EPA-600/1-78-003
January 1978
Environmental Health Effects Research Series
       TERATOLOGY AND ACUTE  TOXICOLOGY OF
                  SELECTED CHEMICAL  PESTICIDES
                    ADMINISTERED BY  INHALATION
                                     Health Effects Research Laboratory
                                     Office of Research and Development
                                    U.S. Environmental Protection Agency
                               Research Triangle Park, North Carolina 27711

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                RESEARCH REPORTING SERIES

Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology.  Elimination of traditional grouping  was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:

      1.  Environmental Health Effects Research
      2.  Environmental Protection Technology
      3.  Ecological Research
      4.  Environmental Monitoring
      5.  Socioeconomic Environmental Studies
      6.  Scientific and Technical Assessment Reports (STAR)
      7.  Interagency Energy-Environment Research and Development
      8.  "Special" Reports
      9.  Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series. This series describes projects and studies relating to the toler-
ances of man for unhealthful  substances or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals — but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia  22161.

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                                         EPA-600/1-78-003
                                         January 1978
 TERATOLOGY AND ACUTE TOXICOLOGY OF SELECTED
CHEMICAL PESTICIDES ADMINISTERED BY INHALATION
                       by
      Gordon W. Jewell and James V. Dilley
           Stanford Research Institute
          Menlo Park, California  94025
             Contract No. 68-02-1751
                Project Officer

                 Neil Chernoff
         Experimental Biology Division
       Health Effects Research Laboratory
       Research Triangle Park, N.C. 27711
      U.S. ENVIRONMENTAL PROTECTION AGENCY
       OFFICE OF RESEARCH AND DEVELOPMENT
       HEALTH EFFECTS RESEARCH LABORATORY
       RESEARCH TRIANGLE PARK, N.C. 27711

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                              DISCLAIMER

     This report has been reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved for
publication.  Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products
constitute endorsement or recommendation for use.
                                   ii

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                               FOREWORD

     The many benefits of our modern, developing,  industrial society are
accompanied by certain hazards.  Careful assessment of the relative risk
of existing and new man-made environmental hazards is necessary for the •
establishment of sound regulatory policy.  These regulations serve to
enhance the quality of our environment in order to promote the public
health and welfare and the productive capacity of  our Nation's population.

     The Health Effects Research Laboratory, Research Triangle Park,
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects.  These
Studies address problems in air pollution, non-ionizing radiation,
environmental carcinogenesis and the toxicology of pesticides as well as
other chemical pollutants.  The Laboratory develops and revises air quality
criteria documents on pollutants for which national ambient air quality
standards exist or are proposed, provides the data for registration of new
pesticides or proposed suspension of those already in use, conducts research
on hazardous and toxic materials, and is preparing the health basis for
non-ionizing radiation standards.  Direct support  to the regulatory function
of the Agency is provided in the form of expert testimony and preparation of
affidavits as well as expert advice to the Administrator to assure the
adequacy of health care and surveillance of persons having suffered imminent
and substantial endangerment of their health,  s

     As part of its overall mission, the U.S. Environmental Protection
Agency is concerned with the effects of pesticides on mammals including
man.  One area of specific concern is the effects  of such compounds on
the developing mammalian organism.  The following report deals with
the potential of selected pesticides to induce toxic effects in embryos
and/or fetuses of animals exposed to such chemicals by the pulmonary
route during gestation.
                                          John H. Knelson, M.D.
                                               Director,
                                   Health Effects Research Laboratory
                                   iii

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                                PREFACE

     The Federal Insecticide, Fungicide,  and Rodenticide Act designates
the Environmental Protection Agency as the governmental body responsible
for the safety of all pesticides used in the United States.   More
recently, the Federal Environmental Pesticide Control Act (PL 92-516)
strengthened EPA's regulatory responsibilities in the area of pesticides
to include intra- as well as inter-state commerce.
     To be federally registered, a pesticide must be determined to not
be hazardous to health or to the environment when used according to
its labeling restrictions.  Thus, relative to the new law as well as
to specific directives included in Public Law 93-135, 1973,  EPA now
is conducting a thorough review of the implications of using alternate
chemicals for pest control, including older registered pesticides.
Stanford Research Institute is contributing to these goals through two
distinct programs engaged in
     • comparative toxicity of pesticides administered by various
       routes, and
     • teratology of inhaled chemicals.
     This report includes the results of both studies mentioned above.
The first compares the acute toxicity of Parathion, Methyl Parathion,
Guthion, Azodrin, and Thimet when administered to male and female rats
by the percutaneous, oral, intravenous,  and inhalation routes.  The
second deals with the potential fetotoxicity of chloroform,  ethylene
thiourea, Thimet, Bromacil, and Simazine administered by daily inhalation
on days 7 through 14 of gestation in rats.  The results and discussion
of both studies are dealt with separately in the following report.
                                   IV

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                               ABSTRACT

     A method was developed for generating pesticide aerosols within
the respirable particle size range of 0.3 to 3.0 urn.  Analytical
methods were established for determining pesticide concentrations in
chamber air samples and in tissues.  A unique chamber exposure system
was developed that permitted the simultaneous exposure of four different
groups of rats to four different concentrations of pesticide from a
single generation source.  Parathion, methyl parathion, Thimet, Guthion,
and Azodrin were administered to rats by the oral, dermal, intravenous
or inhalation routes, and the LD50s or LC5os were compared.  Inhalation
was the most toxic route of administration, followed by the intravenous,
oral, and then dermal routes.  Females were more sensitive than males
to parathion and Thimet by all routes of administration.  Azodrin was
more toxic to females by the intravenous and oral routes, and Guthion
was more toxic to females by dermal application.  No correlation was
found between mortality and cholinesterase inhibition or blood or liver
pesticide content.  No gross or histopathological lesions were
identified that could be attributed to pesticide treatment.
     Timed-pregnant rats were exposed to vapors/aerosols of chloroform,
ethylene thiourea, Thimet, Bromacil, and Simazine for 1 to 3 hours
daily on days 7 through 14 of gestation.  Three different concentrations
of each compound were used for each study.  All animals were sacrificed
on d.ay 20 of gestation and examined for total litter size, fetal weight,
and fetal resorptions.  No dose-related terata were found in any of
the studies.  Chloroform produced an increased embryotoxicity at 20
mg/liter (4100 ppm).   The highest doses of ethylene thiourea and Thimet
produced a small increase in resorptions.
     This report was submitted in fulfillment of Contract No. 68-02-1751
by Stanford Research Institute under the Sponsorship of the U.S.
Environmental Protection Agency.
                                   v

