atmospheric pressure and ready sublimation at 140°C and 1 nun pressure. Using these 2 points and drawing a v a p o r pressure curve parallel to Mirex, the v a p o r pressure of Kepone at 100°C was estimated at 0.0004 ram, equivalent to a b o u t 0.5 p p m (roughly equivalent to 0.01 mg/l or 10 mg/m3). This level is about 30-fold in excess of the TLV o f 0.35 m g / m 3 proposed by K n a p p , "without any consideration of mechanically carried dust f r o m the drier". K n a p p stated that he recently learned " . . . that Kepone is dried in a forced-air drier at 100 °C", and c o m m e n t e d as follows: " . . . The exit air could be a serious inhalation baTard. This calculation (of 10 rag/m s levels) assumes saturation of air with Kepone at the drier exit, which condition may not be achieved but might be approached if air velocity through the drier were low. In any event, in any drier installation, provision should be made for safe exit of drier gases." k. Comments on labeling The Allied labeling properly draws attention to hazards of exposure to Kepone v a p o r or dust by inhalation, ingestion, or skin absorption. It, however, fails to specify the acute toxicity of Kepone, or to make any reference to available information on its neurotoxicity, reproductive toxicity, and carcinogenicity.

6. References 1 E.E. Gilbert and S. L. Giolito, U.S. Patent No. 2,616,825, November 4, 1952; U.S. Patent Reissues 24,435, 1958, and 24,749, December 15, 1959. 2 E.T. McBee, C. W. Roberts, J. D. Idol and R. H. Earle, .i.. Am. Chem. Soc., 78 (1956) 1511. 3 Pesticide Chemical Official Compendium, Association of the American Pesticide Control Officials, 1966.

10 4 W.A. Knapp, Allied Chemical, memorandum to 1. Swisher, Kepone Biodegradability, May 22, 1970. 5 A. A. A. Gawaad, M. A. Hamad and F. H. E1-Gayar, Zentralbl. Bakteriol., Parusitenkd., lnfektionskr. Hyg., 127 (1972) 290. 6 A . A . A . Gawaad, M. A. Hamad and F. H. E1-Gayar, Agrokem. Talajtan, 2 (1973) 161. 7 A. A. A. Gawaad, M. A. Hamad and F. H. E1-Gayar, Zentralbl. Bakteriol., Parasitenkd., Infektionskr. Hyg., 127 (1972) 296. 8 A . A . A . Gawaad, M. A. Hamad and F. H. EI-Gayar, Agrokem Talajtan, 2 (1973) 169. 9 E.G. Alley, B. R. Layton and J. P. Minyard, Jr., J. Agr. Food Chem., 27 (1974) 442. 10 W.B. Wheeler, D. A. Carlson, G. P. Marshall and P. Andrade, J. Agr. Food Chem., (1976) in press. 11 Environmental Protection Agency (EPA), Health Effects Research L a r r y , Preliminary Report on Kepone Levels Found in Environmental Samples from the Hopewell, VA, Area, December 16, 1975. 12 Allied Chemical, Research Informal Memorandum 834, Kepone Production--Semiworks, Hopewell, VA, March 7-May 8, 1966, June 21, 1966. 13 Allied Chemical, Kepone: Technical Receipts at Baltimore, Md, Plants, Appendix A to letter from W. S. Fergnson, to S. It. Wasserug, EPA, Philadelphia, PA, September 12, 1975. 14 It. M. Moore, National Institute for Occupational Safety and Health (NIOSH), Memorandum and attachments from EPA and Allied, to N. P. Page, National Cancer Institute ('NCD, January 8, 1976. 15 Allied Chemical, Jennings, 1958, cited as personal communication in M. N. Gleason, R. E. Gosselin, H. C, Hodge and R. P. Smith, (Eds.), William & Wilkins Co., Baltimore, 1959. 16 M.M. Darley, Allied Chemical, letter to W. F. Huber, Nease Chemical Co., April 15, 1958. 17 Allied Petition to Food and Drug Administration (IDA), Section B. Application, amount, frequency and time, undated. 18 W.A. Knapp, Allied Chemical, letter to J. S. Leary, U.S. Department of Agriculture (USDA), December 12, 1963. 19 Allied Research Informal Memorandum 884, June 21, 1966. 20 L.C. Dostian, Allied Chemical, Memorandum to Gooden, Allied Chemical, with Memorandum 884 attached, and copy to W. P. Moore, Allied Chemical, June 27, 1966. 21 Documentation submitted by L. P. Legrand, Senior Attorney, Allied, to W. P. Moore, LSPC, August 5, 1975. 22 W . A . Knapp, Allied Chemical, Kepone Toxicology and Safety Precautions, Memorandum to I. Swisher, Allied Chemical, July 25, 1975. 23 Allied Draft, U.S. Department of Labor, Material Safety Data Sheet, Kepone, undated. 24 Allied Draft, Kepone Safety and Handling Precautions and First AM Procedures, undated. 25 Allied Chemical, Document, Estimate of Inhalation Hazard of Kepone Vapor and Dust, unsigned, undated. 26 Allied Chemical Sample No. 117593, EPA Register 218-553-AA, 21°-544, 218-566, 218-589, March 3, 1975. 27 W.A. Knapp, Allied Chemical, Kepone Vapor, memorandum to W. S. Ferguson, December 5, 1975.

II. ENVIRONMENTALAND BODY BURDENS

1. Burdens f r o m registered uses The only registered food and feed use o f K e p o n e in the U.S. is f o r b a n a n a s , with a tolerance o f 10 p p b and a limit o f analytical sensitivity o f 5 p p b 1. Residues have b e e n occasionally f o u n d in b a n a n a peel, in a range o f 5-9 ppb, but n o t in the edible f r u i t 1. Theoretically, eating 1 p o u n d (454 g) o f b a n a n a s daily with residues o f 5 p p b i n the fruit would result in an intake o f 5 ttg or 0.07/~g/mg/day f o r a 70 kg adult.

I1 While there have been no FDA market basket surveys for Kepone, human fat residues have not been detected in EPA monitoring surveys 2. The possibility of residues in food crops grown in rotation with Kepone-treated tobacco has been recently raised, particularly in view of evidence of its stability in soil 2. 2. Environmental burdens f r o m production and formulation

With the exception of atmospheric particulates, samples of which had been available from earlier dates, other sample collection was initiated by EPA on August 16, 1975, after LSPC had been closed for approximately 3 weeks3; each sample was prepared for analysis by 2 laboratory groups operating "under a continuous quality assurance program". After clean-up, each sample was measured by electron capture gas-liquid chromatography, EC/GC, on a minimum of 2 different columns. Quantitative confirmation of selected samples was made with GC, using a micro-electrolytic conductivity detector and also by GC interfaced with mass spectrometry. a. Air

High volume air sample filters from Hopewell News, about 200 yards from LSPC, prior to March 1974 when LSPC went into production, contained only nanegram per m 3 levels of Kepone3; subsequent levels ranged from 3.0 to 55 #g/m 3 (Table 1), depending on weather conditions and date of collection. Levels at more distant sites in May 1975 ranged from 1.4-21 ng/m 3 (Table 1). Kepone concentrations ranged from less than 1 to over 40% of total suspended particulates 2, averaging 55% 4. b. Water

Plant drainage waters from Hopewell were discharged into Gravelly Run and thence into the James River 5. Levels of Kepone in river water at the time of sampling, after August 16, 1975, ranged from the non-detectable, 50 ppt, in the York River and Swift Creek areas to levels o f 1-4 ppb in Bailey's Creek, which receives effluent from the Hopewell Sewage Treatment Plant3; levels of up to 0.3 ppb were found in the James River near the mouth of Bailey's Creek, and of 0.1 ppb "m4he Appomatox TABLE 1 KEPONE AIR SAMPLES AS A F U N C T I O N OF COLLECTION DATE

ANDD I S T ~ C ~

VltO~ LSPC ( A u t h o r ' s D a t a )

Location

Distance & direction from LSPC

Sampling date

Atmospheric conc. ng]m8 I~g/m3

Hopewell News Hopewell News Hopewell News Hopewell News Hopewell News Hopewell Airport

0.13 miles south 0.13 miles south 0.13 milessouth 0.13 milessouth 0.13 milessouth 3.24 miles east

Petersburg

8.19 miles S.W.

Byrd Airport South Richmond

14.12 milesnorth 15.6 miles N.W.

1/11/74 1/29/74 3/30/74 2/5/75 5/18/75 5/18/75 5/18/75 5/30/75 5/30/75

0.35 0.18 ---3.63 20.7 1.93 1.41

--3.07 54.8 4.57 ---

---

12 TABLE 2 CONCENTRATIONS OF KEPONE IN WATERS AT VARIOUS LOCATIONS (Author's

Location

James River at confluence with Appomatox River James River a t mouth of Bailey's Creek Bailey's Creek Influent to Hopewell Sewage Treatment Plant Hopewcil chlorine-mixing pond sewage effluent Water from "Kepone Lagoon" LSPC et~uent Ice from cold storage Raw water for HopeweU Treatment Plant Final water from Hopeweil Treatment Plant

Data)

Conc. of Kepone ppb

0.15 0.24 0.84-4.18 2.11

ppm

m

3.46

u 1.91

--

0.36 0.94

0.10

0.05

River. Hopewell drinking water contained no detectable Kepone levels. W a t e r f r o m a holding pond at LSPC and at a land fill contained 2-3 p p m o f Kepone. Efltuent f r o m LSPC contained 0.1-1.0 p p m ; ice samples f r o m an ice plant across t h e street f r o m LSPC contained comparable levels. These data 3 are extended and s u m m a r i z e d in Table 2. Records o f Allied Plant drainage waters in M a y 11-15, 1971, indicate levels o f 50 ppb of total chlorinated hydrocarbons 5. c. Sediments, soils and sources

Levels in river b o t t o m sediments varied f r o m 10 p p b in the m o s t westerly portions o f the A p p o m a t o x River to 1-4 p p m in the Bailey's Creek area. Soil samples adjacent to LSPC on Terminal Street contained 1-2yo Kepone (10,000-20,000 p p m ) . Surface soils from the east edge o f LSPC and up to 3,000 ft. due east contained up to 300 and 2-6 ppm, respectively. Sludge samples from the holding pond and from the land fill near the Hopewell Sewage treatment plant contained 200-600 p p m of Kepone. Allied records indicate that twelve 55-gallon drums o f . a spoiled b a t c h of K e p o n e were disposed o f by burial in early 1973 s. It is further stated: "The buried material was later unearthed, the acid values neutralized and the residues mixed with soil. This disposal site is partially covered by subsequent construction. We have so far been unable to verify press reports that Kepone containing solids were also disposed of in another land fill". The circumstances resulting in the heavy contamination o f sewage and land flU have been recently indicated 5 as follows: "Of all the chemical plants in Hopowell, it (LSPC) was the only one allowed to hook up directly with the city's sewage system. This permission was granted, according to the former City Manager, on the recommendation of the then Director of Public Works for the City, C. L. Jones. Prior to holding that position, Jones had been in charge of the Allied Division that included Kepone production. He was also the area's representative on the Virginia State Water Pollution Control Board...

13 "Reportedly, by the second week after Life Science began production, the bacteriallypowered digesters in the city sewagetreatmentplant no longer functioned.For months, the city public work department's response was to illegallydump undigestedsewagematerial into land fills."

In addition to direct discharge into surface waters, contamination of water from Kepone in soil is a possibility supported by two lines of evidence. Firstly, the high persistence of Kepone in soil is indicated by Allied data, showing no decline in residue levels 154 days after treatment of soils 1. Secondly, substantial leaching of Kepone occurs at rates depending on soil type, as evidenced in model studies, using a Daphnia Bioassay, in which leachates ranging from 1.2-36.8 % in a clay loam were obtained 6 in 24 h.

d. Fish and shellfish Clams and oysters from the James River at sampling locations from 8-64 miles from Hopewell contained 0.2--0.8 ppm of Kepone 3. Drawn carp and catfish from the James River in the Bailey Creek area contained 0.01-0.02 ppm, and drawn shad, bass and bream contained 1-2 ppm; liver and entrails of bass and bream had concentrations up to 14 ppm. It is concluded that: "These results support the hypothesis of bioaccumulationof Kepone in some of the fish of the James River area."

3. Community body burdens from production and formulation A community survey was initiated by EPA on August 18, 1975 "to ascertain if human exposures as reflected by blood Kepone had in fact occurred and if so to attempt to define the geographic limits of the exposure area".

a. Methods Circles with radii equivalent to 0.25, 0.5, 0.75 and 1.0 miles were drawn on a map of HopeweU centered on LSPC. The area within 0.25 miles was primarily commercial, with a small predominantly black residential area in the south and a 7-story relatively new apartment dwelling in the north, predominantly with elderly white; every fifth occupied residence was chosen in these 2 areas. Of the 3 outer circles, a randomly selected single unit dwelling was selected from each residential block intersecting the circle. The survey was comprised of 93 residences and 216 occupants, all of whom were invited to complete a questionnaire dealing with age, sex, race, residential and occupational histories and self-reported neurologic symptoms. A single venous blood sample was taken from each participant; additionally, a sebum sample was taken by wiping the forehead with a gauze pad soaked in acetone. All samples were stored under refrigeration in a limited access system. Blood samples were analyzed by 2 laboratories independently. Following clean-up, each extract was analyzed by EC/GC on one of 2 different columns, with a sensitivity of 1 ppb. Confirmatory analyses were made on samples over 5 ppb on both columns, and additionally by E C / G C following chemical derivatization to Mirex.

14 TABLE 3 BLOOD LEVEI~ OF KEPONE

(Author's Table)

Area

% Blood samples over 5 ppb (Nos.)

Residential area south of LSPC Apartment to north of LSPC One-half mile away Three-quarters mile away One mile away

39 (34/87) 7.7 (2/26) 5.9 (2/24) 2.6 (1/39) 3.3 (1/30)

TABLE 4 SYMPTOMS I N COMMUNITY ADULTS A N D LSPC WORKERS

(Ba$~ on Author's Data)

Symptoms

% Incidence in community adults ( Nos.)

% Incidence in LSPC workers ( Nos.)

Nervousness Tremor Pleuritic pain Weight loss Visual disturbances

20 (31) 12 (18) 11 (17) 10 (15) 13 (20)

42 (48) 43 (49) 24 (27) 19 (21) 30 (34)

b. Results The age distribution o f the survey subjects was as follows: 59 and 150 were under and over the ages o f 18, respectively a. O f the adults, the mean ages o f 50 and 105 males and females were 47.8 and 48.3 years, respectively. None of the s u r v e y subjects ever reported as having worked at LSPC or Allied Semiworks. Within t h e innermost area o f 0.25 miles, Kepone levels were distributed in a bimodal fashion w i t h regard to age, peaking in pre-teenage children and older adults. O f the 216 samples, 40 or approximately 19 ~o were over 5 ppb, none exceeding 50 p p b ; the r e m a i n i n g 81 ~o were less than 5 ppb. Blood levels were approximately linear as a function o f proximity to LSPC 7 (Table 3). N o detectable levels o f Kepone were found in any s e b u m sample. The results o f the questionnaire on the symptomatology o f 156 adults, and a comparison with that o f 113 LSPC workers 6, are summarized in Table 4.

4. References

1 Environmental Protection Agency (EPA) Pesticide Petition, Allied Chemical, OEO 919, Section D. 2 EPA, ~ of Pesticide Programs, Draft Review, Kepone, August 15, 1975. 3 EPA, Health Effects Research Laboratory, Preliminary Report on Kepone Levels Found in Environmental Samplesfrom the HopeweU, VA Area, ~ b e r 16, 1975. 4 A.W. Reitze and G. L. Reitze, Environment, 18 (1976) 2. 5 W.S. Ferguson, Allied Chemical, letter to S. R. Wasserug, Enforcement Division EPA, September 12, 1975. 6 A . A . A . Gawaad, M. A. Hamad and F. H. EI-Gayar, Int. Pest. Control, 13 (1971) 8.

