planning and scientific expertise of the PPL, and the regulatory functions of the Office of Pesticides. The extent of this project will depend on the needs of the regulatory functions of the Office of Pesticides. The extent of the project in 1972 will be limited to the productivity of a senior staff member and technician with all needed support being provided from existing staff. In 1972 the capability of initiating actual treatment of animals for a rodent study will be no greater than two weeks. In 1973 both acute primate and rodent studies would be initiated within one week. When not actually involved in direct support of the regulatory needs of the office of pesticides the group will develop protocol to be utilized in requesting information from industry and in completing toxicity data for compounds which may fall under the jurisdiction of the PPL in the future.

(f) Conduct library research in the scientific fields related to the above studies. Interpret, and subject to statistical analysis, the data obtained from these studies. Prepare and deliver reports at scientific meetings and submit completed work for publication in scientific journals.

(g) Aquisition and cataloging of scientific books, journals and reprints for the maintenance and updating of a scientific library for the use of the entire laboratory is a continuing function of this project.

(h) Develop a "model system" for measurement of enzyme induction in vivo. The substrate would be a substance of low toxicity with measurable blood levels, which is metabolized by several different pathways to metabolites that can be readily detected. Such a system will allow a considerable savings in time, money and experimental animals. It would also permit relatively safe in vivo studies of enzyme induction in selected human populations by community studies groups.

(i) Determine efficacy of antidotes to pesticide intoxication in primates. This could result in improved techniques for treating human intoxication.

(j) Develop new analytical procedures and assay systems necessary for accomplishment of the specific objectives of the above studies.

(k) Develop a suitable assay for competitive inhibitors of cholinesterases, such as carbamates. The methods currently available to our community studies groups for the measurement of irreversible cholinesterase inhibition are not suitable for assessment of competitive inhibition. The anticipated expanded use of carbamates to replace chlorinated hydrocarbons makes development of this assay a priority project.

(1) Standardize the in vitro enzyme assays used to detect induction so that data among various EPA labs and between EPA labs and outside labs can be compared.

(m) Construction of a primate breeding colony will begin to insure a reliable, uniform, and relatively inexpensive supply of experimental animals for our studies.

(n) Complete construction of the first phase primate breeding colony.

(0) Secure equipment to expand our capabilities to utilize radioisotopes for metabolism and induction studies.

(p) Initiate construction of additional temporary quarters to house primates currently occupying space in building #9, that could be used to better advantage as laboratory space.

(q) Purchase expandable electron microscopy unit to allow more detailed examinations of tissues from experimental animals. This service would also be made available to other laboratories within the pesticides office and other units of EPA.

III. Scope:

The research outlined in this work program will be carried out at the Perrine Primate Laboratory. The large numbers of primates required for these studies make the establishment of a primate breeding colony a necessity. Prior experience based on a small pilot colony indicates that the sub-tropical climate found in Perrine is ideal for such a colony.

In the past the vast majority of toxicological research has been carried out using mammals that are not closely related to man. In order to better assess the hazards of pesticides and other environmental contaminants to man, it is necessary to study the effects of these compounds in nonhuman primates. IV. Results:

Reports will be provided to regulatory personnel of the Office of Pesticides containing any data produced that would be of importance in setting standards. This will include data regarding alterations in toxicity due to secondary factors and data of studies requested by the regulatory functions. Liaison reports re

garding research in progress and white papers describing anticipated long term research needs of the regulatory functions will be submitted. Reports concerning antidotal efficacy will be supplied to the label review activities and to the DPCS regarding their poisoning prevention and treatment activities.

FISCAL YEAR 1972

(a) Reports concerning the following studies are expected in FY 1972:

1. A Sensitive Gas Chromatographic Method for Human Cholinesterase Determination.

2. Comparative Stimulation of yHCH Metabolism by Pretreatment of Rats with yHCH. DDT and DDT+ yHCH.

3. Some Effects in Squirrel Monkeys on Repeated Administration of p,p'-DDT. 4. Changes in the Metabolism of Biogenic Amines Following the Administration of Carbaryl to Rats.