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                               CONTENTS


FOREWORD	ill

PREFACE	    iv

ABSTRACT 	     v

LIST OF ILLUSTRATIONS	viii

LIST OF TABLES	    ix

ACKNOWLEDGEMENTS 	   xii

INTRODUCTION 	     1

MATERIALS AND METHODS	     3

     Animals 	     3
     Pesticides	     3
     Administration of Compounds 	     5
     Aerosol Generation	     5
     Chamber Atmosphere Analysis 	     8
     Particle-Size Analysis	     9
     Pesticide Analysis	     9
     Tissue Pesticide Analysis 	    10
     Pesticide Purification	    11
     Cholinesterase Assay	    11
     Animal Sacrifice	    12
     Pathology	    12

GENERAL TOXICOLOGY STUDIES - RESULTS 	    13

     Exposure Chambers 	    13
     Aerosol Particle Size	    15
     Purity of Pesticides	    15
     Acute Toxicity Determinations 	    15
     Pesticide Levels in Tissues 	    19
     Blood Cholinesterase Activity 	    22
     Pathology	    22

DISCUSSION	    24
     Aerosol Generation and Characterization 	    24
     Exposure Chambers 	    24
     Acute Toxicity Studies	    25
     Tissue Distribution 	    26
     Cholinesterase Inhibition 	    28
     Pathology	    28

                                   vi

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SUMMARY	29

FETOTOXICITY STUDIES - RESULTS AND DISCUSSION 	   30

     Chloroform	30
     Ethylene Thiourea	33
     Thimet	  .   33
     Bromacil	36
     Simazine	41

SUMMARY	46

RECOMMENDATIONS 	   47

REFERENCES	48
                                  vii

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                        ILLUSTRATIONS
The Structural Formulae, Trade Names, and the U.S.
Nomenclature of the Ten Chemicals Studied	
Arrangement of the Aerosol Generation and
Distribution System	     6

Arrangement of the Aerosol Exposure Equipment	     7
                            Vlll

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                                TABLES


 1   Conditions for Pesticide Analysis 	    9

 2   Dilution Ratios in the Exposure Chamber 	   13

 3   Concentrations of Various Pesticides Distributed
     to Four Inhalation Chambers Simultaneously by a
     Special Metered Dilutional Delivery System	14

 4   Particle Size Distribution of Aerosols Generated
     from Five Different Pesticides with an Ultrasonic
     Nebulizer	16

 5   Purity of Technical-Grade Pesticides as
     Determined by Gas Chromatography During the
     Inhalation Exposure Periods 	  	   16

 6   Acute Toxicity of Parathion,  Methyl Parathion,
     Thimet, Guthion, and Azodrin to Male and
     Female Rats When Given by Oral, Intravenous,
     Dermal, and Inhalation Routes 	   18

 7   Blood Plasma Levels at Various Time Intervals
     After Oral, Intravenous, Dermal, or Inhalation
     Administration	20

 8   Tissue Levels of Parathion or Methyl Parathion
     Found in Rat Liver After Oral or Intravenous
     Administration	21

 9   Cholinesterase Inhibition in the Whole Blood
     of Male or Female Rats One Hour After Treatment
     with Methyl Parathion, Guthion, or Azodrin	23

10   A Comparison of the Acute Intravenous LD50 and
     the Acute Inhalation LC50 of  Parathion, Methyl
     Parathion, Thimet, Guthion, and Azodrin in Male
     and Female Rats	,	27

11   Average Body Weights of Pregnant Rats Exposed
     to Chloroform Atmospheres for 1 Hour Daily
     During Days 7 Through 14 of Gestation	31

12   Average Daily Food Consumption of Pregnant Rats
     Exposed to Chloroform Atmospheres for 1 Hour
     Daily During Days 7 Through 14 of Gestation	31

                                   ix

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13   Litter Size, Resorptions, Live Fetuses,  and
     Fetal Weights at Day 20 of Gestation in Pregnant
     Rats Exposed to Chloroform Atmospheres for
     1 Hour Daily During Days 7 Through 14 of
     Gestation	32

14   Average Body Weights of Pregnant Rats Exposed
     to Ethylene Thiourea Aerosols for 3 Hours Daily
     During Days 7 Through 14 of Gestation	34

15   Average Daily Food Consumption of Pregnant Rats
     Exposed to Ethylene Thiourea Aerosols for
     3 Hours Daily During Days 7 Through 14 of
     Gestation	34

16   Litter Size, Resorptions, Live Fetuses,  and
     Fetal Weights at Day 20 of Gestation in Pregnant
     Rats Exposed to Ethylene Thiourea Aerosols for
     3 Hours Daily During Days 7 Through 14 of
     Gestation	35

17   Average Body Weights of Pregnant Rats Exposed
     to Thimet Aerosols for 1 Hour Daily During
     Days 7 Through 14 of Gestation	37

18   Average Daily Food Consumption of Pregnant Rats
     Exposed to Thimet Aerosols for 1 Hour Daily
     During Days 7 Through 14 of Gestation	37

19   Litter Size, Resorptions, Live Fetuses,  and
     Fetal Weights at Day 20 of Gestation in Pregnant
     Rats Exposed to Thimet Aerosols for 1 Hour
     Daily During Days 7 Through 14 of Gestation	38

20   Average Body Weights of Pregnant Rats Exposed
     to Bromacil Aerosols for 2 Hours Daily During
     Days 7 Through 14 of Gestation	39

21   Average Daily Food Consumption of Pregnant Rats
     Exposed to Bromacil Aerosols for 2 Hours Daily
     During Days 7 Through 14 of Gestation	39

22   Litter Size, Resorptions, Live Fetuses,  and
     Fetal Weights at Day 20 of Gestation in Pregnant
     Rats Exposed to Bromacil Aerosols for 2 Hours
     Daily During Days 7 Through 14 of Gestation	40