15 7 EPA,Health EffectsResearch Laboratory,Preliminary Report on Kepone Levels Found in Human Bloodfrom the GeneralPopulation of Hopewell, VA, March 3, 1976. $ S.B. Cannon, J. M. Veazey, R. D. Kimbrough, P. J. Landrigan and C. W. Heath, Center for Disease Control (CDC) Atlanta, memorandum,Kepone Poisoning--Virginia, to Director CDC, January 7, 1976.

lII. OCCUPATIONALEXPOSURE 1. Modes o f occupational exposure There are 3 principal modes of occupational exposure to Kepone, as for other pesticides, as follows: to the applicator; to the formulator; and to the manufacturer. The number of U.S.A. applicators of Kepone-containing products is unknown. The major U.S. applications have been for ant and roach baits, with products containing approximately 0.125 ~o Keponel; an additional use was for the control of the mole cricket in Florida grass, using a 50 ~o wettable powder. Kepone was primarily formulated at the Allied Formulating and Shipping plant in Baltimore, where it was diluted to mixtures of varying concentration, for export and, to a much lesser extent, for domestic use. In the U.S., the mixtures were then shipped to formulators for incorporation into various pesticide products. While there are 26 federally registered Kepone formulators, NIOSH 2 has, however, estimated that there are in fact as many as 36. Since 1968, there have been only 2 manufacturers of Kepone. Allied Chemical Co., at its Semiworks in Hopewell, VA, produced approximately 1.6 million pounds of Kepone from 1968 to March, 1974. Life Science Products Co. (LSPC), which was formed in November, 1973 and went out of production on July 24, 1975, produced approximately 1.7 million pounds, which it shipped to the Allied Baltimore plant from November 15, 1974 to July 28, 19753. 2. Exposure at the Allied formulating Plant in Baltimore All the Kepone produced in the U.S.A., at Allied or other plants from 1951 through 1975, was shipped to the Allied Plant, Race Street, Baltimore, MD. a. Clinical evaluation o f employees 4 Clinical evaluation of approximately 30 Allied employees at the Baltimore Formulating and Shipping Plant, was made during October and November, 1975, with particular reference to neurological signs and symptoms4; data on serum Kepone levels were also included in the report. It was claimed that no apparent evidence of disability or impairment o f function was noted 4. b. Analysis o f clinical data and serum Kepone levels 5 The object of this interim analysis was to determine whether there was any relationship between the clinical findings and serum Kepone levels in the Allied

16 TABLE 1 RESULTS OF LILIENFELD's ANALYSIS Neurologicalsigns and symptoms

No. employees

Median Kepone levels (ppb) Serum Blood•

Male

Present Questionable Negative Negative

12 2 13 Female 3

605 805 186

272 362 84

199

90

•" Estimated. TABLE 2 RESULTS OF LEVIN's ANALYSIS Neurological signs and symptoms

No. of individuals

Median Kepone levels (ppb) Serum Blood

Male employees

Objective signs Symptomsonly Questionable Negative Negative

3 654 7 519 4 584 13 186 Female employees 3 199

294 234 263 84 90

employees4; estimates of equivalent whole blood Kepone levels were made by multiplying serum values by 0.45. The clinical data were evaluated independently and "blindly", without initial knowledge of the Kepone levels, by Drs. Lilienfeld and Levin. The possibility of clinical bias, due to knowledge of Kepone levels 4, was stressed s, as was the fact that the "vast majority of the neurological findings were symptomatic without any objective signs". While no conclusions were drawn from this interim analysis, it is of interest to note in both the Levin and Lilienfeld analyses that median Kepone levels in workers with objective or questionable signs and symptoms were approximately 3-fold higher than in those with negative findings (Tables 1 and 2). c. Subsequent hospitalization o f an employee

An Allied employee who had packed Kepone from 1969-1974, besides other pesticides including DDT and lead arsenate, was hospitalized on 2/4/76 complaining o f nervousness, deep bone pain and insomnia 6. His only sign was a fine extension tremor and his blood Kepone was 1.18 ppm. 3. Exposure at the Allied Semiworks Plant, Hopewell', VA

Allied Chemical was the principal manufacturer of Kepone prior to March, 1974,

17 when it ceased production. Apart from minor production in its Claymont, DE and Marcus Hook, PA plants in 1951 and 1962-1963, respectively, all Allied production was located at a plant in HopeweU, VA, from 1966-1967, and at its Hopewell Semiworks from 1968 to 19743 . Information on conditions in the Allied plant has been provided by former employees 6. Kepone was manufactured intermittently for 2-3 months annually, and not on a continuous basis, as in LSPC; additionally, production volumes at the Allied plant were relatively low. Initially, the wet reaction mixture was spread by hand in pan driers, placed on racks in drying ovens, dumped onto a partially enclosed and ventilated screen to break it up, and then allowed to fall into an open drum through a sleeve. Later production processes were modified by installation of a rotary drier which allowed direct feed from the reactors and centrifuge which emptied directly into a sleeve feeding the drums; drums were fitted with covers to cut down dust 7. Workers were required to change clothing before going home and according to information from NIOSH, it was claimed that "better personal protective equipment (than at LSPC) was provided and u s e d . . . , 7 It should, however, be noted that respirators were apparently not provided until 1970, when Allied was required to do so by the State Department of Labor. In fact, other than the lower exposure due to the intermittent and relatively low production at Allied, there seems to be little basis to substantiate inferences as to alleged better work practices at Allied than at LSPC. According to claims made by Allied Chemical 8, " . . . no untoward effects have yet been noted in Allied Chemical Corporation employees who handled Kepone at their Hopewell, VA, or Baltimore, MD, plants". However, according to CDC, an illness attack rate of approximately 31% was found in 38 employees of the Allied, Hopewell plant 9. In this connection, it is of interest to note that higher blood Kepone levels were found in Baltimore plant employees with neurological signs or symptoms, than in those who were asymptomatic 5. 4. Exposure at the Life Science Products Co., Hopewell, VA a. The physical plant The plant comprised a total area of approximately 10,000 square feet and consisted of a renovated gasoline filling station, together with 2 outbuildings grouped around an open paved area, "the pad", where intermediate steps in Kepone production were located. b. Operations and conditions in the plant 7' 9 Most reactions were located in the "pad". Filtration and drying operations were conducted under shed-like enclosures; these operations then fed into the packing areas. The quench tanks were constructed of inadequate material and frequently leaked, allowing a precipitate of wet Kepone to flow almost constantly onto the "pad" area 9. Filtration was accomplished on a belt, which fed automatically into a pan drier. Further operations were described as followsT:

18 "This in turn fed a Holoflite dryer by means of a screw conveyor which emptied through a sleeve into a drum. The last three operations appeared to present the greatest exposure in that there were openings and only minimal local exhaust." After drying, the material was moved by conveyor to the baghouse a n d packaged by handg; an apparatus designed to decrease dust in the baghouse seldom functioned properly. A plant inspection by N I O S H on August 12, 1975 revealed the following conditions: "General impressions were that there was gross contamination in the filtration, drying and packing areas. Material was noted on walkways, floors and walls and overhead structures--especially in the packing area. Outside, in the reaction area, massive amounts of material were noted on the pad . . . . At some points there were almost two feet of re~ted material around the equipment". On the basis of discussions with follows:

workers 6, conditions were further described as

"Workers wore clothing home on many occasions. Respirators were not used on a regular basis until the plant was shut down. Food was also consumed on a regular basis in the dry or packing area due to the work load. When food was consumed in break areas, special precautions were not made to protect against contamination of this area. W~rk clothing was worn into lunch area and a coating of powder was found on lunchroom floor and tables. Although air pollution equipment in the form of a baghouse was installed, this too appears to have added to the occupational exposure in that workers went inside the baghouse on a regular basis to shake down filter bags". The company provided a few respirators, but they were of p o o r quality and were almost never usedg; no protective clothing or boots were provided, n o r was a n y separate clean area provided for eating or for storing clothes. It should a l s o be noted that production ran 24 h a day for 7 days a week 7. The only determination o f Kepone air levels in the plant, made on J u l y 22, 1975 when the plant was operating below full production, revealed levels o f 3 m g / m 3 in the dryer house, and 0.016 m g / m 3 besides the baghouse 9. It may be n o t e d that the K e p o n e standard recommended by N I O S H I°- on January 27, 1976 for o c c u p a t i o n a l exposure to Kepone was 1 #g/m 3.

c. Events leading to the closing of the plant On September 20, 1974 a former employee o f L S P C filed a complaint a t the regional O S H A offices in Richmond, VA, on the grounds that he had been discharged for refusing to work over a dryer with Kepone dust blowing in his face 11. T h e O S H A office ignored the original complaint, handled i t as a discrimination case, and failed to initiate a plant inspection; the case remained inactive until January, 1976, when it was re-opened. Based on information subsequently provided by employees 7, m a n a g e m e n t was aware o f the adverse health effects in the plant, particularly the " s h a k e s " . On being asked by employees to install engineering controls to improve working conditions, m a n a g e m e n t is alleged to have responded to the effect that " . . . there w a s nothing toxic about Kepone and that it was just nerves"~ One worker was subsequently told

19 that the problem was "not the Kepone, but HCP---(and) to keep clean and not say anything to the other workers". According to another account 12: "Workers went to their own doctors with complaintsof uncontrollableshaking and other effects. Local doctors attributed these troubles to 'nerves'. But a still undeterminednumber of employees quit due to the health problems they blamed on the working conditions at the plant; not all these workers have been located". Meanwhile the workers' compensation carrier, Travelers Insurance Co., made two "inspections", resulting in minor electrical repairs 11. The State Bureau of Industrial Hygiene attempted to arrange a voluntary inspection which LSPC repeatedly postponed. From March to July 1975, EPA, which has responsibility for pesticide manufacturing plants, was aware of problems at LSPC, although it was not registered with EPA, but failed to inspect the plant 12. In July, 1975 an Allied employee went to see his private doctor, Dr. Yi-Nan Chou, with a symptom-complex of weight loss, tremors, opsoclonus, and nervousness. Dr. Chou sent a blood sample to the Bureau of Laboratories of CDC which was found to contain 7.5 ppm Kepone. On July 21, 1975, CDC notified Dr. Jackson, Acting Director of Preventive Medical Services of Virginia, who inspected LSPC on July 23, 1975 and examined and took blood samples from 10 current employees, 3 of whom were then hospitalized with severe symptoms. LSPC voluntarily closed on July 24, 1975, apparently forestalling action which had been initiated by Dr. Jackson in the office of the State Attorney General. By executive order of the Virginia State Department of Health, the plant was closed on July 26, 1975. Subsequent events included prohibition by EPA of production, sale and use of Kepone in August, 1975; involvement of epidemiological and analytic services of CDC in August 19752; a NIOSH survey of LSPC on August 12, 1975; initiation of environmental and community Kepone sampling and analyses by EPA in August, 1975; initiation of plans by CDC, NIOSH, and the Virginia State Department of Health to undertake long-term surveillance of LSPC workers for effects including reproductive, neurological, behavioral and carcinogenic; and transmission by NIOSH to OSHA on January 23, 1976 of a recommended emergency standard for occupational exposure to Kepone of 1 /~g/ma of breathing zone air 9. It should be noted that the proposed emergency standard relates only to Kepone particulates and appears to ignore possible vapor phase exposure. The labelling requirements for the proposed occupational standard include the following statements: "Danger! Extreme Health Hazard; Cancer~uspect agent; and nerve destroyingagent."

d. Epidemiological investigation The CDC investigation determined that a total of 148 persons had worked at LSPC since it commenced operations, 32 of whom were still employed when the plant was closed 9.

20 TABLE 3 DISPOSITION OF LSPC EMPLOYEES (based on Author's Data) Category

Nos.

Nos. unavailable

Nos. available

No. cases Kepone poisoning ( ~o available)

Current Former

32 116

0 35

32 81

17 (53) 45(56)

Total

148

35

113

62(55)

TABLE 4 DI~;TRIBUTIONOFSIGNSANDSYMPTOI~IN 62 CASESOFKEPONEPOISONING(Author's Table 1) Signs and symptoms

Nos (%)

Nervousness Tremors Visual deficiencies Pleuritic pain Weight loss Joint pain

49 (79) 49 (79) 25 (40) 20 (32) 17 (27) 11 (18)

CDC interviewed, examined, took blood samples and completed a standardized questionnaire on a total of I 13/148 (76 ~o) LSPC employees 9; additionally some family members of the employees, workers in adjacent businesses and some previous employees of the Allied plant were studied. The CDC report also describes the community survey, undertaken by EPA 13, and contrasts their symptomatology with that of the plant workers. Of the 113 available LSPC workers, neurological signs were found in 36, 26 were symptomatic, and no signs or symptoms were found in 51; thus, Kepone poisoning was diagnosed in 62 cases (55~o), which were approximately equally distributed between: current and former employees (Table 3). None of the 4 females working in LSPC were affected. The predominant symptoms were nervousness and shakiness which increased with activity; irritability, a sense of apprehension, anorexia, weakness, pleuritic pain and arthralgia were also common (Table 4). Symptoms began to appear within 6 weeks of employment and persisted as long as 6 months after termination. Neurological signs included ataxia, opsoclonus, tremors, hyperactive stretch reflexes, weakness, and impairment of recent memory; additional signs included tacJaycardia and hepatomegaly8. A rash consistent with contact dermatitis was also reported by 76/113 workers 14. Testicular atrophy was not noted on clinical examination. Laboratory findings included abnormal liver function tests, with alkaline phosphatase levels increased 1.5-2-fold, and LDH and transaminase levels ranging from

21 the upper limits of normality to the "mildly elevated"; little REM sleep in sleep EEG's; myopathic E M G patterns; demyelination of peripheral nerves; and oligospermia with no motile forms. Delayed wound healing after biopsies was also noted. Illness attack rates were strongly related to job category, of which 6 were recognized: secretarial/office; janitorial; maintenance; pad men; lead operators and foremen; and dryer and filter operators. The attack rate for workers indirectly involved in Kepone production, secretarial and janitorial categories, was 2/15 (13 ~o). The attack rate for the 4 other categories directly involved in production was 58/98 (59 %). Higher, but not statistically significant, attack rates occurred among the 4 categories with the highest exposure to Kepone; a possible factor considered was the mobility of workers in some categories. Rates were similar for current and former workers. The mean latency between start of employment and onset of symptoms was 6 weeks and symptoms persisted as long as 6 months after termination of employment. "Cases" were defined as workers or others in defined groups with symptoms of nervousness and/or tremors. The mean age of non-cases was slightly older than for cases, possibly because older workers were employed in conditions of lesser exposure. The duration of employment was longer for cases than for non-cases; this was statistically significant for former workers. Kepone was found in the blood of all 32 current employees, ranging from 0.165-26 ppm; levels were generally higher in cases than non-cases (Table 5). Kepone levels were statistically higher in cases than noncases, with respective means of 8.48 and 1.57 ppm. Kepone levels in human sebum from 7 hospitalized and 7 non-hospitalized cases were 0.2-3.0 and 0.05-0.8/ag/wipe, respectively. Illness attack rates were investigated in 87 other non-LSPC employees who were examined and bled, and who were categorized as follows: 38 former employees of the Allied plant in Hopewell; 32 people who worked in businesses adjacent to LSPC; 10 workers at the sewage plant; 5 cab drivers who drove LSPC employees to the plant daily; and 2 truck drivers whose route took them past LSPC twice daily. A total TABLE 5 MEANBLOODKEPOh~LEVELSntOM 32 "CUgg~tzr"EMPLOYEES(Author's Table 3) Job category

Cases

Non-cases

No.

Blood level (ppm)

No.

Blood level (ppm)

0 0 3

--3.0

5 3 4

1.1 2.6 1.5

Pad men

1

11.6

0

--

I.cad operators/foremen Dryer & filter operators

7 6

13.25 5.29

2 1

0.7 3.0

Secretarial office Janitorial Maintenance

Mean (range) s p <

0.01.

8.48a (1.35-26)

1.57

(0.165-3.0)

22 attack rate of 31% (27/87) was found; rates in all categories were similar, except for the truck drivers, both o f whom were affected. The mean age of cases a n d non-cases was 42 years. Thirty-two family members o f LSPC workers were also examined. O f the case group, 10 wives or mothers who washed work clothes had symptoms o f rash or nervousness and 1 had a tremor; no children were symptomatic. No f a m i l y members o f non-cases had any symptoms. The possible role o f H C P in Kepone poisoning was considered but d i s c o u n t e d for four reasons 9: (i) HCP levels were found in only few workers and these were negligible. (ii) Measurably high Kepone levels were found in the blood of cases. (iii) Highest attack rates were found in job categories most heavily exposed to Kepone. (iv) Toxicological studies on animals with HCP produced a s y n d r o m e different to that seen in the plant workers. Additionally, Kepone induced a toxicological syndrome in animals similar to that seen in plant workers. Consideration was also given to determining the major route o f exposure to Kepone 9. It was concluded that inhalation, ingestion and skin a b s o r p t i o n were all involved.

e. Comments

It is apparent from the N I O S H inspection 7, and from other information, that the standard of work practices at LSPC was grossly inadequate, and f a i l e d to reflect available information on the toxicology o f Kepone, and even to meet the minimal safety precautions recommended by Allied Chemical Company, recognizing the major deficiences of such precautions and labeling requirements.