5. The Absence of Conversion of o,p'-DDT to p,p′-DDT in the Rat.

6. The Metabolism of Parathion in the Rhesus Monkey.

7. Metabolic Alterations in the Squirrel Monkey Induced by DDT Administration and Ascorbic Acid Deficiency.

(b) Data will be collected from a study involving the effect of ascorbic acid deficiency and varied protein quality on metabolism in the guinea pig. Results should indicate interaction between protein quality and ascorbic acid on various detoxification systems.

(c) Work in this laboratory has resulted in the isolation and identification of several unreported chlorophenol metabolites from the metabolism of lindane in the rat. A study has been proposed and a protocol submitted for the investigation of the progressive excretion of chlorophenols resulting from repeated low level administration of lindane to rats. In addition the simultaneous administration of DDT and lindane to another group of rats will be utilized to study the interaction of a second chlorinated pesticide on the quantity and distribution of the chlorophenol metabolites.

(d) A suitable "model system substrate” is being sought. Landane is being employed in an early attempt to correlate enzyme induction with metabolite excretion. Various contaminants encountered by man in his food, water and elsewhere in the environment will be compared by their influence on lindane metabolism.

(e) Studies on the efficacy of antidotes to pesticide intoxication will result in improved techniques for treating human intoxication.

(f) The continued isolation and determination of the metabolites of lindane and other pesticides will be conducted.

(g) Data concerning the long-term effects of feeding DDT or its metabolite DDE on the total pattern of parathion metabolism by liver microsomal preparations and the effects of these inducers on the LD50 of parathion will be collected, analyzed and put in manuscript form to be submitted for publication.

(h) Data concerning effects of o.p'-DDT on secretion of steroid hormones will have been collected, analyzed and put in manuscript form to be submitted for publication.

(i) Development will begin on a suitable assay system for evaluation of competitive inhibitors of cholinesterases such as carbamates. This will result in a new assay system to replace the present system, which was designed for irreversible inhibitors.

(j) The various conditions necessary for the most reproducible in vitro assay of various enzymes effected by pesticides will be investigated. Optimum conditions will be evaluated so that a standardized protocol can be recommended for collection of data from these enzymes by EPA laboratories and contractors.

V. Approach:

In a typical statistically designed experiment, animals will be exposed for a prescribed period of time with the compound (s) under investigation. During the treatment period excreta and blood will be collected separately and analyzed for metabolites and various endogenous materials. At the end of the treatment period the animals will be sacrificed. Excreta and tissue will be sampled for chemical and enzymatic analyses. In addition, tissues will be examined for gross pathological changes. The collected data will be statistically analyzed, interpreted, put into manuscript form and submitted for publication.

Special techniques that will be employed in the analysis of data are as follows: (1) Gas chromatography using a variety of detectors. (2) Thin-layer chromatography.

(3) Radio-isotope counting.

(4) Electrophoresis.

(5) Spectrophotometry, including UV, IR, NMR, mass spectoscopy, and flame photometry.

(6) Ultracentrifugation.

(7) Computer analysis of data.

(8) Column chromatography employing automatic fraction collector.

(9) Fluorimetry.

(10) Microscopic examination of gross pathological changes.

As indicated by the time schedule, new techniques will be utilized as the equip. ment becomes available.

Another approach that will be used in most in vivo studies is isolation of tissue or sub-cellular fraction to be studied, incubation with pesticide or metabolite, and isolation of metabolites or measurement of effect of tested substance on various enzyme systems. The same special techniques as in the first approach will be used. In studies concerning toxicity of pesticides or the effects of other environmental factors on the toxicity of pesticides, standard FDA toxicological protocols will be used supplemented by improved protocols as they become available.

In all cases the data obtained will be compared to any available human data to ascertain the relevance of our research to human exposure.