23   Average Body Weights of Pregnant Rats Exposed
     to Simazine Aerosols for 2 Hours Daily During
     Days 7 Through 14 of Gestation	42

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24   Average Daily Food Consumption of Pregnant Rats
     Exposed to Simazine Aerosols for 2 Hours Daily
     During Days 7 Through 14 of Gestation	42

25   Litter Size,  Resorptions, Live Fetuses,  and
     Fetal Weights at Day 20 of Gestation in  Pregnant
     Rats Exposed  to Simazine Aerosols for 2  Hours
     Daily During  Days 7 Through 14 of Gestation	43

26   Conversion of Daily Mean Aerosol Concentrations
     to Dosages in mg/kg	45
                                   XI

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                           ACKNOWLEDGMENTS

    Stanford Research Institute would like to acknowledge the following
persons for their excellent'professional support:  Charles Lapple,
assisted by Clyde Witham, in engineering design; Dr. Ronald Spanggord,
assisted by Elaine Shingai, in analytical chemistry; Dr. Daniel Sasmore,
assisted by Barbara Kirkhart, in pathology; Dr. Dale Coulson and
Elizabeth McCarthy in physical chemistry; and David Kay and Neal Winslow
in biology.
                                 xii

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                             INTRODUCTION

     The Environmental Protection Agency is conducting a thorough
investigation of insecticides to determine their effects on health
and the environment.  Through a review of the literature and laboratory
studies, the Agency anticipates that alternative chemicals for pest
control may be recommended.
     To support these aims, EPA commissioned the Toxicology Department
of SRI to assess the acute single-dose toxicity of potent organophosphate
pesticides preliminary to a comparative evaluation of candidate alterna-
tive or substitute agents and to study the fetotoxic potential of
specified pesticides or herbicides to animals after inhalation exposure.
In the first study, we compared the H>5o and LC5Q values of parathion,
methyl parathion, Thimet, Azodrin, and Guthion administered by the oral,
intravenous, percutaneous, and respiratory routes.  Also recorded were
pesticide blood concentrations after exposure and/or blood cholinesterase
values as well as gross and histopathologic changes.
     This study was divided into the following five phases:  (1) the
generation of respirable particles; (2) development of analytical
methods to determine the aero'sol concentration in the exposure chamber;
(3) acute toxicity study in rats; (4) analysis of animal tissue for
insecticide content; and (5) examination of gross and histopathological
changes of animal organs.
     The fetotoxicity studies were divided into three phases.  Pregnant
rats were subjected to:
     (1) Inhalation exposure to chloroform to reproduce results
         reported by Schwetz et al.1
     (2) Inhalation exposure to ethylene thiourea, a potent
         teratogen when given orally in high doses,2 to
         determine its teratogenicity when given by inhalation
         and to verify the methodology.

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     (3) Inhalation exposure to the insecticide Thimet and the
         herbicides Simazine and Bromacil to test their teratogenic
         potential.
     All inhalation exposures were conducted at SRI.  Fetuses were
shipped to the Project Monitor at Research Triangle Park, North Carolina,
for teratogenic evaluation.
     This report describes the inhalation exposures and the observed
effects on the rats and fetal changes in their offspring that had
occurred by day 20 of gestation, when the animals were sacrificed.

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                         MATERIALS AND METHODS

Animals
     Adult male and female Sprague-Dawley rats weighing between 200 and
250 g were obtained from Simonsen Laboratories, Gilroy, California.
The animals were housed in plastic cages with hardwood chip bedding
and were provided with Purina Laboratory Rat Chow and water ad_ libitum.
The animals were isolated upon arrival at the Laboratory and were
allowed to acclimatize before use.  All animals were observed closely
during this time to ensure that only healthy animals were used for the
subsequent studies.

Pesticides
     The pesticides selected for the general toxicity studies were
parathion, methyl parathion, Thimet, Guthion, and Azodrin.  Figure 1
presents their structural formulae, trade names, and chemical names.
All the pesticides were supplied as technical-grade material from the
Battelle Memorial Institute Repository, Columbus Laboratories, Columbus,
Ohio.
     The chemicals selected for the fetotoxicity study were chloroform,
ethylene thiourea, Thimet, Bromacil, and Simazine.  Chloroform (analytical
grade) and xylene were obtained from Mallinckrodt Chemical Works,
St. Louis, Missouri.  DMSO (dimethyl sulfoxide) was obtained from Crown
Zellerbach, Camas, Washington.  Ethylene thiourea (2-imidazolidinethione),
practical grade, was obtained from Matheson, Coleman and Bell, East
Rutherford, New Jersey.  Thimet, Bromacil, and Simazine were obtained
from the EPA pesticide repository at Battelle Memorial Institute,
Columbus, Ohio.

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                                                                                                          PT
                                                                                                          J     M/TH
         CH3fr  >=o
          Br
PAHATHION:  O.O-OIETHYLO-B-NIT3OPHENYLPHOSPHOROTHIOATE
                                                                                              BROMACIL; 5-8ROMO-3-SEC-BUTYL-8—METHYLURACILI8CII
           \L
METHYL PAHATHION: O. O-OIMETHYL O-O— NITRCPHENYL PHOSPHOROTHIOATE
              Ct

SIMAZINE.-6—CHLORO—N.N-DIETHYL-1.3.5-THIAZ1NE-2.4-DIAMINEOCI]

            H

     CH-	N
                                                                                                            \

                                                                                                          ./
                c —s
THIMET: O.O-OI ETHYL S-IETHYLTHIOI METHYL PHOSPHOROOITHIOATE
                                                                                               ETHYLENETHIOURgA
           M
GUTHION: O. 0 -DIMETHYLS - :4-OXO-I,2.3-aENZOTRlAZIN-3 (4H| -YLMSTHYL) PHCSPHOROOITHIOATS
                                                                                              CHLOROFORM
               CH3
 CH3   °\ j      I   H  O    H

         /P-O—C = C —C	 N	CHn
AZCORIN: CH-3-IOIMETHOXY PHOSPHINYLOXY)-N—VIETHYUCF!OTONAMIOS
                      FIGURE 1.  THE STRUCTURAL FORMULAE, TRADE NAMES, AND THE U.S. NOMENCLATURE OF THE
                                   NINE CHEMICALS STUDIED