5. References

1 R.M. Moore, National Institute for Occupational Health and Safety (NIOSH), Memorandum and Attachments to N. P. Page, National Cancer Institute, January 8, 1976. 2 J. M. Finklea, Director NIOSH, Testimony Before the Subcommittee on Manpower, Compensation, and Health Safety, House Committee on Education and Labor, January 30, 1976. 3 Allied Chemical, Kepone Technical Receipts at Baltimore, MD, Plant, Appendix A to letter from W. S. Ferguson to S. R. Wasserug, Environmental Protection Agency (EPA), Philadelphia, PA, September 12, 1975. 4 E. Cotter, October-November, 1975, data cited in ref. 5 (see below). 5 A.M. Lilienfeld, Report on Analysis of Results of Physical Examinations of Race Street Plant Employees and Serum Kepone Levels, May 8, 1976. 6 H. L. McMartin, NIOSH, Memorandum for the Record, Allied Employee Hospitalized--Kepone, February 10, 1976. 7 W. Straub, NIOSH, Field Trip to Life Science Products Co., HopeweU, VA, Summary, Memorandum to W. A. Felsing, NIOSH, August 18, 1975. 8 J.W. Lloyd, NIOSH, Contact with Allied Chemical Corporation Representative Concerning Toxicity of Kepone, memorandum to W. A. Felsing, NIOSH, August 29, 1975.

23 9 S.B. Cannon, R. D. Kimbrough, J. M. Veazey, P. J. Landrigan and C. W. Heath, Center for Disease Control (CDC), Kepone Poisoning Virginia, memorandum to Director CDC, January 7, 1976. 10 Recommended Standard for Occupational Exposure to Kepone, NIOSH, U.S. Department of HEW, January 27, 1976. 11 AFL/CIO, Industrial Union Department Spotlight, The Work Environment: Life is Cheap in Hopewell, VA, 5, 1976, pp. 3--4. 12 A.W. Reitze and G. L. Reitz¢, Environment, 18 (1976) 2. 13 EPA, Health Effects Research Laboratory, Preliminary Report on Kepone Levels Found in Human Blood from the General Population of Hopeweil, I/A, March 3, 1976. 14 D. J. Sencer, Director CDC, Testimony Before the Subcommittee on Agricultural Research and General Legislation, Senate Committee on Agriculture and Forestry, January 22, 1976. 15 EPA, Preliminary Report on Kepone Levels Found in Environmental Samples from the Hopeweli, VA, Area, December 16, 1975.

IV. TOXICOLOGY--IN VITRO EFFECTSOF KEPONE

1. Freedland and McFarland 1, 1965 Commercial formulations of Kepone, and three other pesticides, were found to be inhibitory for beef liver glutamate dehydrogenase. Kepone inhibition was completely reversed by ADP. While the physiological significance of this inhibition is unknown, its mechanism may reflect the lipid solubility of Kepone and the localization of glutamate dehydrogenase o f most cells within lipid-rich mitochondria. 2. Desaiah and Koch 2, 1975 Kepone and a reduction product were shown to induce marked in vitro inhibition of ATPase activity in channel catfish brain. Both inhibitory effects were of similar magnitude and were dose-dependent. Inhibition of oligomycin-sensitive, mitochondrial, ATPase was greater than of oligomycin-insensitive enzyme. These results suggest that the initial toxic action of Kepone may be a reduct!o~r i n ATP synthesis by inhibition of mitochondrial oxidative phosphorylation. Similar effects have been demonstrated in vitro and in vivo for other organochlorine insecticides. 3. Hendrickson and Bowden a, 1975 Rabbit muscle lactate dehydrogenase is competitively inhibited in vitro by Mirex and by Kepone, its carbonyl analog, at levels as low as 0.01 mM (10 ppm). This inhibition occurred at test levels well below the toxic, and at levels comparable to accumulation concentrations of Mirex in fish and other non-target organisms.

4. References 1 R . A . Freedland and L. Z. McFarland, Life Sci., 4 (1965) 1735. 2 D. Desaiah and R. B. Koch, Bull. Environ. Contam. ToxicoL, 13 (1976) 153. 3 C.M. Hendrickson and J. A. Bowden, J. Agric. Food Chem., 23 (1975) 407.

24 V. TOXICOLOGY IN PHYTOPLANKTON, AQUATIC INVERTEBRATES AND FISH

1. Butler ~, 1963 In an investigation on the lethal effects of approximately 46 pesticides, Kepone was shown to be highly toxic. Concentrations of 1.0 ppm decreased the productivity, C 14 uptake, of natural phytoplankton by 95 Yo within 4 h. The toxicity of Kepone was higher than that of any of 12 organochlorine pesticides tested and the second highest of any of the 46 pesticides tested. Kepone was also comparatively toxic to oysters, causing an approximate 50 Yo reduction in shell growth during a 96-h exposure. However, Kepone was among the least toxic of the organochlorines to adult shrimp, juvenile blue crabs, juveline white mullet, and long nose killifish.

2. Mulla 2, 1963 Kepone applied at a rate of 0.1 and 0.5 lb/acre on shallow ponds caused 2 and 8 % mortality, respectively, in mosquito fish at 24 h, with no increase in mortality up to 14 days. Kepone at these rates of application was ineffective against mosquito larvae and bullfrogs. 3. Bridges a, 1965 The duration of exposure of red-ear sunfish to Kepone was shown to be a more important determination of toxicity than temperature; mortality was 34-fold greater for 96- than 6-h exposure, as compared with a 7-fold excess at 85 than 45 °F. The same time factor also appears to operate for sunfish 4, which are approximately 10-fold less sensitive than rainbow trout (Table 1). TABLE

1

TOXIC EFFECTSOF KEPONE ON SUNFISHAND TROUT

Fish

LCSO at specified hours (ppm)

Sunfish Trout

TABLE

24

48

96

0.62 0.066

0.27 0.038

0.14 0.02

2

TOXIC EFFECTS OF KEPONE ON TROUT

Species trout

Bluegill Rainbow

LCSO (mg/l)

No discernible effect

24h

96h

level (rag/l)

0.257 0.156

0.051 0.036

0.024 0.04

25 4. Bionomics Inc. 5, 1974

The susceptibility to Kepone of the bluegill and rainbow trout was determined under static bioassay conditions for 96 h. The results are summarized in Table 2.

5. Bionomics Inc. 6, 1975

The calculated 24- and 96-h LC50 values for fiddler crabs under conditions of static bioassay were > 1.6 and 1.47 mg/1, respectively; the no discernible effect level was 0.32 mg/1 (320 ppb). There is no description of toxic effects other than mortality.

6. E P A 4, 1975a a. Ecological hazards

Kepone appears hazardous in the marine environment only if present in concentrations greater than 0.09 ppm, which levels would be toxic to trout, or from prolonged periods of exposure up to 96 h, at concentrations greater than 0.031 ppm. Reduction in phytoplankton populations by short exposures to high concentrations, 4 h at 1.0 ppm, could destroy the lowest level of the food chain. It is thus concluded 2, that Kepone is "highly toxic to estuarine phytoplankton... (and) that aquatic environment may become contaminated and seriously disrupted by Chlordecone residues". b. Bioaccumulation

It was considered that Kepone would tend to bioaccumulate less than for other organochlorines, because of its lower partition coefficient 7, expressed by its relatively greater and lesser solubility in water and oil, respectively. However, levels of Kepone in sturgeon, oysters, dams, bass and bream in the James River constituted suggestive evidence of bioaccumulation 7.

7. References

1 P. A. Butler, Pesticide-Wildlife Studies: A Review of Fish and Wildlife Service Investigations During 1961 and 1962, Fish and WildlifeService, Circ. 169, 1963,pp. 11-25. 2 M. 8. Mulls, Mosquito News, 23 (1963) 299. 3 W. R. Bridges, Biological Problems in Water Pollution, Third Seminar, August 13-17, 1962, USDHBW, Div. Water Supp. Poll. Centr. Cincinnati,OH, 1965, pp. 247-249. 4 EPA,Officeof Pesticide Programs, Draft ReviewKepone, August 15, 1975. 5 BionomicsInc., Laboratory for Environmental Rcseaxch Wareham, MA, Acute Toxicity of Kepane to Bluegill and Rainbow Trout, unpublished,Allied ContractReport, February, 1974. 6 Bionomics,Inc., Marine Research Laboratory, Pensacola, FL, Acute Toxicity of Kepone to Fiddler Crabs, unpublished, Allied Contract Report, April, 1975. 7 EPA, Health F_HectsRescaxch Laboratory,Preliminary Report on Kepone Levels Found in Environmental Samples from the Hopewell, VA, Area, Dccemlmr16, 1975.

26 VI. AVIAN TOXICOLOGY

1. DeWitt and George 1, 1961 The approximate chronic oral LD50 for quail and pheasants was 500 and 1000 mg/kg, respectively. The maximum dietary concentration o f Kepone allowing normal survival o f pheasants was < 200 ppm. It was concluded that the c h r o n i c toxicity o f Kepone for quail and pheasants was similar to D D T . 2. Sherman and Ross 2, 1961 This report describes the acute and sub-acute oral toxicity o f - ~ e p o n e , and 21 other insecticides, in chickens as part of a program for screening candidate compounds for potential larvicidal action in poultry manure. Male and female New Hampshire chickens o f 7-14 days age were used in these studies. Technical grade Kepone was supplied by Allied Chemical Co. a. Acute toxicity Acute toxicity tests, in general, were based on single oral administration o f single doses of the insecticide at 4-5 dosage levels to groups o f 5--30 chicks, which were then observed for one week. Kepone was administered in gelatin capsules a t unspecified dose levels to unspecified numbers of chicks. Data are reported for females only, the LD50 of which was 480 mg/kg. The authors described the toxic effects o f K e p o n e as follows: "Kepone-affected chicks exhibited a typical syndrome characterized by violent shaking of the entire body. The onset of the syndrome was rapid, the intensity varying with dosage level. At the highest levels the chicks were unable to walk; they just sat and trembled. At the lower dosage levels the animals walked with an ataxic gait, yet appeared to feed and drink at the normal rate. The intensity of the syndrome gradually diminished with time. The survivors among the chicks given 400 mg/kg still exhibited some symptoms 5 days after treatment and were still slightly irritable, although the syndrome was absent on the sixth day. Despite the severity of the syndrome caused by this material, the growth of the surviving animals was normal."

b. Sub-acute toxicity Sub-acute toxicity tests, in general, are based on continuous feeding o f the insecticides for 1-2 weeks to 7- or 14-day old chicks, in groups o f 10 females for most o f the insecticides and to groups of 4 males for others. Kepone was fed over 2 weeks at 4 dose levels, 22.0, 110.0, 220.0, a n d 440.0 p p m to groups o f 10 or I1 females, with 9 untreated controls, following w h i c h animals were observed for a further 2 weeks. Mortality at the 440 and 220 ppm levels was 100 and 10Yo, respectively; the sub-acute LD50 is thus < 440 mg/kg. M e a n weight gain was significantly reduced (p = 0.05) at the highest feeding level o v e r the 2-week period; feed consumption was also increased, expressing reduced efficiency o f f o o d utilization at this level. The development o f the Kepone syndrome was described as follows: "The birds fed the highest level of Kepone (440 ppm) showed initial symptoms o f the syndrome after 5 days on this feed. Although the affected birds trembled constantly, their normal activities

27 appeared unrestricted. At the end of 7 days, the affected birds could no longer stand, but sat trembling with their heads down . . . . The birds given Kepone at a concentration of 220 ppm exhibited only slight symptoms until the sixth day, but the tremors became progressively intense until by the fourteenth day all the chicks exhibited a well-developed syndrome. The birds given Kepone at 110 and 22 ppm did not exhibit the Kepone syndrome until the 10th day. The intensity of the syndrome was not as great as that caused by the more concentrated Kepone-feeds, yet the birds were highly excitable. Ten days after being placed on the untreated feed, the chicks which had previously received Kepone at concentrations of 22 and 110 ppm appeared much calmer, but those which had previously received 220 ppm were still highly nervous. By the sixth day, however, all surviving chicks appeared to have completely recovered." Based on the above observations, it was concluded that the toxic effects of Kepone were cumulative at the concentrations tested; these acute effects appeared to be gradually reversible. It was further concluded that the chick is less susceptible to Kepone toxicity than the rat a.

3. DeWitt et aL 4, 1962 Dietary LC50 values for young and adult bobwhite quail are 600 and 530 ppm, respectively; for young and adult ring-necked pheasants, 606 and 115 ppm, respectively; and for young mallards, 400 ppm. In field tests, Kepone was mixed in a peanut butter bait that was applied to fire ant nests and trails over 480 acres at about 3.5 g/acre. Dead bobwhite quail and birds with severe body tremors were soon noted; similar effects were also induced in meadow larks. Feeding Kepone at 50 p p m inhibited reproduction in quail. Feeding of pheasants at levels as low as 1 p p m during the winter and spring reproductive periods markedly reduced reproduction; at other times 25 p p m almost completely inhibited reproduction. Pheasants fed 5 p p m produced 5070 fewer viable chicks than controls. Males fed concentrations of 50 p p m developed female plumage, and histologically abnormal testes and malformed sperm; these were ascribed to an "estrogen-like effect" of Kepone.

4. DeWitt et al. 5, 1963 This study confirmed previous results on the toxicity and inhibitory effects of Kepone on the reproduction of quail and pheasants 4, and extended these findings to ducks. The effects of K e p o n e on plumage and reproduction appear to be reversible within 60-90 days following return to control feeding.

5. Naber and Ware 6, 1965 This report describes the reproductive effects of Kepone, and Mirex, on the laying hen. Kepone was fed at dietary levels of 75 and 150 p p m to groups o f 15 hens for 16 weeks, following which the birds were returned to control diets; control groups consisted of 12 hens. Eggs were saved for hatchability tests at 0, 6, and 12 weeks; hens were inseminated with pooled semen from males on control diets. Kepone levels in eggs were determined b y GLC, following hexane--dimethylformamide extraction.

28 While Kepone had no effect on fertility, both dietary levels, however, reduced egg production significantly during the initial 12 weeks of the test (p < 0.05). While feed intake was not affected, marked weight loss occurred at the 150ppm level. Hatchability or embryonic survival was markedly reduced (p < 0.05) at the 150 ppm level. Additionally, it was noted that "Most of the chicks hatching in this group exhibited a nervous syndrome characterized by quivering of the legs, wings and head. These chicks were unable to walk and many were unable to stand upright". A similar "nervous syndrome" was also seen in a hen in the 150 ppm after her egg production ceased during the tests. None of the chicks from the groups of hens fed 150 ppm survived until 14 days; survival was also markedly reduced in the chicks from the 75 ppm hen groups, fed 6 for 12 weeks, although body weights were not reduced. Yolk residues reached their maxima, 163 and 336 ppm in eggs from the 75 and 150 ppm groups of hens, respectively, by a second hatch in the fifth week and did not increase further up to 16 weeks of feeding and over a third hatch; residues of approximately 23 and 78 ppm persisted in the 75 and 150 ppm groups 3 weeks after return to control diets. It was concluded that the inhibitory effects o f Kepone on reproduction in hens involves all phases including egg production, hatchability, and survival of the hatched chicks. 6. McFarland and Lacy 7, 1969 This study was designed primarily to investigate the reproductive effects of Kepone in Japanese quail. a. Effects on immature males A group of 10-week old males was fed 300 ppm Kepone for 3 weeks and then killed. Liver weights were significantly increased (p < 0.01) and testicular weights were significantly reduced (p < 0.01) from control values. Testes were described as atrophic. Additionally, reduction in hematocrit values and increased refractive index of plasma (p < 0.01) were noted. b. Effects on mature males In the first experiment, a group of I0 adult males, 9-12 weeks old was fed 400 ppm o f Kepone. The median time to death was 10 days, with a range f r o m 7-15 days. Death was characterized by extensive tremors progressing through ataxia to complete paresis. No weight loss occurred. Findings of note included enlarged livers with plurivacuolar lipid inclusions in liver cells, decreased width of the cloacal gland (p < 0.01), suggestive of LH and testosterone deficiency, and a variable effect on the testes. While mean testicular weights were within control limits, testes of several Kepone-treated animals were enlarged, with dilated and thinned seminiferous tubules. These changes were described as follows:

"The testes of adult quail fed Kepone were affectedin a bizarre manner---some were atrophic and contained degeneratingseminiferoustubules; others were enlarged as much as twice that of

29 controls; the tubular diameters were increased, and the epithelium was partly degenerate. Such a change could possibly be associated with estrogenic activity affecting Sertoli cell function and absorption of seminal liquors." In the second experiment, birds were killed, together with controls, when tremors commenced, approximately from 6-7 days after feeding commenced. Decreased cloacal gland width was again noted (p < 0.01), as was liver enlargement (p < 0.01), and dilated and thinned seminiferous tubules. Reduction of hematocrit and increased refractive index of plasma (p < 0.01) were also noted. c. Effects on immature females