Narrative: Fiscal Year 72

Project Title: Physiological Effects of Pesticides on the Nervous System of Primates and Other Mammals.

Project Director: J. A. Santolucito

Justification:

Human populations are exposed to a complex of environmental stressors including pesticides. Individuals are exposed to these agents, most of which are neurotoxins, throughout their lifetimes either chronically or repeatedly and most often in very low concentrations. The magnitude of the insult may be too slight to manifest itself as an obvious functional deficit yet it might result in significant changes in EEG or behavior. The impact of such behavioral changes on the wellbeing and productivity of a human population could be considerable. In order to evaluate the potential hazard to human health resulting from this situation, there is a need for maintaining a non-human primate research program in which studies on the effects of prolonged, low-level exposures to selected pesticides and/or other chemical toxicants can be carried out using controlled animal experimentation methods. Data so obtained is unattainable from epidemiological studies on human populations alone and is essential for the establishment of criteria used for determining relative risks associated with the use of these compounds. This project will fulfill an important role in meeting these needs during FY 72.

Objectives:

The ultimate objectives of this project are to determine:

(1) what effects prolonged exposures to pesticides have on the function of the primate nervous system;

(2) whether these effects are transient or persistent:

(3) the mechanisms by which the effects are produced; and

(4) estimates of potential hazards to human health. These objectives will remain essentially unchanged during the period FY 72. The focal point of the project is the study of the effects of chronic pesticide exposure on the electroencephalogram (EEG) of monkeys, initiated in 1968, which will continue throughout the period. Associated with this are studies on the behavioral and neuroendocrine effects of pesticide exposure as well as the use of model systems to explore the mechanisms of action.

FISCAL YEAR 1972

Studies on the effect of pesticide exposure on pituitary hormone activity will be initiated and continued throughout the five-year period. Similarly, studies on the effects of pesticides on synapses using the superior cervical ganglion of the rat as a model system will be initiated.

Scope:

This project is necessarily carried out at the Perrine Primate Research Laboratory which has the only on-going, in-house primate research facility of the Pesticides Division of the Environmental Protection Agency. Considerable data has accumulated on the toxic effects of pesticides in several species of mammals including man. However, very little information is at hand concerning the neurophysiologic effects of prolonged exposure to sub-toxic quantities of these agents particularly in primates. The uniqueness of this research project relates to its investigations on the effects of low-level, chronic pesticide exposure in non-human primates.

Results:

FISCAL YEAR 1972

Sufficient behavioral studies in rats will be completed to initiate development of protocols for primate behavioral studies and for reporting.

It is anticipated that the rat superior cervical ganglion will provide an important and useful model system for elucidating some of the electrophysiological findings in the chronically-exposed monkey study. These investigations will yield a research report during this fiscal year.

The study of chemically induced defects is essential in the protection of human health. Teratology, mutagenesis, carcinogenesis and pathology are considered to be interrelated disciplines for the study of abnormal growth and development. The project will be developed one area at a time, and integrated to provide maximal efficiency. Teratology will be developed first.

Non-human primates are closer to humans in their embryological development and responses than other experimental animals presently used. The compounds studied will be those to which susceptible pregnant mothers are most exposed as determined by DPSC.

II. Objectives:

To study the teratological effects of environmental pollutants with special emphasis on pesticides. This project will methodically test environmental pollutants first singularly and then in combination and establish dose response data to allow an appropriate extrapolation of human risk. FY-72 objectives will be to develop a screening procedure in rodents which will be used until an appropriate gang cage breeding facility can be constructed. FY-72 objectives include the construction and stocking of the facility with appropriate animals and the initiation of the study of the teratogenic effects of the "dioxin" component of 2,4,5-T. Mutagenesis will be initiated in FY-72 and become functional in FY-73. Carcinogenesis will be initiated in FY-73 and made functional in FY-74.