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Administration of Compounds
     In the general toxicology studies, compounds were administered
intravenously via Ae tail vein, orally via an intragastric tube,
dermally on an area of the back that had been clipped free of hair,
or by inhalation.  Dilutions of compounds for intravenous, oral, or
dermal application xyere made with propylene glycol.  Technical-grade
compounds vere used for inhalation without being diluted.
     In the fetotoxicity studies, all compounds were administered daily
during day 7 through day 14 of gestation by inhalation using the
methodology, inhalation chambers., and equipment described herein.
Daily exposure durations were 1 hour for chloroform and Thimet, 2 hours
for Bromacil and Simazine, and 3 hours for ethylene thiourea.
Aerosol Generation
     Raspirable aerosols were generated with either an ultrasonic or
a pneumatic generator.  As the aerosols were generated, the particulates
were swept into a dilution chamber with intake air so that only a
narrow range of particle sizes were carried into the aerosol distribution
system.  Aerosol concentrations needed for each chamber were obtained
by regulating the exhaust and dilution air.  Figure 2 is a schematic
of the aerosol generation and distribution system.  As the aerosol
passed through the chamber, it was exhausted through a metering orifice
that controlled the ratio of the concentrations between chambers.
The exhaust was collected in a manifold and passed through a vacuum
pump that maintained a negative pressure of about 0.5 inches of water
in the chambers.  The exhaust passed through an absolute filter and
then through an incinerator, which burned at approximately 1100°C.
The exhaust was then routed through an exhaust ejector and out the
stack.  Figure 3 is a schematic diagram of the arrangement of the
entire exposure facility.
     Chloroform vapor was generated by passing a small part of the
chamber air intake over a chloroform surface in a 250-ml side-arm flask
that was temperature-stabilized with a water bath.  The air into the
flask was regulated with a critical orifice so that flows were kept
constant.
                                   5

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           TO CHAMBER 4
           TO CHAMBER 3
            TO CHAMBER 2
            TO CHAMBER 1
                                (VACUUM)
                               EXHAUST AIR
                                   t
                               GENERATOR
                                  AND
                                DILUTION
                                CHAMBER
                                                 DILUTION AIR
                                                 DILUTION AIR
•DILUTION AIR

• EXHAUST AIR
                                                 DILUTION AIR
                                                  INTAKE AIR
FIGURE 2   ARRANGEMENT OF  THE AEROSOL GENERATION AND DISTRIBUTION
           SYSTEM

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FIGURE 3   ARRANGEMENT OF THE AEROSOL EXPOSURE EQUIPMENT

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     Thimet aerosols were generated from a 1% solution of Thimet in
xylene, using a pneumatic aerosol generator.  The dilution of Thimet
was necessary because of its acute toxicity (11 mg/m3 for a 1-hour
exposure in female rats) and because we desired to have some survivors
after 8 days of daily exposure to the highest dose of the aerosols.
     Bromacil, Simazine, and ethylene thiourea aerosols were generated
with four DeVilbiss nebulizers operated at 15 psi.  Water was used
as the solvent for ethylene thiourea, but DMSO was used for both Bromacil
and Simazine.  We chose this method of generation because of a need
to obtain high concentrations of aerosol in the exposure chambers.
Chamber Atmosphere Analysis
     Chloroform was collected from the animal breathing area in the
chambers by means of polyethylene PE-50 tubing and transferred into a
1.0-ml gas-tight syringe.  This sample was injected directly into a
Varian Model 1520 gas chromatograph equipped with a flame ionization
detector.  The column used was a 3/16" by 48" glass column packed with
3% OV-17 on Gas Chrom Q 100-120 mesh.  Injector and column temperature
were ambient and N2 was the carrier gas.
     Samples of all the other compounds tested were collected on nucleo-
pore filters.  A gravimetric determination was made on all samples
with a Perkin-Elmer Model AD2 microbalance capable of weighing with an
accuracy of ± 1 microgram.  Gas chromatographic (GC) analysis of the
material eluted from the filter was made with the Varian Model 1520 GC.
For ethylene thiourea, we used a 3/16" by 48" glass column packed with
3% OV-17 on Gas Chrom WH; column temperature was 150°C and the retention
time was 150 seconds.  N2 was used as the carrier gas.  The detector
was a photometric detector equipped with a sulfur filter.
     The GC analysis for Thimet is described below.
     Bromacil and Simazine were analyzed by GC using a flame ionization
detector and a 3/16" by 48" glass column packed with 3% OV-17 on Gas
Chrom Q.  Methyl parathion was used as an internal standard for
Bromacil, which had a retention time of 255 seconds at a column tempera-

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ture of 210°C.  Thimet was used as an internal standard for Simazine,
which had a retention time of 755 seconds at the same column temperature.
Particle-Size Analysis
     The particle sizes of the aerosols generated for these studies
were detemined using a seven-stage cascade impactor.  The particle
sizes listed below are the mean and standard geometric deviation of
the aerodynamic size (in microns).
                              Aerodynamic          •  Standard Geometric
	Compound	              Mean (yi)               Deviation (ag)
Thimet                            0.44                      2.50
Simazine                          0.50                      2.4
Bromacil                          0.44                      2.2
Ethylene thiourea                 0.82                      2.9
Pesticide Analysis
     Pesticide analysis was performed with a Varian model 1520 gas
chromatograph equipped with a flame photometric detector.  The column
used was 2" x 1/4" glass packed with 3% OV-17 on 80/100 mesh Gas Chrom
Q.  Detector flow rates were 20 ml/min 62, 200 ml/min H2i and 50 ml/min
synthetic air.  The carrier gas used was nitrogen; the carrier gas
flow, the column temperature, and the solvent and internal standard
used were different for each pesticide studied,.as shown in Table 1.