A group of 10-week old females was fed 300 ppm Kepone for 2 weeks and then killed. Body and liver weights were increased (t7 < 0.01), and oviduct weights were greatly increased (p < 0.001). Generalized hypertrophy and hyperplasia, together with a prominent secretory epithelium, was noted in the oviduct. d. Effects on mature females

A group of ten 3-week old females, just beginning to lay eggs, was fed 200 ppm Kepone for 30 days and then killed. Liver weights were significantly enlarged (p < 0.01), as were ovarian weights (p < 0.05), due to increased numbers of developing and of large yellow follicles (p < 0.05). There were no changes in oviduct weight or in egg production. e. Effects endocrine ablation Two-week old females were ovariectomized, fed 300 ppm Kepone for 10 days and then killed. While body weights were within control values, liver weights were increased (p < 0.01) and oviduct weights were greatly increased (p < 0.1301). Three-week old females were hypophysectomized and on reaching 5 weeks of age were fed 300 ppm of Kepone for 10 days and then killed. While body, liver and ovary weights were within control limits, marked oviducal hypotrophy and hyperplasia persisted (p < 0.01). While the oviducts of Kepone-fed ovariectomized quail were more than 100-fold larger than controls, those of hypophysectomized birds were only about 25-fold greater than controls. f. Discussion

Whole body tremors induced in Japanese quail were similar to those previously noted in chickens 2' ~. Male quail fed 400 ppm Kepone had a median time to death of 10 days, whereas female chicks fed 440 ppm died between 6 and 15 days 2. Thus, the susceptibility of quail and chickens appears similar, and is much less than that of the rat a. Estrogenic activity appears to be induced by Kepone in the immature female, but not in the laying quail. Similar effects on immature chicks had been previously noted 2. Ovariectomy did not reverse the oviducal stimulation by Kepone; hypophysectomy reduced it somewhat, indicating that while the major effect of Kepone was mediated through the pituitary-hypothalamic axis, presumably due to release of FSH, it also

30 appears to act directly on the oviduct. The increased number of developing follicules in ovaries of mature quail fed Kepone is also consistent with FSH stimulation. This is consistent with findings in mice that Kepone prolongs FSH release and ovarian estrogen secretion, while blocking LH release s. Estrogen-like effects induced by Kepone in males are suggested by the induction of abnormal seminiferous tubules, some of which were degenerating, and atrophic and others dilated and enlarged, stimulation of reproductive organs of immature and mature females, depression of cloacal gland activity 9, and shift of the hematocrit and refractive index of plasma towards normal female levels lO. Enlarged fatty livers were rapidly induced in immature and mature females and males; this effect was reversed by hypophysectomy but not ovariectomy. Lipid inclusion bodies, but no other pathology, was noted. Increased body weights were induced by Kepone in immature females only. 7. McFarland et al. 11, 1971 Japanese quail were fed Kepone for 21 days. Levels as low as 5 ppm induced teratogenic egg shell deposition. This was characterized by major defects in the cuticle and vertical crystal layers, increased thickness of the spongy layer, and blockage of pores leading to suffocation. 8. Atwal 12, 1973 This paper reports on an electron microscopic study of the liver of Japanese quail following administration of Kepone. Female quail were injected intramuscularly with 0.5 mg Kepone in corn oil daily for 10 days and then sacrificed. Disappearance of glycogen granules was rapid. Rough endoplasmic reticulum was moderately dilated, particularly around mitochondria which were enlarged and swollen. Large cytoplasmic lipid vacuoles were seen, grouped circumferentially by mitochondria. Autophagic vacuoles developed, particularly in the pericanalicular region, and were extruded into canaliculi and vascular channels. Such morphological evidence of excretion of Kepone appears consistent with the suggestions of rapid clearance of Kepone residues from the mouse liver following cessation of treatment 8. 9. Eroschenko and Wilson t 3, 1974 This study was undertaken to investigate the "estrogen-like effects produced by feeding the insecticide Kepone" on various organs of Japanese quail, and to compare these with the effects of gonadotrophins produced by photostimulation. a. Effects o f Kepone in immature quail Kepone, supplied by Allied Chemical Co., was administered at 200 ppm in diets for 3 weeks to groups of approximately 7 male and female 3-week old quail, o n a photoperiod of 16 h light and 8 h darkness (16L:8D), which were sacrificed at subsequent intervals of 0, l, 2 and 3 weeks; additional groups of 17-21 birds were allowed to survive from 6--10 weeks after Kepone treatment in the reduced photoperiod.

31 Significant increases in weights of oviducts, adrenals and liver occurred within I week, and of ovaries and testes by 3 weeks. Further reduction in the photoperiod to 6L: 18D, after the 6th week, resulted in severe tremors, ataxia, marked cachexia and high mortality, particularly in males, with a mean time to death of approximately 3 days.

b. Effect of Kepone on immature quail with reduced photoperiod Kepone was administered at 200 ppm for 3 weeks to groups of 7 male and female 6-weeks old quail, whose sexual development had been inhibited at three weeks by reduction of the photoperiod to 6L: 18D, which were sacrificed at 10 weeks. Significant increases in oviduct and liver weights occurred within 1 week of feeding and of adrenals by the 2nd week. The effect on oviduct weights indicates a direct action of Kepone, in addition to a gonadotrophin-mediated effect, and is consistent with similar findings in hypophysectomized quail 2. There was no increase in ovarian and testicular weights, presumably as such Kepone effects are gonadotrophin-mediated. No tremors or ataxia were noted in any group. c. Effect of Kepone on mature quail Kepone was administered at 200 ppm for 3 weeks to 24-week old quail, following which the photoperiod was reduced to 6L: 18D. Groups of 7-12 males and egg-laying females were then sacrificed at 4 subsequent weekly intervals. The 3 weeks of Kepone feeding induced significant increases in weights of testes, but not of oviducts and ovaries; weights of livers were increased in males but not in females. Subsequent reduction in the photoperiod markedly reduced the weights of control oviducts, ovaries and livers in females; in groups previously treated with Kepone, ovarian weights were unchanged, but oviduct and liver weights were increased. Reduction in the photoperiod markedly reduced testicular weights; in groups previously treated with Kepone, liver weights remain increased, but testicular regression, as evidenced by weight loss, was noted. No tremors or mortality were noted during the reduced photoperiod in previously treated birds. d. Discussion Reproductive activity in the quail depends on photostimulation, and a light regime of 16L:8D is stimulatory. Exogenous estrogens increase oviduct weights and size of immature quail and chicks; Kepone is also highly stimulatory, particularly in immature quail on a reduced photoperiod, while having no "estrogenlike" effect in egglaying birds. Previous studies suggest that Kepone stimulates FSH release, thereby stimulating ovarian estrogen release, and also acts directly on the immature oviduct in the absence of the pituitary 7. Sim~ar,vidence of induction of prolonged FSH and estrogen stimulation, with blocking of LH release preventing ovulation and corpora lutea formation, has been reported in mice a. Administration of Kepone to maturing and adult males under a 16L :8D light cycle produced heavier testes. Reference is made to a manuscript submitted for publication, in which histologic examination demonstrated that the testes were

32 edematous, with greatly distended seminiferous tubules. However, following light reduction, Kepone reduced testicular weights to below control values. The high mortality in Kepone-fed immature quail subject to subsequent reduced photostimulation appears to be associated with rapid fat mobilization and presumably Kepone release. Similar light reduction had no effect on mortality in Kepone-fed adults. Additionally, Kepone feeding during reduced photostimulation of immature quail induced no mortality. Immature birds have less body fat than adults and decreased light thus would cause higher mobilization of Kepone than in adults. The higher fat content of adults, and better detoxification systems, in addition to excretion of Kepone via egg laying, probably accounted for the improved viability of adults. 10. Eroschenko and Wilson 14, 1975 The purpose of this study was to investigate the histological effects of Kepone in quail of both sexes maintained under short and long photoperiods.

a. Experimental groups In the I st experiment, 3-week old birds under "normal" photostimulation (16L: 8D) were given control or 200 ppm Kepone diets until 6 weeks. At 4 weeks and at subsequent weekly intervals until 6 weeks, 6-8 test and control birds were sacrificed for histology. In the 2nd experiment, 6 week old birds previously maintained at reduced photostimulation (6L: 18D) for three weeks were fed control and 200 ppm Kepone diets until the 9th week; 6-8 test and control animals were sacrificed weekly from 6-9 weeks. In the third experiment, 24-week old birds under normal photostimulation (16L: 8D) were fed test and control diets for 3 weeks, when 6-8 birds from each group were sacrificed. b. Histological effects Kepone The majority of liver cells from all Kepone fed birds had large plurivacuolar lipid cytoplasmic inclusion bodies, creating a "Swiss-cheese like" appearance. Adrenals from treated birds exhibited cortical and medullary hypertrophy. One week treatment of 3-week old birds under 16L:SD produced marked differentiation, secretory and ciliary patterns with branched tubular glands; control birds had no evidence of secretory activity or ciliogenesis until 5 weeks. While Kepone had no effect on the oviduct of 6- and 27-week old laying quail kept under 16L:8D, however it induced marked stimulatory effects in 3-9 week old quail under reduced photostimulation (6L:18D). No difference was noted between ovaries of test and control birds kept under reduced light as well as from adult laying birds; however at 6 weeks, Kepone treated birds had multiple primary oocytes with a poorly developed follicular hierarchy. Testes of Kepone-fed birds under 6L: 18D were similar to controls. Testes from 6 and 27 week old birds under 16L:8D revealed striking abnormalities.

33 " . . . greatly distended seminifexous tubules and interstitial spaces occupied by fluid of undetermined origin. Seminiferous tubules of these edematous testes contained flattened, reduced and generally disrupted germinal epithelium. Many of the lumina in these tubules contained desquammated cellular debris consisting of germ cells in various stages of maturation. Curled, abnormal sperm as well as maturing spermatids appeared distorted within the germinal epithelium and in the desquarnmated debris of the lumina. The sperm bundles were now less compact, due possibly to the decreased number of sperm and to a reduced number of maturing spermatids." c. Discussion Differentiation of the immature avian oviduct is rapid and depends on photostimulation and hormonal stimulation. Administration of Kepone induced general hypertrophy, hyperplasia and secretory activity in oviducts of immature quail, even when kept under reduced light. Ovaries f r o m maturing Kepone-fed birds under 16L:8D light had heavier ovaries than controls ~3; these were shown to have a greater number of primary oocytes and developing follicles, but with a Iess evident follicular hierarchy. This is consistent with the findings of fewer eggs in the uterus o f Keponefed quail 7, and with the constant estrus and absence of corpora lutea in Keponetreated mice 4. The heavy lipid accumulation in the livers o f Kepone-treated quail, in the absence o f other pathology, is consistent with previous short-term findings in quailT; longer term exposures in mice, however, revealed focal necrosis and hyperplasia s. The testes of maturing and mature quail under 16L:8D light were severely affected. Similar effects have also been recorded in Kepone-fed pheasants 5 and are consistent with the changes induced by estrogens in avians 15; contrastingly, Kepone failed to induce testicular abnormalities in mice s. 11. Hazleton Laboratories, lnc. ~6, 1975 Groups of 10 mallard ducks o f both sexes were tested with a range of dietary concentrations o f Kepone f r o m 100 to 2150 p p m over a 5-day feeding period, with a subsequent 3-day observation period. The LC50 was calculated to be 497 ppm. Toxic symptoms included dose-related reduction in body weight and food consumption, hyperactivity, loss of coordination and righting reflex, salivation and convulsions.

12. References 1 J.B. De Witt and J. L. George, 1960 U.S. Dept. Interior, Bur. Sport Fisheries and Wildlife, Circ. 84, Pesticide--Wildlife, Review, 1959. 2 M. Sherman and E. Ross, Toxicol. AppL PharmacoL, 3 (1961) 521. 3 Allied Chemical Co., Kepone (insecticide-fungicide) decachiorotetracyclodecanone, Develop. Information Bull. 6-58, New York, 1958, p. 2. 4 J.B. DeWitt, D. G. Crabtree, R. B. Finley and J. L. George, Effects on Wildlife in U.S. Dept. of Interior, Bur. Sport Fisheries and Wildlife. Circ. 143, Effects of Pesticides on Fish and Wildlife in 1960, 1962. 5 J.B. DeWitt, W. H. Stickel and P. F. Springer, Wildlife Studies, Patuxent Wildlife Research Center, 1961-1962, in U.S. Dept. Interior, Fish and Wildlife Service, Circ. 167, Pesticide-Wildlife Studies During 1961 and 1962, A review of Fish and Wildlife Service Investigations, 1963. 6 E.C. Nabcr and G. W. Ware, Poultry ScL, 44 (1965) 875. 7 L.Z. MeFarland and P. B. Lacy, Toxicol. Appl. Pharmacol., 15 (1969) 441.

34 8 9 10 11 12 13 14 15 16

J.J. Huber, Toxicol. Appl. Pharmacol., 7 (1965) 516. L.Z. McFarland, R. L. Warner, W. O. Wilson and F. B. Mather, Experientia, 24 (1968) 941. O. Atwal and L. Z. McFarland, Am. I. Vet. Res., 27 (1966) 1059. L.Z. McFarland, R. L. Garrett and J. A. Nowell, Normal Eggshells and Thin Eggshells Caused by Organo-chlorine Insecticides Viewed by the Scanning Electron Microscope, Proc. Fourth Ann. Sem. Symposium, Chicago, 1971. O.S. Atwal, J. Comp. PathoL, 83 (1973) 115. V.P. Eroschenko and W. O. Wilson, Toxicol. Appl. PharmacoL, 29 (1976) 329. V.P. Eroschenko and W. O. Wilson, Toxicol. Appl. Pharmacol., 31 (1975) 491. B. Lofts and R. K. Mutton, in D. S. Farrier and J. R. King (Eds.), Avian Biology, Vol. HI, Academic Press, New York, 1973, pp. 1-107. Hazelton Laboratories Inc., Eight-Day Dietary LCSO-Mallard Ducks: Technical Kepone, unpublished Allied Contract Repor$, March 27, 1975.

V. MAMMALIANTOXICOLOGY 1. Allied Chemical Co. 1, 1949 This report, in the f o r m o f a brief m e m o r a n d u m , presents the results o f preliminary toxicity tests with C o m p o u n d 1189 in rats, which are summarized in T a b l e 1. The toxic effects induced are described as follows: "The test animals developed tremors similar to those produced by DDT. On the basis of these results, it would appear that the MLD to rats (4~aLadministration) is approximately 250 mg/kg." a. Comments N o indication is given as to the purity o f the c o m p o u n d tested. It m a y be noted t h a t this report contains the first available reference to the experimental induction o f tremors by Kepone. It further indicates an oral LD50 between 100 a n d 250 mg]kg, with an observation period o f 10 days. 2. United States Testing Company, Inc. 2, 1949 This unpublished report by United States Testing Company, Inc. was p e r f o r m e d u n d e r contract to Allied Chemical Co.

TABLE 1 ACUTETOXXCrrYIN ~TS (Author's Table) Dosage (mg/kg)

No. rats killed

Survival times

500 250 100 75 50

2/2 2/2 0/2 ] 0/2 ~ 0/2 /

12 h; 36 h 28 h; 5 days All animals still alive at the end of ten days.

35

a. Acute oral toxicity Compound 1189 in olive oil was administered orally at a single dosage of 50 mg/kg to groups of 10 mice, rats, and rabbits and observations were made after 24, 48 and 72 h; procedures were based on 1948 USDA specifications. Five mice died within 24 h and the remaining 5 from 24-48 h. Two rats died within 24 h, 2 more rats died from 24 a.8 h and the remaining 6 "seemed to be well after 72 h". All rabbits were alive and well after 72 h. The report concludes that Compound 1189 " . . . is toxic when fed orally to laboratory mice and rats, but not to laboratory rabbits". A supplementary report of March 8, 1949, however, stated, in apparent contradiction to this report, that the fifth rat died within 72 h. b. Acute percutaneous toxicity Compound 1189 in olive oil at a concentration of 200 mg/kg was tested for percutaneous toxicity in rabbits; procedures were based on 1948 USDA specifications. One inch gauze squares were dipped in an oil solution of Compound 1189 and placed on the shaved skin of I0 rabbits for 24 h; the squares were secured with adhesive tape. The patches were removed after 24 h and additional observations were made after 48 and 72 h, when all animals were reported as "alive and well". A supplementary report of March 8, 1949, however, in apparent contradiction to this report, stated: "The degree of irritation caused by Compound 1189 after 24 h in contact with bare skin ranged from very severe irritations to definite irritations. Observations made after 72 h showed that the irritations are healing and the rabbits are well." A further supplementary report dated May 12, 1949, however, stated that by 5 weeks, 2 of the rabbits had lost considerable weight~ 1 developed severe facial erythema, and 1 killed her 3-week old progeny, noting that "this is a very rare occurrence". The report further states: " F o r these reasons the four test rabbits have been killed." A hand-written note on the report by EEG also states: "These four rabbits would certainly have died."

c. Comments This appears to be the first toxicological test on Kepone. It established an acute oral LD50 for both mice and rats of < 50 mg/kg. The report also demonstrates that oily solutions of 200 mg/kg were highly toxic to rabbit skin, and also induced delayed toxicity and mortality over an observation period of 5 weeks. Delayed toxicity was also seen in rats following single oral dosage.