The project will study the effects of specific dioxins and heavy metals found in pesticides formulations. The morphological expression of this effect will be described. After completion of the animal facility the study of teratogenic effects in animals other than primates will be used only as a screening study will all definitive work being done in non-human primates. The results of these studies will be compared to investigations in "occupationally” or “highly" exposed humans to determine the human health hazard.

III. Scope:

The research outlined in this work program will be carried out at the Perrine Primate Laboratory. The large numbers of primates required for these studies make the establishment of a primate breeding colony a necessity. Prior experience based on a small pilot colony indicates that the sub-tropical climate found in Perrine is ideal for such a colony.

In the past the vast majority of toxicological research has been carried out using mammals that are not closely related to man. In order to better assess the hazards of pesticides and other environmental contaminants to man, it is necessary to study the effects of these compounds in non-human primates.

IV. Results

Reports will be produced on each compound 1 year after the work begins. Recommendations will be available to the petition review activities for possible regulatory actions when a compound is found to be teratogenic. Epidemiological studies in appropriate human populations will be initiated as a result of positive findings and human hazards assessed. Better human protection will result from use of the more appropriate experimental animals and the associated information obtained.

V. Approach:

Compounds will be administered to pregnant rhesus monkeys at various doses to determine the dose response characteristics. If a compound is found to be teratogenic another group of animals will be treated at an appropriate dose with the terata produced being described in detail to allow for possible detection in human populations.

510101005

DIVISION OF PESTICIDES COMMUNITY STUDIES: TOXICOLOGY BRANCH
FISCAL YEAR 1972 PROGRAM PLAN

Project Title: Biological Effects

Prepared by Joyce Goldstein and J. E. Suggs-April 1971

Program Justification:

Liver enzymes. Exposure to pesticides alters the metabolism of chemicals and endogenous substances by the liver via induction and inhibition. This can result in increased susceptibility to the toxic effects of drugs or other environmental compounds. In addition, the metabolism of endogenous compounds can be altered, resulting in physiological or pathological changes. For example, the disease porphyria, which can be acquired by exposure to certain chemicals, seems to be related to induction of drug-metabolizing enzymes and heme synthesis through mechanisms not yet completely understood. Increased metabolism of steroids by the liver in birds after DDT exposure has contributed to decreased eggshall thickness and the subsequent ban of DDT.

Data is needed on the extent of exposure necessary for induction and inhibition of this system with various pesticides, and particularly on the relationship between enzyme induction and resulting physiological changes which may be harmful or endanger a species for evaluation of the overall significance of this phenomenon.

Biochemical Mechanisms. Toxic agents and stress agents are manifested by visible alterations in cell structure as observed by microscopy or by cell function as measured by various chemical analyses. These manifestations are always preceded by some basic enzymatic or biochemical abnormality. Since a compound is metabolically converted to a variety of products, those products should be examined as an agent which affects a biological system. Objectives:

In FY 1972, determine the effects of dietary levels of piperonyl butoxide on six pathways, microsomal protein and P-450. These are to be examined in the rat after varying dosages and feeding times. Changes will be correlated with the cellular anatomy (proliferation of endoplasmic reticulum) and in vivo metabolism by the liver. Similar experiments with Arochlors will be undertaken, and findings correlated with possible changes in heme metabolism, porphyria, and liver damage.

Biochemical Mechanisms. To determine the effects of selected chlorinated hydrocarbons on free amino acid levels of the central nervous system. Any observed change should be investigated as an alteration of enzyme activity or as a result of alteration of active transport mechanisms. Any change in amino acid levels will also be an indication for expanded study of the effects of the chlorinated hydrocarbon on nitrogen balance.

In FY 1972, the effects of hexachlorophene on brain amino acid levels will be investigated. The proposed metabolite of DDT, DDCHO, will be sought in vivo as a product of oxidation of DDOH.

Results:

Liver Enzymes. Although changes in ability to metabolize chemicals are not given much weight in pesticide regulation at the present time, it is recognized