                                Table 1
                   CONDITIONS FOR PESTICIDE ANALYSIS
Pesticide
Methyl parathion
Parathion

Thimet

Azodrin

Guthion
Solvent
Acetone
Acetone

Acetone

Acetone

Benzene
Column
Temp. (°C)
200
200

200

225

245
N2 Flow
(ml/min)
60
60

80

60

110
Internal
Standard
Parathion
Methyl
parathion
Methyl
parathion
Methyl
parathion
7 Co-Ral

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     Pesticide samples were collected from each chamber on preweighed
nucleopore filters.  The filters were reweighed at the end of the
exposure period to determine the total mass generated during the
exposure.  The pesticides were then eluted from the filter with a
solvent and analyzed by gas chromatography.  An Autolab 6300 digital
integrator was used for analysis of peak area and retention time.  The
amount of pesticide on each filter was determined by comparison with
an internal standard.
Tissue Pesticide Analysis
     Pesticide concentrations in plasma were determined according to
the method of Vukovich et al.   By this method, 1 ml of plasma is
mixed with 20 yl of concentrated hydrochloric acid and 1.5 ml of
n-hexane.  The mixture is shaken for 20 minutes on a mechanical shaker
and then centrifuged for 10 minutes to break up any emulsion formed.
An aliquot is then injected into the gas chromatograph.  Recoveries of
radiolabeled samples of methyl parathion, parathion, and Thimet were
96 to 106%, 88 to 104%, and 82 to 102%, respectively.  When radio-
labeled methyl parathion samples were refrigerated for 3 days at 4°C,
the recovery was 96 to 107%, indicating that refrigerated plasma
could be stored for up to 3 days without loss or destruction of the
compound.
     To determine pesticide concentrations in frozen liver, about 4 g
of tissue was placed in a Sorval Omni-mixer with an equal weight of
anhydrous sodium sulfate and 20 ml of 10% isopropanol in hexane.  The
sample was blended for 5 minutes and then the extract was decanted through
filter paper (Eaton-Dikeman, grade 512) into a Kuderna-Danish assembly
that contained 6 g of anhydrous sodium sulfate.  The extraction was
repeated twice, and the pooled sodium sulfate was rinsed with 10 ml
of 10% isopropanol in hexane.  All the extracts were then.concentrated
down to a volume of approximately 3 ml over a steam bath.
                                   10

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Pesticide Purification
     For parathion and methyl parathion, a column measuring 0.5" in
diameter was packed to a depth of 10 cm with 60/100 mesh Florisil
that had been maintained at 130°C for at least 24 hours.  One inch of
anhydrous sodium sulfate was added to the top of the column.  The
column was washed with 10 ml of hexane, leaving a minimal amount of
hexane above the sodium sulfate at the top of the column.  The pesticide
concentrate was quantitatively transferred to the column using two
2.5--ml volumes of hexane.  Elution of the column began using 15 ml
of 10% diethyl ether in hexane, followed by 15 ml of 50% diethyl ether
in hexane.  The eluant was collected in a Kuderna-Danish assembly
and concentrated in a steam bath to a few milliliters.  Final evaporation
to the desired volume was obtained using a gentle stream of nitrogen.
     The purification of Thimet was essentially the same as for the
parathions except that the Florisil column was packed to a depth
of 5 cm, and elution was accomplished using 15 ml of 10% diethyl ether
in hexane.
     The purification of Guthion and Azodrin was somewhat different.
A 0.5"-diameter column was packed with Florisil to a depth of 5 cm
and topped off with anhydrous sodium sulfate.  The column was washed
with 20 ml of hexane, and then 9 ml of hexane was added to 1 ml of
benzene extract of Guthion and put on the column.  The pesticide was
eluted using 100 ml of chloroform.  The eluant was steam-evaporated to
a small volume and then taken to dryness with a stream of nitrogen.
The residue was dissolved in 1 ml of acetone for gas chromatography.
The recoveries of Guthion and Azodrin averaged 94% and 65%, respectively.
Cholinesterase Assay
     Cholinesterase activity was determined by the Hesterin method,
which is a modification of the procedure of Fleisher and Pope.   In the
modified procedure, we used a 0.5-ml blood sample and a reagent blank
in place of a water blank for the spectrophotometric determinations.
The absorbance was read at 515 ym using a Beckman Model B spectro-
photometer.

                                   11

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Animal Sacrifice
     All animals in the fetotoxicity study were sacrificed on the
twentieth day of gestation by asphyxiation in a chamber filled with C02-
Each animal was examined for gross pathology, and the liver and gravid
uterus were removed and weighed.  The live fetuses then were removed
and weighed, the uterus was examined, and the number of resorptions
was noted.  After the litters were weighed, each was divided into two
equal groups of fetuses, one group fixed in Bouin's solution for
necropsy and the other fixed in 70% ethanol for eventual clearing,
staining, and skeletal analysis.
Pathology
     Tissues taken for histopathological examination were immediately
fixed in 10% neutral buffered formalin and later stained routinely
with eosin and hematoxylin.
                                   12

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                 GENERAL TOXICOLOGY STUDIES - RESULTS

Exposure Chambers
     Four exposure chambers, each of which can accommodate up to ten
rats, were assembled in a series so that a single aerosol generator
could provide exactly the same test material to each chamber.  The
aerosol concentrations in each chamber were altered by introducing
metered dilutional air into the induction system before its entry into
the inhalation chamber.  The dilution ratio was governed by a metering
orifice so that one of the ratios shown in Table 2 was theoretically
possible at a constant pressure.


                                Table 2
               DILUTION RATIOS IN THE EXPOSURE CHAMBERS
Dilution
Factor
/2
2
5

I
1
1
1

II
0.70
0.50
0.20

III
0.50
0.25
0.04

IV
0.35
0.12
0.008
   Orifice
Diameter (in)
  0.081
  0.052
  0.025
In practice, these ratios were achieved within a reasonable, expected
concentration.  Table 3 gives the concentrations achieved during some
of the inhalation exposures to each of the five pesticides used in
this study.
                                  13

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                                Table 3

  CONCENTRATIONS OF VARIOUS PESTICIDES DISTRIBUTED TO FOUR INHALATION
CHAMBERS SIMULTANEOUSLY BY A SPECIAL METERED DILUTIONAL DELIVERY SYSTEM
Dilution
Pesticide Ratio
Parathion
Methyl
parathion
Azodrin