3. United States Testing Company, lnc. a, 1949 This unpublished report, a follow-up to the previous report 2, describes the testing of a 25 ~o formulation of Compound 1189 in olive oil. Acute oral toxicity was tested in 5 mice at a total formulation dosage of 50 mg/kg, equivalent to a dosage of 12.5 mg/kg of Compound 1189. No toxicity was observed by 48 h. Acute percutaneous toxicity was also tested in rabbits at a total formulation

36 dosage of 200 mg/kg, equivalent to a concentration of 50 mg/kg. No toxicity was observed after 24 h, and it was noted that " . . . none of the animals showed any evidence of skin irritation." a. Comments The duration of these tests on mice and rabbits was briefer than in the earlier report z, and the concentration of Compound 1189 tested orally was only 12.5 mg]kg. N o skin irritation was reported in rabbits at the lowest concentration tested, 50 mg]kg. 4. United States Testing Company Inc. 4, 1950 Acute oral toxicity tests were made in groups of 10 mice, rats and rabbits with 50 mg/kg olive oil of a 50~o formulation, equivalent to a 25 mg/kg dosage. All animals were alive and well 72 h later. Thus, the acute oral LDS0 of Kepone for mice and rats is between 25 and 50 mg/kg. However, a supplementary report, dated June 5, 1950, stated that a total of 8 mice had died by 5 days, at which time all rats and rabbits were still alive. Thus, the acute oral LDS0 for mice is < 25 mg/kg. Acute percutaneous tests in rabbits of a 200 mg/kg formulation, equivalent to a concentration of 100 mg/kg, producedno skin irritation or mortality within 72 h. a. Comments A memo of August 14, 1957 from M. M. Darley, Allied Chemical, claimed that Compound 1189 used in test 4299 was "pure material"; the 50 ~o compound used in test 20337 was stated to contain 50.5 % technical compound in an air milled wettable powder, and with regard to test 6254, "it can be safely assumed that it contained 25 ~o technical Compound 1189". However, a subsequent memo of August 16, 1957 from W. A. Knapp, Allied Chemical, referring to Darley's memo, stressed the need to develop a "reasonably accurate assay (of the purity) of technical product before we can proceed with toxicity tests". Reference is also made to the possibility that H C P and its well-defined low polymers could be "major impurities" in Compound 1189, and that I.R. should be used as a first analytical approach. The memo also discusses the need to- develop analytical procedures for residues "sensitive to a fraction of a ppm, probably to 0.1 ppm", in view of the high toxicity of Compound 1189, whose LDS0 is < 50 mg/kg. These data established that Kepone was highly toxic within the criteria of the Code of Federal Regulations, necessitating appropriate warning labelling. However, discrepancies between the results of the preceding and subsequent toxicity tests are likely to reflect differences in the purity of the various formulations tested, quite apart from the possible presence of interfering "inert ingredients". Results of the 3 U.S. testing company studies are compared in Table 2. The above tests, based on a batch of Compound 1189 of unspecified purity, indicate an acute oral LD50 for mice of between 12.5 and 25 mg/kg, and for rats and rabbits between 25 and 50 mg/kg. The rabbit skin irritation dose for oily solutions is between 100 and 200 mg/kg.

37 TABLE 2 COMPARISON OF RESULTS OF THE U.S, TESTING CO. ORAL AND PERCUTANEOUS TOXICITY TESTS (based

on

U.S. Testing Co. Inc. data)

Test No.

4299~ 203374 62548

% For- Formulation Keponedosage Acute oral LDSO mulation dosage (mg/kg) (rag/kg)

100 50 25

Rabbit skin irritation

Oral Skin

Oral

Skin

Mice

Rats

Rabbits

50 50 50

50 25 12.5

200 100 50

< 50 < 25 > 12.5

50 > 25 > 12.5

> 50 > 25 > 12.5

200 200 200

+ -

5. Allied Chemical Co. mammalian toxicological studies 5' 6, 1961-1962 a. Organization o f data These studies were u n d e r t a k e n by Dr. P. S. Larson, o f the Medical College o f Virginia, u n d e r contract t o Allied Chemical. These studies c o m m e n c e d on January, 1958 and were completed b y O c t o b e r 24, 1961; the results are organized in 3 volumes, as F D A petitions for a t e m p o r a r y tolerance on potatoes, labeled "Section C. Toxicological Studies", as follows:

Date submission

Contents

1. March, 1960

Acute and sub-acute toxicity; 1 year rat studies; preliminary dog study (this petition is subsequently referred to here as P. 1) Chronic rat studies (P. 2) Chronic dog study (P. 3)

2. July, 1961 3. February. 1962

Additionally, these d a t a are summarized in an undated MS, prepared but n o t subsequently submitted f o r publication 6. The MS is entitled "Acute, Subacute and Chronic Toxicological Studies on . . . (Kepone, Cpd. 1189)". The authors and their affiliations are listed as follows: P. S. Larson, Gordon R., Hennigar, Jr., E. M. Crawford, R. Blaekwell Smith, Jr., Susan J. Melette, W. A. Knapp, J. K. Finnegan and H. B. Haag. From the Departments of Pharmacology and Medicine, Medical College of Virginia, Richmond, 19, the Department of Pathology, College of Medicine, State University of New York, Brooklyn 3, and the General Chemical Division, Allied Chemical Corporation, Morristown, N.J.

b. Material tested " C o m p o u n d 1189" is u s e d s y n o n y m o u s l y with K e p o n e t h r o u g h o u t the studies. I n the case o f the chronic r a t study on 1 ppm, the term " C o m p o u n d 1763" is used, apparently s y n o n y m o u s l y w i t h K e p o n e 6.

38 Two formulations were used in various tests, as follows: (i) Powdered technical grade Kepone in corn oil of the following percentage composition: Water 3.3- 3.8 Methanol 2.4- 2.5 Hexachloropentadiene 0.1 Compound 1189 93.7-94.1 An average Kepone assay of 94.0 ~o was used for diet calculations. (ii) An aqueous solution of the following percentage composition: Compound 1189 22.3 Triton X 151 5.0 Triton X 171 5.0 Isophorone 20.0 Hi Sol #2309 47.7

c. Acute toxicity (Table 3) Severe tremors developed in all species tested; these generally reached a maximum within 2-3 days, gradually subsided over a week or more, and were exacerbated by excitement. Tremors in rabbits were more persistent following percutaneous than oral exposure, apparently due to a "repository" in the skin which is gradually absorbed into the body. No evidence of skin irritation occurred with oil as the solvent in percutaneous tests; aqueous solutions, however, induced marked irritation, edema and scab formation. Deaths generally occurred within 1-2 weeks following dosage; however, deaths were delayed up to I month in rabbits following percutaneous testing o f aqueous solutions. Gross autopsy findings were otherwise negative in all tests. The results of these tests, although indicating acute toxicity levels somewhat lower than those reported in 1949 (Table 2), nevertheless, establish that K e p o n e is highly toxic by standard definitions (see e.g. Gleason et al., Cli~¢al Toxicology o f Commercial Products, 1968; Sax, Dangerous Properties of Industrial Materials, 1975). TABLE 3 RV.SU-LTSOr ACUTe TOxJcrrY TESTS (based o n Allied data)

Species

Rats Rats Rabbits Rabbits Rabbits Rabbits Digs

Route

Oral Oral Oral Oral Percutaneous Percutaneous Oral

a 1 additional death at 11 days.

Solvent

Oil Aqueous Oil Aqueous Oil Aqueous Oil

LDSO in mg/kg (No. animals) Males

Females

132 4- 8 (40) 96 4- 1 (40) 71 4- 6 (40) 65 4- 0 (40) 410 4- 65 (40) 435 4- 11 (40) ca. 250 (16)

126 -4- 12 (40) ------

Range of days to death 2- 7 1- 9 2 - 6a 1-10 3-13 1-30 2- 8

39 TABLE 4 RESULTSOF20-DAYORALTOXICITYTEST(P.h'.4) Group

Control Test

Av. wt. on specified day (g)

0

5

12

22

160 160

207 196

239 196

279 197

No. dying

No. doses preceding death

0/10 10/20

10,11,12,13,14,17,17,18,19,20

d. Sub-Acute 20-day oral toxicity test in rats (Table 4) Twenty young male albino rats were gavaged for 20 daily successive doses, except for Sundays, with 0.1 LD50 (9.6 mg/kg) of aqueous Kepone; 10 controls were used. By the fifth dose, all rats had tremors; survivors to 20 days were observed for an additional 10 days during which the tremors subsided. The tremor effect is described as follows:

"The characteristic effect of this compound is the development of severe DDT-like tremors, the severity of which depends upon dosage level and duration of exposure. Tremors may persist for a week or more following single exposures and cumulatively develop from daily repeated, individually ineffective doses." e. Sub-Acute and chronic oral toxicity in rats In the first study, groups o f 40 young male and female rats were administered Kepone in their diets for 2 years at concentrations of 5, 10, 25, 50, and 80 ppm; controls consisted of groups o f 40 animals. Animals were individually caged and weighed weekly; food consumption was measured over a 3-day period at the end of 1, 3, 6, 12 and 24 months. Animals dying in the first 3 weeks were replaced by rats of the same age and sex that had been maintained on control diets. Pooled urine samples from 5 rats of each group were collected at 3 monthly intervals. Five animals from each group were sacrificed at 13 weeks for analysis of fat, and also at 52 weeks, when an additional 3-5 rats o f each sex were returned to control diets for 4 weeks and sacrificed at 56 weeks (Table 5). In addition to complete histology, major organs and body weights were determined at each period o f sacrifice. Hematological studies were made on 5 rats o f each group at 3 monthly intervals; coagulation tests were also made on some survivors at the end of 3 months. Metabolic rates were determined at 9 months. In a subsequent study, conducted because increased liver weights had been noted in 5 ppm females by 3 months, groups o f 40 animals were placed on 0 and 1 ppm diets for 2 years; apart f r o m omission of metabolic rate determinations, fat analyses, coagulation, and withdrawal studies, the procedures were as those described in the first study. (1) Survival. All rats o n 80 and 50 ppm died by weeks 17 and 25, respectively. While it is stated that the 2-year survival was reduced in 25 ppm females, however, it appears that survival was reduced in a dose-dependent manner in all treatment groups, including the 1 p p m , particularly in females (Table 5).

40 40 40 40

0 5 10 25

40 40 40 40

0 5 I0 25

5 5 5 5

5 5

5 5 5 5

5 5

5 5 5 5

9 7

5 5 5 5

9 7

4 4 4 4

0 0

4 5 3 3

0 0

14 14 14 14

14 12

14 15 13 13

14 12

14 10 9 3

14 11

7 3 4 3

12 9

53.9 38.4 34.5 11.5

53.9 39.2

26.9 12.0 14.8 11.1

46.1 34.5

~o=

Nos.

Total

Survivors at 2 yrs.

56

13

52

Sacrificed at specified weeks

17 17 12 14

(3) (1) (4) (5)

17 (NS) 13 (NS)

8 (5) 6 (3) 9 (6) 4 (3)

14 (NS) 11 (NS)

Effective b

0 6 25 0

0 0

13 33 22 50

7 0

nephritis

% With grade 1II glomerulo-

-----

---

12.5 16.6 44.4 0

0 0

% With testicular atrophy

0 0

0 0 2 0

1 0

0 5.8 8.3 0

0 0

0 0 22.2 0

7.1 0

0 0 3 1

0 0

0 0 0 2

0 0

0 0 25 25

0 0

0 0 0 50

0 0

%

No.

No.

%

Hepatocellular carcinoma

"Hyperplasia"

Animals with liver lesions

0 1 1 0

(Based on Allied data)

• Based on initial numbers minus the numbers sacrificed, b Numbers animals surviving from 1~-2 years, excluding those sacrificed at 56 weeks, and excluding those additional animals, in parentheses, in which autolysis precluded histological examination. NS = not stated.

40 40

0 1

Females

40 40

Initial

Number animals

0 1

Males

G ro up and dose (ppm)

SURVIVAL AND THE INCIDENCE OF TESTICULAR, RENAL AND HEPATIC LESIONS IN KEPONE-TREATED RATS

TABLE 5

g

41 (2) Tremors. Tremors developed in the 80 and 50 ppm groups, by 2 and 3 weeks, respectively, and became progressively severe till death. In the 25 ppm group, tremors developed by 3 and 5 months in females and males, respectively, being particularly evoked by excitement; in a few females, the tremors developed progressively till 6 months, when they tended to regress. While animals at lower feeding levels had no frank tremors, both males and females on 10 ppm were highly excitable by 7 months. (3) Food consumption. A dose-dependent increased food consumption, which was progressive with time, was noted in males and females at all concentrations except 1 ppm. (4) Oxygen consumption. This was measured at 9 months and was significantly increased in males, at levels over 10 ppm; an elevated trend was also noted in females. Trends in the increases in metabolic rate and food consumption were of a similar order. (5) Body weight. Depressed growth was evident between 5 and 10 ppm in females, and between 10 and 25 ppm in males; at 5 and 10 ppm growth stimulation was however, noted in males, from 26-78 weeks. Growth depression occurred early, as described below: "In keeping with the cumulative action of this material, as found in earlier studies, no effectis apparent in the first week. Depression in females was evident after first weeks and in males after third weeks." (6) Haematology. Anaemia was found in rats at 25 ppm by the fifteenth month; this persisted till the termination of the studies. A haemorrhagic diathesis, without objective findings, was noted during blood collection from animals in the 50 and 80 ppm groups; this appeared to be due to vasodilatation. Additionally, some animals on the 50 and 80 ppm doses dying early in the study developed subcutaneous hemorrhages. (7) Proteinuria. At concentrations of 5 ppm and above, pr0teinuria, as measured by semiquantitative and quantitative tests, was markedly increased and its development with age accelerated from 6 months onwards; contrary to statements in the text, the tables demonstrate excess proteinuria at 1 ppm at various intervals over and at 2 years. (8) Fat storage. Fat analyses involved extensive extraction procedures with final potentiometric determination of chloride. Fat storage of Kepone was seen at all feeding levels; the ratio of fat/feeding levels increased linearly with increasing feeding levels over a range from 1 to 80 ppm; this relation is contrary to that seen with other organochlorines such as D D T 7. These data are summarized in Table 6. (9) Organ weights. Significant and persistent elevations of organ/body weight ratios occurred at lower Kepone concentrations in the liver than other organs (Table 7). An effect of 1 ppm on the female liver and kidney at 3 months was small but statistically significant. It was, however, claimed that: " . . . the magnitude of the differences (increases) found are not greater than those seen from year to year in control rats in this laboratory". (10) Withdrawal studies. In general, withdrawal of Kepone from diets at 12 months for 1 month prior to sacrifice had relatively little effect on various toxic

42 TABLE 6 FAT ANALYSES OF KEPONE AT THREE MONTHS

(P.2, Table XVI)

Feeding level (ppm)

Kepone in fat (ppm)

Ratio of fat to feeding levels

5 10 25 50 80

10.0 14.9 46.3 192 383

2.0 1.5

1.9 3.8 4.8

TABLE 7 CONCENTRATIONS KEPONE SIGNIFICANTLY INCREASING ORGAN TO BODY W E I G H T RATIOS

Organ

Concentrations (ppm) including significant weight increase at specified month 3

Liver Male Female ~y Male Female Heart Male Female Testes

15 5.3 41 12

12

13 (withdrawal)

9 8.7

3.7 10

(Based on P. 2)

Significant effect at I ppm (month)

24

12.5 5.4

17 > 25

10 > 25

> 25 25

16 25 24 > 25 50 (decrease) 25

> 25 > 25 > 25

No data 25 > 25

+(3) +(3) w

manifestations. Some regression of protcinuria was noted in the 5 p p m group. N o consistent effect was noted on body weight. However, in some animals with mild tremors in the 25 p p m male group, this effect disappeared. Thus, "changes induced b y twelve months exposure to C o m p o u n d 1189 are not readily reversible." (11) Histology. At 3 months, congestion o f the liver was seen a t and above 10 ppm; at 50 and 80 p p m liver cells were swollen with margination of granules, but n o frank necrosis. Testicular atrophy, ranging from slight to severe, w a s f o u n d in 80~o (12/15) of males in the 25, 50 and 80 p p m groups; atrophy was m o s t severe at the higher feeding levels, and was often accompanied by calcification of t h e testicular artery. N o significant lesions were reported in animals sacrificed at 12 m o n t h s and after the withdrawal period o f 1 month; no reference is made to testicular p a t h o l o g y in these groups. However, a high incidence o f testicular atrophy, 44~o in the 10 p p m group, was noted in the tabular listings o f the petitions, but not in the text, in animals dying between 1 and 2 years and in survivors sacrificed at 2 years (Table 5). In rats surviving between 1 and 2 years, including survivors sacrificed at 2 years,

43 TABLE 8 HISTOLOGICAL DIAGNOSES ON SIX "POSSIBLY CARCINOMATOUS)) LIVERS

Sex and feeding level (No.)