Thim'et
Guthion
(20% in DMSO)
/2
/2 .
/2
2
2
2
5
/2
Actual
Expected
Actual
Expected
Actual
Expected
Actual
Expected
Actual
Actual
Actual
Actual
Expected
sLLCLlllU CJ-
I
193
214
447
500
308
300
740
710
129 •
114
266
250
214
V^Wllt- CL1 l_
II
142
151
302
353
210
211
321
355
59
53
28
155
150
JL a. L. _H_/LI;D
III
98
107
221
250
151
149
162
178
34
20
8
106
107
V'"&/ ill /
IV
63
75
143
176
97
104
90
89
12
10
70
75
                             Actual
86
24
13
                                   14

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Aerosol Particle Size
     Aerosols were generated with an ultrasonic generator and passed
through a cyclone separator and then into the distribution/dilution
system, which was regulated by a 0.081-inch ratio orifice.  The samples
were taken from Chamber IV through the Royco particle counter.
     All the aerosols were generated from neat technical-grade pesticides
except Guthion.  Guthion aerosols were generated from a 20% solution
in DMSO.  Table 4 shows the particle size distribution by count, the
count median diameter, and the standard geometric deviation.  The
particle sizes generated were well within the respirable range of
0.3 to 3.0 ym, and the standard geometric deviation indicates that
the distribution was not too heterodisperse for inhalation studies.
Purity of Pesticides
     All pesticides used for these studies were provided by Battelle
Memorial Institute, Columbus, Ohio.  They were all technical-grade
material.  The purity of each compound was determined by gas chromato-
graphic analysis after each inhalation exposure.  Table 5 presents the
results of these analyses.  Parathion, Azodrin, and Guthion analyses
were consistent and reproducible, whereas analyses of methyl parathion
and Thimet were variable.  No satisfactory explanation has been found
for this apparent discrepancy.
Acute Toxicity Determinations
     All the animals that received toxic or lethal doses of these
organophosphate pesticides exhibited the common signs of cholinergic
poisoning, regardless of the route of administration.  That is, all
exhibited salivation, lacrimation, exophthalmos, defecation, urination,
and muscle fasciculations.  The duration of the cholinergic signs was
dose dependent with each compound but was not comparable among the
several compounds tested.  For example, a sublethal dose of parathion
caused signs that lasted much longer than those produced by a
comparable sublethal dose of Azodrin.  Survivors of toxic doses of
all pesticides had recovered completely 10 to 14 days after dosing.
                                   15

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                                Table 4

         PARTICLE SIZE DISTRIBUTION OF AEROSOLS GENERATED FROM
        FIVE DIFFERENT PESTICIDES WITH AN ULTRASONIC NEBULIZER
                    Percentage of Particle Size Distribution (ym)2
Compound1
Parathion
Methyl
parathion
Guthion
Thimet
Azodrin
0.3-0
16.
13.
23.
23.
17.
.4
0
4
7
0
7
0.4-0
27.
25.
37.
34.
28.
.6
6
2
0
0
8
0.6-1
43.
50.
38.
35.
35.
.4
0
0
2
6
2
1.4-3.0
13
11
1.
7.
18
.3
.4
1
4
.1
>3.0
0.1
0
0
0
0.1
CMD3
0.68
0.71
0.54
0.57
0.69
ag4
1.8
1.7
1.5
1.8
1.9:
  Particles were generated from neat solutions except Guthion,  which was
  20% in DMSO.

2 Determined by the five stages of the.Royco Particle Counter.

  Count median diameter.
  Standard geometric deviation.
                                Table  5

          PURITY OF TECHNICAL GRADE PESTICIDES AS DETERMINED
     BY GAS CHROMATOGRAPHY DURING THE  INHALATION EXPOSURE PERIODS
                   Compound	      Range of Purity (%)

                Parathion                  88-94
                Methyl parathion           70-80
                Thimet                     78-90
                Azodrin                    61-64
                Guthion                    72-73
                                   16

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     Acute Oral Toxicity
     Groups of ten male or female rats were treated with varying single
doses of one pesticide by oral intubation.  The animals were observed
for the following 14 days for mortality and toxic signs.  Table 6
presents the LD50 and 95% confidence limits calculated for each
pesticide.  Thimet was the most toxic of the five pesticides studied,
with LD50s of 3.7 and 1.4 mg/kg in male and female rats, respectively.
Azodrin, with LD5QS of 35 and 20 mg/kg, respectively, for males and
females, was the least toxic.  Parathion, Thimet, and Azodrin were
more toxic to females than to males, whereas Guthion and methyl parathion
were about equitoxic to each sex.
     Acute Intravenous Toxicity
     Groups of ten male or female rats were treated with varying single
doses of one of five pesticides by intravenous injection through the
tail vein.  The animals were observed for 14 days after treatment for
toxic signs and mortality.  Table 6 presents the LD50 and 95% confidence
limits for each pesticide.  Thimet was the most toxic of the pesticides
by the intravenous route, having LDsgS of 2.2 and 1.2 mg/kg in male
and. female rats, respectively.  Although the difference in LD50s was.
very slight between the five pesticides, a small but significant
difference between the LD50s in males and females treated with Thimet'
and. Azodrin was noted.
     Acute Dermal Toxicity
     Groups of ten male or female rats were treated with varying
single doses of one of five pesticides by dermal application.  The
animals were observed during the treatment period for toxic signs
and mortality and for 14 days thereafter.  Table 6 presents the LD50
and. 95% confidence limits for each pesticide.  Thimet was the most
toxic of the compounds when given by dermal application, having LD^QS
of 9.3 and 3.9 mg/kg in male and female rats, respectively.  Parathion
was the next most toxic compound, with LD50s of 49.4 and 19.5 mg/kg
in males and females, respectively.  Guthion was the least toxic of
                                   17

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                                Table 6

   ACUTE TOXICITY OF PARATHION, METHYL PARATHION, THIMET, GUTHION,
AND AZODRIN TO MALE AND FEMALE RATS WHEN GIVEN BY ORAL, INTRAVENOUS,
                    DERMAL, AND INHALATION ROUTES

                95% Confidence Limits in Parentheses
                            LD50 (mg/kg) or LC50 (mg/m3)
Compound
Parathion
Male '
Female
Methyl parathion
Male
Female
Thimet
Male
Female
Guthidn
Male
Female
Azodrin
Male
Female
Oral

14
(11-19)
7.9
(6.3-9.8)

12
(8-16)
18
(14-24)

3.7
(2.6-5.3)
1.4
(0.8-2.5)

16
(13-19)
18
(14-22)

35
(30-40)
20
(17-23)
Intravenous

6.4
(5.0-8.0)
4.5
(3.1-6.6)

9.0
(6.6-11.3)
14.5
(5.0-41.9)

2.2
(1.9-2.6)
1.2
(0.8-1.6)

7.5
(5.1-11.1)
7.5
(5.8-9.8)

11.9
. (10.4-13.7)
9.2
(8.7-9.7)
Dermal

49.4
(39.8-61.2)
19.5
(17.0-22.4)

110
(91.8-131.8)
120
(80-180)

9.3
(7.9-11.0)
3.9
(3.4-4.4)

455
(301-687)
222
(181-271)

210
(104-269)
206
(104-407)
Inhalation

1070
(754-1519)
137
(125-151)

257
(188-352)
287
(243-339)

60
(52-69)
11
(7-15)

69
(62-77)
79
(68-93)

162
(142-185)
176
(159-195)
                                  18

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the pesticides in male rats, with an 11)50 °f ^55 mg/kg.  The LD,-ns
of Guthion in female rats and of Azodrin in male and female rats were
nearly identical, ranging from 206 to 222 mg/kg.  Differences between
sexes in tolerance to the toxicity of Thimet, parathion, and Guthion
were apparent, whereas no difference between sexes existed with Azodrin
and methyl parathion.
     Acute Inhalation Toxicity
     Groups of ten male or female rats were exposed for 1 hour to
atmospheres containing aerosols of one .of five pesticides.  The animals
were observed for toxic signs and mortality during exposure and for
14 days thereafter.  Chamber concentrations were verified during each
exposure period for each pesticide by the appropriate analytical
methodologies.  Table 6 presents the acute LC50S for a 1-hour exposure
of the pesticides.  Thimet was the most 'Coxic of the pesticides when
given by inhalation for 1 hour; the LCsgS were 60 and 11 mg/m3 in.male
and female rats, respectively.  Parathion was the least toxic in male
rats, having an LC5Q of 1070 mg/m3.  Methyl parathion was next, with
LC5QS of 257 and 287 mg/m3 in ma]e and female rats.  The LC^Q of methyl
parathion in female rats is in .excellent agreement with the value f.ound
in another laboratory several  years ago.6  Females were much more
susceptible than ma.l u ral's l:o toxic1 elfects of pnrathion and Th.imel .
No difference in tolerance helwe.en sexes was observed with methyl
pa.r.'ithlon, (iuMiLon, or A/.odrin.
Pesticide Levels in Ti .ssues
     We attempted to determine pesticide levels in blood plasma and
liver at various times after various doses of pesticide administered
by the intravenous, oral, dermal, or inhalation routes.  Tables 7 and
8 summarize these data.  The tables show that the amount of unchanged
pesticide in plasma and liver is extremely variable under similar
conditions of dose, time, and route of administration.  We had antici-
pated that,tissue levels of pesticide would correlate with mortality,
but,, in view of the consistently poor results obtained with these
analyses, we abandoned this phase of the study.

                                   19

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                                Table 7

     BLOOD PLASMA LEVELS OF PARATHION, METHYL PARATHION,  OR THIMET
      IN RATS AT VARIOUS TIME INTERVALS  AFTER ORAL,  INTRAVENOUS,
                 DERMAL, OR INHALATION ADMINISTRATION
Compound
Parathion
Methyl
  parathion
Thimet
Number
of Rats
   6
   5

   4
   7
   3
   5
   5

   5
   5
   3
   4
   3
   6
   9

   7
   5
   6
   5

   12
    8
    7
    6
    7

    5
    6
    6
    6
  Route of
Administration
Intravenous
Intravenous

Oral
Oral
Oral
Oral
Oral

Dermal
Dermal
Dermal
Dermal

Inhalation

Intravenous
Intravenous
Intravenous

Oral
Oral
Oral
Oral

Dermal

Inhalation

Intravenous
Intravenous
Intravenous
Intravenous

Oral
Oral
Oral
Oral

Inhalation

Dose
(mg/kg or
mg/m3)
2.0
5.1
6.0
10.0
11.0
13.5
16.5
50
50
50
50
346
8
8
10
6
8
8
13
85
152
1.0
1.6
2.0
2.5
1.0
2.7
3.7
5.0
12
Time
After
Dosing
(min)
30
60
30
30
60
60
60
120
240
360
24 hrs
60
5
60
20
30
30
120
30
—
155
30
30
30
30
30
60
60
60
60

Pesticide
Tissue Level
ppb (Range)
69 (15-166)
208 (60-330)
203 (14-287)
< 4
7.3 (3-20)
6.8 (4-12)
7.4 (3-11)
62 (38-90)
60 (30-79)
84 (76-102)
46 (35-56)
24 (18-46)
A (3-5)
88 (60-120)
3 (0.7-10.6)
13 (<4-27)
1.4 (0.9-2.2)
41 (1.6-201)
278 (61-518)
109 (59-246)
29 (15-60)
29 (6-114)
16 (6-30)
27 (7-49)
32 (7-49)
69 (23-92)
13 (7-22)
14 (5-33)
23 (5-41)
18 (3-38)
                                    20

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                                Table 8

            TISSUE LEVELS OF PARATHION OR METHYL PARATHION
      FOUND IN RAT LIVER AFTER ORAL OR INTRAVENOUS ADMINISTRATION
Compound
Number
of Rats
  Route of
Administration
 Dose
(mg/kg)
Time
After
Dosing
(min)
 Pesticide
Tissue Level
ppb (Range)
Parathion
          Intravenous
                    5.06
  60



Methyl
parathion






3
5
5
5
2
7
7
6
6
6
2
Oral
Oral
Oral
Intravenous
Intravenous
Intravenous
Oral
Oral
Oral
Oral
Oral
11.0
13.5
16.5
8.0
8.0
8.0
8.0
8.0
9.0
12.0
15.0
60
60
60
5
15
60
30
120
60
60
60
                    2.7 (<0.3-10.2)

                    10.5 (6.5-16.2)
                    21.9 (0.7-69.6)
                    11.8 (<0.3-37.6)