M, 25 ppm (23) M, 25 ppm (39) F, 10 ppm (20) F, 10 ppm (29) F, 10 ppm (38) F, 25 ppm (32)

Histological diagnoses~ Outside consultants Hennigar

Stewart

Steinberg

Peck

Suspect Ca Evolving Ca Suspicious Ca Evolving Ca Evolving Ca Evolving Ca

Ca No Ca No Ca Suspoet Ca Suspect Ca --

Ca At Ca Ca Ca --

Hy Hy Hy Hy Hy --

a Ca = Carcinoma; Hy = Hyperplasia; At = Atypical, "not quite enough to call carcinoma". special attention was directed to the kidney and liver. There appeared to be a trend towards increasing incidence and severity o f chronic glomerulonephritis (Grade III), particularly in females, with increasing dietary concentration o f Kepone (Table 5); an increased incidence was noted in both sexes at 5 p p m but not at 1 ppm. The question of "a possible carcinomatous nature of (liver) lesions in 6 rats" was raised by the pathologist, Dr. Hennigar. At his request the histology o f 5 of these 6 liver lesions was reviewed by three outside consultants (Table 8), who, in general, confirmed these suspicions. Further review of these 6 sections by F D A pathologists, Nelson, Long and Kent Stone resulted in the conclusion that there were no unequivocal carcinomas, although "liver damage was severe ' ' s . On the basis of these findings, it was concluded as follows (132. p12): "From the lack of uniformity of interpretation as to the nature of these liver lesions, it would appear that the question of whether or not Kepone has potential hepatoearcinogenic properties remains equivocal. "Thus, the possibility has been raised that lesions seen in the livers of 3 females on 10 ppm and 1 female and 2 males on 25 ppm are carcinomatous in nature and lack of uniformity of interpretation leaves the answer equivocal. However, the hepatotoxic potency of Cpd. 1189 at these concentrations appears clear." The incidence o f neoplastic, and "hyperplastic", liver lesions is listed in Table 5, on the basis of the m o s t advanced diagnosis of any of the 4 pathologists identified in Table 8, in relation to the "effective" number o f animals, being the number of animals sacrificed between 1 and 2 years, which were not too autolyzed for histology, together with the survivors sacrificed at 2 years. As can be seen, the incidence o f hepatocellular carcinomas was 50 % in the male 25 p p m group, and 25 % in both the female 10 and 25 p p m groups. The senior investigator o f this study, P. S. Larson, quoting the views of an F D A statistician, B. Vos, c o m m e n t e d on the significance o f the carcinomas in the test groups as follows9:

44 "... when the tumor is one that one hardly ever sees in a certain tissue, then the sudden appearance of an incidence of even 2 or 3 arouses suspicion. I am afraid that the latter describes our experience with liver cancer." Recent detailed re-evaluation o f these, and other, data by the E P A Carcinogen Assessment G r o u p led to the following conclusions1°: "Male and female B6C3F1 mice ingesting either the low or high dose levels of Kepone in the diet develop carcinomas of the liver. Carcinomas of the liver were found in Osborne Mendel male and female rats ingesting the higher dose level of Kepone. Albino male and female rats ingesting high doses of Kepone also had carcinomas of the liver. Such carcinomas of the liver not only have the morphologic characteristics of malignant tumors but they also kill the host animal and axe capable of invading and metastasizing to the lungs and of growing on transplantation to animals of the same strain. The overall incidence of all malignant tumors was increased in both Osborne Mendel and albino male and female rats. "The conclusion drawn from these studies is that Kepone is definitely a carcinogen in animals. There is nothing about the studies (treatment of animals, excessive carcinomas in controls, premature death due to other causes) that would cause any doubt about the validity of these studies."

f. Chronic dog study Groups of two 6-month old beagles o f each sex were placed on diets o f 0, 1, 5, and 25 p p m Kepone, totalling 16 animals, for a period o f 128 weeks. T h r e e animals (M, 0 ppm; M, 5 p p m ; F, 1 ppm) died f r o m unrelated causes. One male a n d 1 female on 25 p p m was sacrificed at 124 weeks; the surviving 11 dogs were sacrificed at 128 weeks. N o tremors were observed in any dose group during the study. W e i g h t gain was reduced in 25 p p m animals in the second year. Decreased food efficiency was noted at all treatment levels; particularly in females, at 13 and 26 weeks. T h e r e was a statistically significant increase in organ/body weight ratios for liver, kidney and heart of 25 p p m males and females. Excess proteinuria was not noted in a n y treatment group. Liver function tests at 2 years on 25 p p m dogs were normal. W h i l e it is stated that there were no treatment-related histological abnormalities, focal liver necrosis was, however, noted in 1/2 males and 1/2 females on 1 p p m Kepone.

6. Arant and Arthur 11, 1962 This is a detailed unsigned and undated report on a dairy cow feeding study done under contract to Allied Chemical Co. The feeding studies were p e r f o r m e d at A u b u r n University, Alabama and the milk residues were determined by Allied C h e m i c a l Co. Five pairs of cows were fed 0, 0.25, 0.5, 1.0 and 5.0 p p m Kepone f o r 58 days and then returned to normal diets for a further 85 days, when only "traces o f K e p o n e could be found in milk". "Barely trace amounts", 6-10 ppb, appeared by the sixth day of feeding a t 0.25 p p m a n d leveled off to approximately 23 p p b by 1 month. Residues o f a p p r o x i m a t e l y 10 p p b were found by 57 days in both 0.25 and 0.5 p p m feeding levels; n o detectable residues, less than 5 ppb, were found by 85 days. A t a 5.0 p p m feeding level, residues o f a b o u t 20 ppb had appeared by the first day o f feeding, and leveled o f f to 260-350 p p b by 1 month. Residues were persistent following returning to n o r m a l feed; by

45 85 days, approximately 10 and 15 ppb could be found in the 1.0 and 5.0 ppm feeding levels, respectively. The discussion of the report attempts to calculate what milk residues could be theoretically expected from a 0.1 ppm feeding level, which "probably represents the maximum, or at least a safe mean, level at which Kepone is likely to occur in the daily rations of dairy cows from use in the feed ration of dried citrus pulp produced from citrus sprayed in the prebloom (March-April) period with a single application of Kepone 50~o W.P. at 1/2 or 1 lb per 100 gallons". It was claimed that maximum levels of approximately 12 ppb would be expected during a 2-month feeding at 0.1 ppm, and that residues of 3 ppb would be found 85 days after feeding stopped.

a. C o m m e n t

This report, while indicating an approximate relation between Kepone feeding levels and residues over a narrow dose range, is of limited value as it provides no quantitative data on metabolism, possible toxicity, feed intake, milk production, and body residues, other than stating that these were not affected. Additionally, the low dose range tested, the brief duration of feeding, 60 days, and the small number of animals per group render this study inadequate for the development of toxicological inferences.

7. Larson

12,

1963

This undated and unpublished study was undertaken by Dr. Larson at the request of Dr. W. A. Knapp, Allied Chemical (see letter of August 1, 1963 from Knapp to Larson) in response to a request from Dr. J. Leary, USDA "for data demonstrating the safety of 2 % Kepone bran bait for use on lawns to control mole crickets".

a. Percutaneous toxicity

Kepone-free bran bait at 500 mg/kg, and 2 ~o Kepone bait at concentrations of 250 and 500 mg/kg, equivalent to Kepone concentrations of 5 and 10 mg/kg, were tested on groups of 6 male albino rabbits. The bait was applied under elastic bandages to the shaved trunk for 8 h a day and 5 days a week for 3 weeks, totalling 15 applications; animals were sacrificed 2 weeks following termination of the exposure period. No significant difference was noted in body weights of test and control groups. It is stated by Dr. G. R. Hennigar, the pathologist who examined the sections, that no consistent or significant pathological changes were noted in the Kepone groups, and that no dermatitis was seen "although there was a bit of variation in the thickness of the epidermis". Reference is made to "light staining cytoplasm (with) no cellular reaction round liver cells" in unspecified groups, which effect was discounted. In the author's Table 2, testicular atroph.y~is notedqn 2/6 and 1/6 animals in the low and high Kepone groups, respectively. The text of the report makes no reference to this effect.

46

b. Comments The total dosage o f Kepone in bran bait used in these tests, 15 successive daily applications o f 5 and 10 mg/kg, is less than the single dosages tested in the Allied 1960-1962 studies, in which an LD50 o f 410-435 m g / k g was determined. While the author concludes that no toxic effects were induced, his own Table 2, however, demonstrates a 25 ~o incidence of testicular atrophy in the 12 animals in the 2 Kepone test groups within 2 weeks of the termination o f the 3 weeks exposure. 8. Lester 13, 1964 This unpublished study on the inhalation toxicity o f Kepone dust was conducted by Dr. David Lester o f the Center o f Alcohol Studies, Rutgers State University, New Jersey, under contract to Allied Chemical. This report was submitted to Allied on N o v e m b e r 25, 1964, who subsequently submitted it to the USDA. a. Material tested F o u r groups o f 10 male Sprague Dawley rats were exposed to test and control dusts (Table 9) for 2 h daily for 10 days, and sacrificed 2 weeks later. Exposures were through a glass manifold attached to a feeder mechanism, with an air flow o f 10-12 l/min. Test groups were exposed to 10 % Kepone dust at dust levels of 37 and 154/~g/1, and Kepone concentrations of 3.7 and 15.4/zg/l; these concentrations were selected as being 2-fold and 10-fold, respectively, in excess of the 1-6/zg/l levels "likely to occur in h u m a n usage" 14, under specified conditions o f banana dusting. b. Toxicity N o significant effect on body weight was noted. The only organs weighed and examined histologically were lungs and livers. As noted in footnotes to the author's Table 1: "If certain extreme values are excluded from the means of organ weights, then the means show no significant differences among the groups".

TABLE 9 SUB-ACUTEINHALATIONTOXICITYOF KEPONE(based on Author's Table 1)

Group

Mean body weights (£) 0 Weeks 2 Weeks

Liver % body weight (No. animals)

Lung ~ body weight

Dust-free control

175.0 195.0 172.3 174.4

4.21 4.17 4.46 4.44

0.62 0.66 0.66 0.62

Dust control

Kepone, 3.7 pg/l Kepone, 15.4 pg/l

220.7 242.2 212.9 217.3

4- 0.30 (9)• 4- 0.28 (10) 4- 0.20 (8)s,b 4- 0.17 (8)c.d

4- 0.05 (9)* 4- 0.07 (9) e -4- 0.09 (7p.f 4- 0.05 (7)c.g

1 Animal excluded from autolysis, b Exclusion 1 animal with liver weight of 5.72 %, and with liver lesions, c 1 Animal accidentally killed, a Exclusion I animal with liver weights of 5.22%, and with liver lesions, e Exclusion 1 animal with lung weight of 2.59~o and with marked pneumonitis, t Exclusion 2 animals with lung weights of 2.50% and 0.96%, both with acute bronchitis, J Exclusion 2 animals with lung weights of 1.34% and 1.13 ~, the former with advanced bronchopneumonia.

47 One of the animals in the low Kepone group had a liver weight of 5.72~o; this liver "had many diffusely distributed parenchymal cells presenting definite activity of regeneration". One of the animals in the high Kepone group had a liver weight of 5.22%; this liver had "moderate passive congestion". With the exception of these lesions, it is stated that "no liver lesions were seen". Lung weights were excluded for 1 animal in the dust controls, 2 in the low-dose Kepone group and in the high-dose group; all these lungs exhibited bronchitis or bronchopneumonia. Lung pathology is presented semiquantitatively, including the scores o f lungs excluded from Table 9, without any description; it was stated that "there are no significant differences among the groups". The author concludes as follows: "Inhalational exposure to the levels of 10% Kepone dust used here caused no pathology or other untoward effects."

c. Response of the USDA In a letter of February 4, 1965 from Dr. Coleman of the USDA to Mr. Knapp of Allied Chemical, it is stated: "After review of the inhalation data presented for Kepone 10% dust, it is our conclusion that inhalation of this product presents a hazard and the caution statements providing for inhalation protection should be retained". In this connection, it is of interest to note that the U S D A had previously cautioned Allied Chemical in 1958 against the inhalation toxicity of Kepone. In an internal Allied memorandum, the recommendations and views of Dr. Ligon, USDA toxicologist, were quoted as follows14: " . . . inhalation toxicity.., would become necessary if and when we propose to use Compound 1189 in space sprays". The memo further states: "In any case he (Dr. Ligon) suggested a caution against inhalation for our products".

d. Comments This study is of limited value for the following reasons: (i) The study excludes from major consideration those animals with advanced pathology in the liver and lungs, the weights of which are "extreme values" in excess of means in remaining animals in the affected group. (ii) There is no description of liver pathology, apart from references in the footnote to exclusions in Table 1. (iii) The lung pathology scores are difficult to interpret in the virtual absence o f an accompanying description. Additionally, 4 slides out of an approximately 37 effective animals were "unaccountably lost in transit to the pathologist". (iv) The parameters o f toxic effects examined, are crude and limited; illustratively, no tests were made on lung or liver function.

48 In spite of the limitations of this study, and the contrary conclusions o f the author, it demonstrates that both levels of Kepone tested produced toxic effects, as indicated by increased liver weights and by histopathological lesions induced in both livers and lungs. 9. Huber 1~, 1965 This study, a summary of a 1963 PhD thesis, describes a wide range of effects of Kepone in mice. Inbred BalB/c Jax mice and technical grade Kepone, incorporated in Purina diets, were used in these studies. a. Sub-acute and chronic toxicity These tests were initiated on male and female weanling and adult mice in groups of varying numbers, which were fed Kepone at levels of 0, 10, 30, 40, 60, 70, 80, and I00 ppm for periods up to 12 months. The LD50 values for weanling and adult mice ranged from 50-60 and 60-70 ppm, respectively; no sex differences were observed. No mortality occurred in any mice fed 40 ppm up to 12 months; at this level food and water consumption were increased by 20--40 ~o, without any corresponding increase in body weight. It was additionally noted that: "A constant tremor syndrome appeared within 4 weeks in all mice fed 30 ppm or higher. These tremors terminated within 4 weeks followingwithdrawal of the treated diet". With the exception of the liver, no increase was noted in any organ weights at any dose level. Increased liver weights were noted by 7-14 days on 40 ppm diets, and were doubled, from control values of 6.0 ~o body weights, by 56-60 days, after which they remained approximately constant. While liver weights decreased following return to control diets for 120 days, however, they remained elevated by 18-46 ~o over control values. Histological examination revealed focal necrosis, hypertrophy, hyperplasia and congestion, the degree of which _depended on the length of treatment. Electron microscopy revealed liposphere formation and reduction of mitochondria. b. Kepone residues At a dietary level of 40 ppm, rapid and maximum accumulation o f Kepone was found by GC in the liver, brain, kidneys and body fat by 5 months, the liver being the major organ of accumulation, and in other organs such as gonads, adrenals, uterus, spleen, heart and muscle by 1 month. Kepone residues decreased rapidly from these organs following return to control diets. Based on comparisons with Kepone levels in female mice and then sacrificed, 56-95 ~o of the organ residues were lost in 24 and 150 days, respectively, following cessation of feeding; rates of loss were maximal in the liver. These determinations were based on analyses of single organs of single animals (Table 10). Transplacental passage of Kepone was demonstrated in a single animal fed 40 ppm Kepone for 6 days; 7 embryos averaging 0.3 g contained 5 ppm, and 4 placentas averaged 12 ppm. Passage through milk was demonstrated in a 6-day-old suckling

49 TABLE 10 TOTALESTIMATEDKEPONEIN MOUSEOROANS (based on Author's Table 9) Days on 40 ppm Days on control kepone diet diet

Kepone (#g/g) Liver

Brain

Kidney

Total

90 90 90

160 125 59

12 5.4 5.4

6.0 6.5 1.0

177 131 71

0 14 24

TABLE 11 Erwcrs oF Km~o~ ON ~RODUCnON (based on Author's Table 3) Kepone (ppm)

Pairs

Litters

Mean No. young per litter

~ Survival

Pair days per litter

Pair days per young

89 87 26 42

67 80 200 267

8.7 11.3 42.1 66.7

50

7.0

1st

2nd

8 8 8 8

7 6 4 3

5 4 0 0

7.7 7.1 4.7 4.0

14 14

14 0

14 0

7.1 .

Group A

0 19 30 37.5 Group B

0 40

89 .

.

.

mouse whose mother had been fed 40 p p m Kepone for the previous 6 days; a total K e p o n e level of 3 p p m was found in the suckling, with levels of 16.8 p p m in stomach contents, 33.6 p p m in liver and 16.8 p p m in brain. Analysis of a 15-day-old littermate revealed levels of 36 and 47 p p m in brain and liver, respectively. N o indication of K e p o n e metabolites was found in these studies. c. Reproductive studies

In a pilot study (Group A) with r a n d o m bred pairs of mice fed 19, 30 and 37.5 p p m K e p o n e for 1 m o n t h before mating and 100 days thereafter, the progeny yield was reduced 24, 79 and 87 ~o, respectively, over control values. At the lowest concentration tested, 10 ppm, while survival to weaning was normal, litter size was reduced and pair days/litter and pair days/young were increased. In a similar test (Group B), with BALB/c Jax mice fed Kepone 2 months before the 100-day mating period, no litters at all were produced (Table 11). Matings between G r o u p B females and control males were sterile, but matings between G r o u p B males and control females produced litters. Reproductive abnormalities, however, persisted in males and females from G r o u p B following withdrawal o f K e p o n e for 7 weeks with subsequent marines for 100 days. Litters of males and females previously fed with Kepone were smaller than untreated

50 TABLE 12 REPRODUCTION FOLLOWING WITHDRAWAL OF 4 0 p p m K ~ O N E FROM DIET

Category

Control females × kepone-fed males

Control males Controls × kepone-fed females

No. Pairs 1st Litter- Nos. Avg. No. Young ~. Survival 2rid Litter - Nos. Avg. No. Young y. Survival Pair days/litter Pair days/young y. Reproduction

10 10 5.5 88.6 6 5.7 87.9 62.5 11.5 65.1

10

5

8

5

4.3 64.7 5 4.6 87.5 76.9 17.3 42.4

7.4 89.1 4 7.0 86.2 55.6 7.6 100.0

controls; additionally, mortality in 1st litters from females previously fed with Kepone was increased (Table 12). Histological examination of the testes revealed no abnormality. Histological examination of ovaries demonstrated a virtual absence of corpora lutea, while control ovaries averaged 3.8 corpora. Vaginal smears demonstrated constant estrus in Group B females and in females fed 30 ppm in Group A; smears from Kepone-treated females revealed 81 ~o estrus over a 4-week period, compared to 24~o in controls. FSH activity in pituitary extracts of Kepone-fed females was the same as in controls; however, LH activity in Kepone-fed females was reduced by 25 ~o. It was thus concluded that the reproductive failure induced by Kepone in mice was largely due to an effect in females characterized by prolonged FSH and estrogen stimulation, inducing constant estrus, large follicles, and absence of corpora lutea, but with levels of LH subminimal for ovulation. 10. Good et al. 17, 1965 This study on reproductive effects in mice was undertaken in an attempt to investigate the relevance to placental mammals and man of the previous findings by others on the reproductive effects of Kepone in birds. This study was based on four sequentially conducted tests. In the first preliminary test, separate groups of male and female mice of unspecified strain were fed Kepone in their diet, at concentrations from 10-37.5 ppm (Table 13), for one month and then randomly paired with animals at the same feeding level and then maintained on their original diet for an approximate period of 4 months. The results indicate that Kepone induces a dose-dependent reproductive inhibition at all dose levels tested, as demonstrated by reduction in mean numbers of litters/pair and young/litter, and by increase in mean pair days• litter and pair days/young. The effect on litter size was noted at even the lowest concentration tested; no other adverse effects were noted at the 10 ppm level. BALB/c mice were used in Tests 2 and 3, which were based on Kepone levels of 10 and 5 ppm, respectively, for 4 months. In Test 2, there was a marked reduction

51 TABLE 13 TEST 1. E ~ c r KL~O~rEON REPRODUCTION (based on Author's Table 1)

Kepone (ppm)

No.

No.

Mean No.

Mean No.

pairs

litters

litters per pair

young per litter

0 I0 17.5 25 30 37.5

9 13 16 11 7 I0

15 26 25 12 2 2

1.66 2 1.56 1.I 0.29 0.20

7.93 7.62 7.00 6.08 3.00 5.00

Mean pair days Mean pair days per litter per young

65.3 54.5 72.2 100 241 555

8.3 7.2 13.1 16.5 80.5 111

TABLE 14 TESTS2 A~D 3. v~t~-'rs 5 AND 10 PPM KEvo~re ON REPRODUCTION ( b l ~ d O11Author's Tables 2 and 3)

Kepone No. (ppm) pairs Test 2 0 10 Test 3 0 5

Mean No. % Females producing litters/pair 1st litter 2nd litter

Size

Pair days/ litter

Pair days/ young

1st litter

2nd litter

24 24

1.96 0.92

100 70.6 •

95.6 20.8 ~

6.7 6.2

6.4 6.2

51.2 75.2

7.6 12.1

24 36

1.66 1.44

95.8 80.6 c

78.2 50.0 d

6.2 6.2

7.3 5.7

70.1 85.8

10.2 14.2

Significant, p ----0.05. b Sitmificant, p = 0.01. e Significant, p = 0.2. d Significant, p = 0.05. TABLE 15 4. REPRODUCTIONIN PROGENYOF KEPONE-FEDMICE(Author's Table 4)

Treatment group (No. pairs)

No. litters

No. % Producing progeny Ist 2nd litter litter

Size

Pair days/ Pair days/ litter young

Ist litter

2nd litter

Progeny control and control diet (21) Progeny kepone and control diet (23) Progeny Kepone and kepone diet (20)

21

123

71.4

28.6

5.6

6.5

120.0

20.5

9

42

30.4 s

8.7

4.3

6.0

306.7

65.7

10

40

25.4•

15.0

4.4

5.3

240.0

60.0

Significant, p = 0.05. in t h e n u m b e r s

o f f e m a l e s p r o d u c i n g first a n d , e v e n m o r e so, s e c o n d l i t t e r s ; this

a c c o u n t e d f o r t h e f a c t t h a t t h e t o t a l n u m b e r o f p r o g e n y f r o m t h e t e s t g r o u p was 46 ~o t h a t o f t h e c o n t r o l . T h e a v e r a g e n u m b e r o f p a i r d a y s / l i t t e r a n d p a i r d a y s / y o u n g w e r e m a r k e d l y i n c r e a s e d , t h e l a t t e r reflecting t h e c o m b i n e d effects o f r e d u c t i o n in

52 litter size and numbers of litters produced. Effects at 5 ppm in Test 3 were similar but less marked, the principal effects being a reduction in the numbers of females producing first, and, even more so, second litters; this may reflect the additional duration of exposure of test animals to Kepone (Table 14). Test 4 was based on the progeny of 5 ppm Kepone-treated BALB/e mice and of controls. One group of Kepone-treated progeny was maintained on 5 ppm Kepone diets and the other was given control diets. All groups were paired from 3 months of age for a 3-month period. As can be seen, the principal effect was reduction in fertility in progeny of Kepone-treated mice, whether on control or Kepone diets; additionally, the size of the first and second litters was reduced and the pair days/litter and pair days/young were increased (Table 15). The authors conclude that reproductive impairment is induced by Kepone at levels well below those producing chronic toxicity; the authors refer to data by Huber 16, indicating that mice tolerate 40 ppm levels of Kepone over 18 months without significant mortality. It was also concluded that: "If a threshold exists below which no such (reproductive) effect will be detectable, it must be considerably belowthe levelsstudied in these tests, and a greatlyincreased sample may be necessary to detect it." 11. Smith and Arant is, 1967 This paper reports the occurrence and persistence of Kepone in the milk of dairy cows following its administration in feed. The experimental work reported was undertaken in 1961. Ten dairy cows, of 3 different breeds, weighing 790--1150 lbs were used in these tests. Each cow was fed from individual water tubs and feed buckets and was handmilked. Kepone was administered in feed concentrate, of which each cow was given 15 lbs together with 15 Ibs of grass concentrate daily, at concentrations o f 0.25, 0.5, 1.0 and 5.0 ppm; each dose level was given to 2 cows. The 8 cows, with their 2 untreated controls, were given test diets for 60 days following which they were returned to normal diets. Milk was collected daily for 5 days prior to test on alternate days for the first 2 weeks of the test, weekly thereafter during the remainder of the test period, and for a further 85 days following the cessation of Kepone feeding. No observable toxic effects on weight gain or mild production were noted, and 3 of the 10 cows, in unspecified groups, "dropped normal calves". Kepone residues in milk were determined using hexane-benzene extracts with GLC sensitive to 5 ppb. Detectable levels of Kepone in milk were found approximately 1 week after test feeding commenced and levels gradually increased to their maxima by approximately 1 month. The highest Kepone level in an individual cow was 440 ppb. D a t a on milk levels were presented in graphic form only. At the end of the 60-day test feeding, milk residues of approximately 340, 140, 40, and 20 ppb were found in cows receiving 5.0, 1.0, 0.5 and 0.25 ppm, respectively, of Kepone in diets. Kepone levels during the 60-day test feeding tended to be directly related to butterfat content of milk. Thirtyfive days after return to normal diets, cows fed with 5.0 and 0.25 ppm Kepone had

53 milk residues of 90 and less than 10 ppb, respectively; by 85 days only traces were found in cows on the 5.0 ppm feed level. a. Comment A more detailed account of this study, including tabular listings of Kepone levels is also available in an undated and unsigned report 11, which was submitted to the FDA in 1962.

12. Gaines 19, 1969 The acute oral and dermal toxicity of technical grade Kepone, and 97 other pesticides, was tested in adult Sherman rats under standardized conditions. For oral toxicity testing, a range of doses was administered by single garage of peanut oil solutions to groups of male and female rats, totalling 50 and 60, respectively, in a volume of 0.005 ml/g. The acute oral LD50 in both males and females was 125 mg/kg; the LD10 in both males and females was 92 mg/kg. Thus, the dose response slope for acute oral toxicity of Kepone is steep. For dermal toxicity testing, Kepone solutions in xylene were applied singly to the shaved lower dorsal region. Acute dermal LD50 values in both males and females were > 2000 mg/kg. 13. National Cancer Institute 2°, 1976 This report is a synopsis of results of a carcinogenesis bioassay conducted at Gulf South Research Institute (GSRI), La, initially under direct contract to the Carcinogenesis Bioassay Program o f the National Cancer Institute (NCI), and from November 7, 1974 under a sub-contract to Tracor Jitco, Inc. This preliminary report, dated January, 1976, was made available on April 8, 1976. a. Materials and methods Technical grade hydrate of Chlordecone was purchased in a single lot from City Chemical Co., New York, having been manufactured by Allied Chemical Co. As determined by GSRI, using gas chromatography and an electron capture detector, the material was 92 ~o pure, the other major components being water of hydration and two unidentified contaminants totalling 1.5-1.8 %; Chlordecone readily hydrates at room temperature and humidity, and is normally used as a mono- to tri-hydrate. All diets were formulated with finely-ground Wayne rat feed, to which the required amount of Chlordecone in acetone was added for each test level; diets were mechanically mixed. Additionally, 2 % corn oil was added to final formulations as a dust suppressant. Water and food were made available ad libitum and were replaced 3 times weekly. Chlordecone treatment was terminated at 80 weeks when all animals were returned to control diets until the end of the test period. Osborn Mendel rats and B6C3F1 hybrid mice were used in these tests. Animals were randomized into experimental and test groups, and tests were initiated with 6-week old rodents. The Chlordecone-treated rats were housed alone in individual suspended wire cages, whereas the mice were housed in plastic cages with filter caps,

54 5/cage and 2-3/cage for females and males, respectively, in a room where Toxaphene and Chlordane were also under concurrent test. Cages on individual racks and racks in each room were rotated weekly. Animals were observed twice daily, weighed monthly and sacrificed when moribund or at the termination of experiments, which was at 90 weeks for mice and 112 weeks for rats. Histopathology was conducted by pathologists at GSRI, and was subsequently reviewed by pathologists at Tracor Jitco, with particular reference to liver lesions, several of which were also subsequently reviewed at the NCI. All pertinent data were recorded using a standardized NCI automated processing system. Maximally tolerated doses (MTD) were determined on the basis Of weights and survivals from preliminary sub-chronic 6-week toxicity tests. These were estimated as 30 and 60 ppm for male and female rats, respectively, and 40 and 80 ppm for male and female mice, respectively. These MTD values were the basis of high and low dietary test levels in both mice and rats, of MTD and MTD/2. However, the report states: " . . . these dose levels were overestimated and they were found not to be adequately tolerated, necessitating reductions at least once in all groups".

For this reason, dose levels were changed at least once and sometimes as many as 3 times in the course of the tests. The report thus gives the doses as a calculated "time weighted average", and these are approximate only. The calculated doses for male rats were 8 and 24 ppm, instead of the intended 15 and 30 ppm; the calculated doses for female rats were 18 and 26 ppm, instead of the intended 30 and 60 ppm. The calculated doses for male mice were 20 and 23 ppm, instead of the intended 20 and 40 ppm; the calculated doses for female mice were 20 and 40 ppm, instead of the intended 40 and 80 ppm (Table 16). b. Rat carcinogenicity test Survival in the low dose groups was comparable to that of pooled controls, whereas survival in both males and females at the high dose level was reduced (Table 17) mortality in males was approximately evenly distributed between 52-78 TABLE 16 DOSE LEWtS OF r . ~ O ~ mR RATS AND MXCE(based on Report Table 1)

Species

"MTD"

and sex Rats M F

Intended dose

Calculated dose

Low

High

Low

High

30 60

15 30

30 60

8 18

24 • 26

40 80

20 40

40 80

20 20

23 40

Mice M F

• This group received Kepone on alternate weeks only during the final 75 days of treatment.

17

100 10 49 45

w 100 86 84

61 70 56 40

0 1 0 2

0 0 2 0

0 0 1 10

0 0 1 3

0 0 2 22

0 0 2 7

. -NS NS

. -NS d NS

.

.

Matched controls

neoplastic nodules No. ~

Significance

. -NS 0.0000

. -NS 0.0490

Pooled controls

Animals with hepatocellular carcinoma

No. animals with

. 0 7 9

. 0 7 5

No.

.

.

0 14 20

0 14 12

%

Animals with extrahepatic carcinomas

.

.

0 8 19

0 8 8

No.

0 16 42

0 16 18

Animals with all malignant tumors

Total number o f animals in initial test group, minus number missing or autolyzed, b pooled controls within 3 months o f age o f matched controls and maintained in different rooms, c pooled controls for males with half matched for each dose. d NS = not significant.

Females

63 90 60 42

Pooled controlsb Matched controls Low dose, High dose,

-100 96 84

105 10 50 44

Pooled controlsb Matched controls e Low dose, 8 High dose,

Males

Effective a % Survival weeks No. animals 52 112

Group and calculated dose (ppm)

Rv.stu.zso r CARC'~OG~NICrrY T~ST m RATS (based on Report Table 11)

TABLE

56 and 78-112 weeks, whereas in females maximum mortality occurred in the latter interval. By week 5, a majority of the high level females had developed generalized tremors and some had weight loss; by week 16, epistaxis occurred in high level males and 4 animals died or were sacrificed. Generalized tremors were seen in low level females by 28 weeks. Rough coats, dermatitis, anemia, tremors, vaginal bleeding and cachexia were noted in low to moderate incidence towards the end of the first year and progressed in severity till termination. The report states: "Surviving animals at termination of study generally exhibited a very poor physical condition". As can be seen (Table 17), an increased incidence of hepatocellular carcinomas (HCC) was found in high-dose males and females; this was statistically significant in females and in males when comparisons are based on pooled controls. The report notes the rarity of HCC in Osborne-Mendel rats in justification of the use of pooled controls. The carcinomas were large and well differentiated, with occasional pseudoglandular patterns. No vascular invasion or metastasis was noted. The time to the detection of the first HCC was inversely related to dose; for the low- and high-dose males this was 112 and 108 weeks, respectively, and 87 and 83 weeks for the low and high dose females, respectively. Additionally, "extensive liver hyperplasia, fatty infiltration and degeneration were also found in an unspecified number of rats of both sexes in both dose groups"; none, however, was seen in matched controls. Neoplastic nodules were found in 2 low-dose males and high-dose females and in 1 matched female control. Benign endocrine adenomas were found in test and matched control groups in comparable numbers; no histological data, other than for liver tumors, were cited for pooled controls. However, an excess of extrahepatic malignant tumors was identified in test groups, particularly thyroid carcinomas in low-dose males and reproductive tract carcinomas in both low- and high-dose females. c. M o u s e carcinogenicity test

Survival in all groups was good and comparable, ranging from 84-92 ~o, with the exception of males in the high- and low-dose groups, which had survivals of 58 and 50%, respectively (Table 3); mortality in the male high-dose group was maximal between 52-78 weeks, and in the low-dose group between 78-90 weeks. Generalized tremors developed in high-dose females in the first week which persisted with decreasing intensity throughout the study; some individual males in the high-dose group also developed severe tremors. Approximately 20 ~o of the high-dose males were highly excitable during the second study year. Abdominal distension was noted in high-dose males and females by weeks 45 and 68, respectively. Abdominal masses were noted in some high-dose males and, to a lesser extent, in low-dose males in the second year. As can be seen (Table 18), an increased incidence Of HCC was noted in all treatment groups, particularly in males; this was statistically significant when comparisons are based on pooled or matched controls, even though the incidence of carcinomas in the male matched controls was significantly higher than the usual rate of 8 ~o in males of

57 TABLE 18 RESULTSOF CARCINOGENIC1TYTESTS1N MICE(Based on Report Table II) Group and calculated dose (ppm)

Effective s ~ Survival weeks No. animals 52 90

Weeks Animals with hepatocellular carcinoma to Significance tumor d No. % Matched Pooled controls controls

49 19 48 49

-100 96 90

92 90 58 50

87 87 70 62

8e 6 39 43

16 32 81 88

40

-100 98 96

85 90 84 84

--87 76

0

0

0

0

26 23

52 47

Males

Pooled controls• Matched controlsb Low dose, 20 High dose, 23

--0.0004 0.0000

--0.0000 0.0000

Females

Pooled controlsa Matched controls Low dose, 20 High dose, 40

I0

50 49

-~

0.0031 0.0081

0.0000 0.0000

Matched controls and other controls with birthdate within 4 months of matched controls and maintained in same room as treated animals, b half matched for each dose. c includes 6 carcinomas in the matched controls, d time to detection of first hepatocellular carcinoma at necropsy.

this strain of mouse. The carcinomas were well differentiated, ranging from welldemarcated nodules to large masses. N o vascular invasion or metastases were noted. It may be noted that the time to detection of the first H C C observed at death (Table 18) was shorter for treated than control male mice, and was inversely related to dose in both sexes. Additionally, a high, but unspecified, incidence of extensive liver hyperplasia was noted in males and females of all treatment groups;/t low, but unspecified, incidence of hyperplasia was noted in male and female matched controls. There was no significant difference in the type and incidence of extrahepatic tumors between treated and matched control groups; no data, however, are cited for the pooled controls. d. Discussion

The possible role of minor impurities in determining the carcinogenicity of the technical grade Chlordecone tested, amounting to less than 2 % excluding water, could not be assessed from this study. The possible role of extraneous factors must be recognized especially in the mouse tests, as two other agents were also under test in the same room. However, as all agents were of low volatility and as animals were maintained in enclosed, filtered cages, the potential for contamination was restricted. The diagnosis of H C C "was based upon the cytologic and histologic features o f the neoplasms and knowledge that lesions with the same morphologic characteristics as those observed have exhibited malignant biologic behavior", including progressive growth, invasion, metastasis and death of the host. The terms "neoplastic nodule" and "hepatocellular carcinoma" in rats were based on morphologic criteria and

58 nomenclature reported from a recent workshop on specific hepatocellular lesions in rats 21. (See Appendix 2.) The Osborne-Mendel strain o f rat tested has been extensively used b y the F D A as a general purpose test animal for many years. While it is susceptible to the carcinogenic effects of CCL4 by subcutaneous injection 22, it is h o w e v e r refractory to the hepatocarcinogenic effects o f 2-acetylamino fluorene 23. The B6C3F1 mouse strain tested has been extensively used in the N C I carcinogenesis bioassay program, current experience o f which indicates an overall incidence o f H C C in control males o f 7-10~o and in females of 1 ~o- While the incidence of H C C in m a l e matched controls was thus abnormally high in this study, 6/19 (32 ~o), nevertheless the increased incidence of H C C in male mice at both dose levels was statistically significant with matched besides pooled controls; contrastingly, the incidence of H C C in other pooled controls was within overall current ranges, 2/30 (8 ~o). Additionally, the time to first t u m o r detection was decreased in low- and high-dose males, compared with controls• The absence o f spontaneous H C C in both male and female rats and in female mice o f matched or pooled controls and the significant incidence o f H C C in high-dose female rats and female mice in all treated groups reinforces the findings o f a statistically significant hepatocarcinogenicity in treated male mice, as does the extensive liver hyperplasia in male and female mice and rats at b o t h dose levels, with only a low incidence in male and female matched control mice and none in control rats. The report recognizes the following limitations in the study, some aspects of whose procedures vary from current N C I practiceZ4: . . . While rats were housed in a room where no other chemicals were under concurrent test, Chlordane and Toxaphene were also under test in the mouse room. However, opportunities for cross contamination were minimized by the use of filter cap cages, and also by the use of 2 % corn oil in diets as a dust suppressant. •.. Preliminary toxicity tests for the determination of MTD levels were based on 42, rather than the usual 90 days; this resulted in an underestimate of chronic toxicity. •.. Changes in dosage of Chlordecone during the cour-~ of the study, together with the use of only 2 dose levels limit assessment of dose-response relationships. •.. The number of matched controls was 10-20, rather than the usual 50; however, the use of pooled controls, particularly room controls, compensated for the small numbers of matched controls• Even using the small matched control groups, the carcinogenicity of Chiordecone was statistically significant in all treatment groups, except male rats. •.. The treatment period was for 80 weeks, rather than the usual 2 years; testing over a 2-year period would probably have increased the carcinogenic response, especially in rats. Furthermore mice were sacrificed at 90 weeks, rather than at the usual 2 years. In spite of these limitations, the report concludes that the study d e m o n s t r a t e s the hepatocarcinogenicity o f Chlordecone in both sexes o f rats and mice. The accompanying press release emphasizes that... "The findings are considered definitive for animal studies." e. C o m m e n t s

This study is likely to have underestimated the carcinogenicity o f Chlordeeone as its relatively high dosage, particularly in high-dose male and female m i c e and b o t h dose groups o f male rats, resulted in competing toxicity and high mortality, thus

59 decreasing the number of animals at risk in the latter part of the study. An additional factor was the use of a relatively short duration of treatment, 80 rather than 112 weeks. The apparent absence o f vascular invasion and pulmonary metastases does not militate against the diagnosis of HCC in these studies; serial liver sections and multiple whole-lung sections, which are not done in routine carcinogenicity tests and apparently not in this study, are required to demonstrate these additional parameters of carcinogenicity. Detailed re-evaluation o f these data by the Carcinogen Assessment G r o u p of EPA led to the following conclusion: "... Kepone is definitely a caxcinogen in mice and rats. There is nothing about the study (treatment of animals, excesscarcinngenicity in controls, premature death due to other causes) that would cause any doubt about the validity of the study". 14. E P A 25, 1976 In a preliminary report on blood Kepone levels in a community survey in Hopewell, VA, reference is made to results of preliminary studies on reproductive and other effects of Kepone in mice and rats. Adult male rats were given 10 mg/kg Kepone orally daily, initially for 10 days; this was subsequently reduced to 6 days, because of "severe neurological symptoms before the 5th day". They were then mated with virgin females. An immediate and significant loss o f fertility lasting up to 3 weeks was noted. No indication is given as to whether this was due to a dominant lethal effect. Additionally, infant mice were given 12 and 24 mg/kg for 4 consecutive days after birth, resulting in dreduction of litter viability"; in rats 10 mg/kg/day, resulted in reduction in litter weight.

15. References 1 J.F. Adams, Consultant, Allied Chemical Co., memorandum to E. P. ~ t n ~ , Allied, April 21, 1949. 2 United States Testing Co., Inc., Report of Test Number 4299, February 28, 1949. 3 United States Testing Co., Inc., Report of Test Number 6524, April 22, 1949. 4 United States Testing Co., Inc., Report of Test Number 20337, May 26, 1950. 5 Allied Chemical Co., FDA Petitions for a Teraporary Tolerance on Potatoes. Section C: Acute and Subacute Toxicity; 1-year Rat Studies; Preliminary Dog Study, March, 1960. Chronic Rat Studies, July, 1961. Chronic Dog Study, February, 1962. 6 P.S. Larson, G. R. Henningar, E. M. Crawford, R. BlackwellSmith, S. J. Melette, W. A. Knapp, J. K. Finnegan and H. B. Haag, Acute, Sub-acute and Chronic Toxicological Studies on . . . ( Kepone Cpd. 1189).

7 E.P. Laug, A. A. Nelson, O. G. Fitzhugh, F. M. Kunze, J. Pharmacol. Exp. Ther., 98 (1950) 268. 8 W.A. Knapp, Allied Chemical, Assignment Report No. 18, FDS, Washington, D.C., March 9, 1961. 9 P.S. Larson, letter to W. A. Knapp, Allied Chemical, March 11, 1963. 10 The Carcinogen Assessment Group, Environmental Protection Agency 0EPA), Analysis of Kepone, July 27, 1976. 11 F.S. Arant and B. W. Arthur, Dairy Cow Feeding Study, unpublished Allied Contract Report, 1962.

60 12 P. S. l.au'son, Percutaneous Toxicity to Rabbits of 2% Kepone Bran Bait and 2 ~ Kepone Bran Bait Containing no Kepone, unpublished Allied Contract Report, 1963. 13 D. Lester, KeponeInhalation Studies, 10~ Dust, unpublished Allied Contract Report, November 27, 1964. 14 W.A. Knapp, Allied Chemical Co., letter to J. S. Lear,/, USDA, December 12, 1963. 15 W.A. Knapp, Allied Chemical Co., memorandum to File, Compound1189--Toxicity, January 13, 1958. 16 J.J. Huber, Toxicol. AppL Pharmacol., 7 (1965) 516. 17 E.F. Good, G. W. Ware and D. F. Miller, J. Econ. EntomoL, 58 (1965) 754. 18 J.C. Smith and F. S. Arant, J. Econ. EntomoL, 60 (1967) 925. 19 T.B. Gaines, Toxicol. Appl. Pharmacol., 14 (1969) 515. 20 Report on Carcinogenesis Bioassay of Technical Grade Chlordecone (Kepone), Carcinogenesis Program, Division of Cancer Cause and Prevention, NCI, January, 1976. 21 R.A. Squire and M. H. Levitt, Cancer Res., 35 (1975) 3214. 22 M.D. Reuber and E. L. Glover, J. Natl. Cancer Inst., 44 (1970) 419. 23 A. Syrneonidis, J. Natl. Cancer Inst., 15 (1954) 539. 24 J. Sontag, N. Page and U. Saffiotti, Guidelinesfor Carcinogen Bioassay in ~ 1 l Rodents, NCI Carcinogenesis Technical Report No. 1, April 30, 1975. 25 Environmental Protection Agency, Health Effects Research Laboratory, Preliminary Report on Kepone Levels Foundin Human Bloodfrom the GeneralPopulation of Hopewell, VA, March, 3 1976. APPENDIX The salient conclusions of this report, with particular relevance to the toxicology o f Kepone and its presumptive and observed effects on the Life Science Products Company (LSPC) employees and litigants, are summarized below. The references in parentheses relate to specified page numbers in the text of this report. 1. Toxicity

The high acute toxicity o f Kepone was established in studies contracted by Allied Chemical Company in 1949-1950, in which the oral LD50 in rabbits, mice, and rats was found to be < 50 mg/kg (p. 34--39); the dose-response slope for acute toxicity in rats was subsequently found to be steep (p. 53). In the 1949-1950 Allied studies, percutaneous administration o f Kepone was found to induce delayed toxicity and mortality over a several week period (p. 35-36). The cumulative toxicity o f Kepone in rats was established in subsequent sub-acute oral toxicity tests, contracted by Allied Chemical Company, in which excess mortality was noted at the lowest dose level tested, 1 ppm (p. 39); excess proteinuria and increased liver and kidney weights were also noted in chronic feeding studies in rats on the 1-ppm dose level (p. 41). In the same rat sub-acute feeding studies, fat storage of Kepone was seen at all feeding levels (p. 41); the ratio of fat-feeding levels increased linearly with increasing feeding levels over a range from 1 to 80 ppm. Similarly in mice, Kepone was found to accumulate rapidly and maximally in body fat, liver, and other organs (p. 48). 2. Neurotoxicity

Kepone was first demonstrated to be neurotoxic in rats, in studies contracted by Allied Chemical Company in 1949 (p. 34), in whom DDT-like tremors were induced. In subse.quent Allied studies, tremors were induced in rats within a few days following

61 a single dose of Kepone (p. 38), and within weeks to months following sub-acute or chronic administration of sub-toxic doses (39, 41); these effects were poorly reversible (p. 41). Tremors were also induced in other species including rabbits, following oral or percutaneous administration (p. 35), mice (p. 48, 56), and birds, including chicks, hens and quail (p. 26, 29). In addition to tremors, the neurotoxic syndrome was also characterized by excitability, incoordination, convulsions, and paralysis. There is a clear consistency between the experimental neurotoxic effects of Kepone and the effects subsequently reported in LSPC employees (p. 20, 21).

3. Male reproductive toxicity Kepone was first reported to induce reproductive impairment in male rats in 1961 in studies contracted by Allied Chemical Company (p. 42); a high incidence of testieular atrophy was induced by sub-acute or chronic administration of Kepone. Similar effects were induced in rabbits following percutaneous administration of Kepone (p. 45). Reproductive abnormalities were found in Kepone-treated male mice, ineluding reduction in litter size, which persisted following withdrawal of Kepone (p. 49-52); loss of fertility was noted in Kepone-treated male rats (p. 59). Studies in male birds, in which Kepone induced female plumage, abnormal seminiferous tubules, and malformed sperm (p. 27, 28, 33), have suggested that Kepone induces an estrogenlike effect, which is FSH-mediated. 4. Carcinogenicity Kepone was first demonstrated to be hepatocarcinogenic in male and female albino rats, in studies contracted by Allied Chemical Company in 1961 (p. 43, 44). These findings were subsequently confirmed in National Cancer Institute (NCI) tests, in which hepatocarcinogenie effects were induced in male and female OsbornMendal rats, and in male and female B6C3F1 hybrid mice (p. 53-59); the mouse and rat hepatocellular carcinomas are comparable and valid indices of carcinogenicity. Carcinogenic effects were also induced in extra-hepatic sites in rats of both sexes. Based on these data, the NCI and the Carcinogen Assessment Group of the Environmental Protection Agency independently concluded that Kepone is an unequivocal carcinogen. There is an overwhelming consensus in the scientific literature that carcinogenicity data derived from experimental studies, particularly when based on more than one strain and more than one species, have a high degree of presumptive human relevance and may thus be properly extrapolated to humans. Concerns as to the carcinogenicity of Kepone in LSPC employees are further emphasized by their high exposure levels (p. 18), and by their high body burdens of Kepone (p. 2). 5. Exposure to Kepone of life science products company employees The manufacture of Kepone at LSPC appears to have been characterized by high production volumes (p. 4), and grossly inadequate work practices (p. 17, 18). Work practices at LSPC failed to reflect the substantial available information on the toxicology and carcinogenicity of Kepone, and also failed to even approach the level

62 of labelling requirements and of precautions recommended in Allied Chemical Company documentation. The only determination of Kepone air levels in the plant, made on July 22, 1975 when the plant was operating below full production, revealed levels o f 3,000/zg/m a in the dryer house (p. 18); this level is approximately 10-fold in excess of the TLV proposed by Allied (p. 7-9), and 3,000-fold in excess of the subsequently proposed NIOSH standard of 1/zg/m a (p. 19). It is of further interest to note that air levels of 55 /~g/m3 were found 0.13 miles south of plant when it was in full production (p. 11). Based on the observed Kepone level of 3,000 /~g/ma, assuming complete absorption of Kepone and an inhalation rate of 10 m a over an 8-h working day, the intake of Kepone in an employee in the dryer house would be approximately 430 /zg/kg/day. The symptoms and signs noted in LSPC employees (p. 18-21) were, in general, consistent with those previously described in the toxicological literature on Kepone; particular parallelism extends to neurological signs of irritability, tremors, weakness, and ataxia and to ligospermia, and hepatomegaly. Kepone was found in the blood o f all LSPC employees at levels from 165 to 26,000 ppb (p. 21); it is o f interest to note that Kepone blood levels in the surrounding community were approximately linear as a direct function of proximity to LSPC, with a maximum of 50 ppb within a radius of 0.25 miles (p. 13-14).