                    8.9 (1.1-29.0)
                    5.6 (2.9-8.2)
                    0.7 (<0.2-1.3)

                    11.9 (<0.3-46.7)
                    16.2 (<0.3-51.8)
                    12.9 (<0.3-34.4)
                    8.5 (1.6-26.5)
                    10.6 (7.6-13.5)
                                   21

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Blood Cholinesterase Activity
     Blood was taken from groups of animals 1 hour after treatment
with an approximately lethal dose of methyl parathion, Guthion, or
Azodrin by various routes of administration.  Table 9 summarizes the
results of these studies.  Blood cholinesterase inhibition was greatest
after Azodrin treatment by all routes of administration.  Slightly
less inhibition was seen after methyl parathion treatment.  Guthion
was not as inhibitory as Azodrin or methyl parathion; the greatest
inhibition obtained with Guthion was after inhalation exposure.  The
most surprising result was that little inhibition occurred with Guthion
even after intravenous administration.
Pathology
     No consistent pathological pattern was observed in animals given
lethal doses of individual pesticides, although the rats that received
methyl parathion and died within 1 hour of dosing exhibited hemorrhages
in the thymus and lungs as well as dilation of the cerebral blood
vessels.  Because of the consistent absence of characteristic gross
changes, only kidney, lung, brain, and skin of animals given the high-
dose level by all routes of administration were examined microscopically.
Of the 102 rats examined, no characteristic or recurrent histopathologic
changes were found.
     Lungs from animals that inhaled pesticide in the 1-hour
exposures were fixed in 10% neutral buffered formalin, sectioned, and
examined histologically.  Seventy-five lungs were examined, and no
specific recurrent changes were found.  If hemorrhage, congestion,
and edema are considered to represent pulmonary irritation, and if
attention is focused on hemorrhage, Azodrin was the least irritating,
since no hemorrhages were noted.  Methyl parathion was intermediate,
and the greatest irritation was observed with Thimet, Guthion, and
parathion.
                                   22

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                              Table 9

CHOLINESTERASE INHIBITION IN THE WHOLE BLOOD OF MALE OR FEMALE RATS
ONE HOUR AFTER TREATMENT WITH METHYL PARATHION, GUTHION, OR AZODRIN
Average
Percentage of
Cholinesterase
Route of
Compound Administration
Methyl Oral
parathion Intravenous
Dermal
Dermal
Dermal
Inhalation
Guthion Oral
Intravenous
Intravenous
Inhalation
Azodrin Oral
Oral
Intravenous
Intravenous
Inhalation
Inhalation

Sex
Male
Male
Male
Male
Female
Male
Female
Male
Female
Male
Male
Female
Male
Female
Male
Female
Dose
(mg/kg)
11.7
6.6
110
85
85
264
13
5.6
5.6
39
25
15
12
9
156
192
Inhibition
(range in
59
76
84
64
57
59
8
26
14
41
82
89
92
79
74
69
parentheses)
(52-73)
(67-81)
(78-93)
(50-73)1
(49-67) l
(53-61)
(7-20)
(9-38)
(7-19)
(27-59)
(63-89)
(83-94)
(91-93)
(65-90)
(73-77)
(62-68)
Blood taken six hours after treatment.
                                 23

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                              DISCUSSION

Aerosol Generation and Characterization
     The generation of a well-defined, compatible, and reproducible
aerosol is a critical requirement for attaining reliable and meaningful
inhalation toxicity data.  Such generation was achieved in these
studies by use of the ultrasonic generator.  The aerosol generated
was passed into a cyclone separator, and only the desired particle
sizes were routed into the inhalation chambers.  This was confirmed
by analysis of the aerosols of each of the five pesticides with the
Royco particle counter.  All the aerosols had a count median diameter
of 0.5 to 0.65 ym.  The respirable range is considered to be 0.3 to
3.0 urn.   In addition, the standard geometric deviation (ag) of less
than 2.0 indicates that, although the aerosols were heterodisperse,
nearly all the particles were well within the respirable size range.
Exposure Chambers
     A unique exposure chamber system was constructed for these studies.
It allowed simultaneous exposure of up to four groups of animals to
an aerosol from a single generator.  This was made possible by serial
dilution of the generated aerosol just before its entry into each
chamber.  The dilution system was regulated by a fixed orifice, which
gave a constant dilutional ratio between each chamber when operated
at a constant flow pressure.  By careful selection of ratioing orifices,
we could obtain an LC5Q for a compound in one sex of one species of
animals from a single 1-hour exposure.  However, a much more useful
application of this exposure system is in studies requiring repeated
exposures at multiple dose levels.  The researcher can easily duplicate
these exposures from day to day by merely setting the flow rates and
pressures to predetermined positions each day.
                                   24

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     Moreover, these exposure chambers provide nose-only exposure to
the aerosol.  This factor is important if the compound being studied
can easily be absorbed through the skin.  Nose-only exposure is also
important if the mechanism of action or the metabolism of a compound
is different when administered by different routes.
Acute Toxicity Studies
     Thimet was the most toxic of the five pesticides studied, regardless
of the route of administration.  It was also two to three times less
toxic to males than to females by all routes except inhalation, by
which route it was four times less toxic.  Parathion was less toxic
to males than to females when administered orally, dermally, or by
inhalation but not by intravenous injection.  Guthion was less toxic
to males only after dermal application, and Azodrin was less toxic to
males only by oral or intravenous administration.  No difference in
tolerance between sexes was noted with methyl parathion.
     Comparison of the intravenous, oral, and dermal toxicity of each
compound reveals that, on a mg/kg basis, the intravenous route was
most toxic, followed by the oral and then the dermal routes.  It is
difficult to compare the LC50 of the inhalation exposures with the
11)50 °f the intravenous dosing studies.  However, certain assumptions
can be made that permit a conversion of the LC$Q data from a mg/m3
to a mg/kg basis.  For example, we know that a 200-g rat has a
respiratory minute volume of about 80 ml/min.  Therefore, a 200-g rat
will breathe about 4,800 ml in 60 minutes, or a total of 4.8 liters
of the test atmosphere.  A reasonable assumption is that about 20%
of an aerosol with a particle size range of 0.3 to 3.0 ym will be
deposited in the bronchial or alveolar area of the lung.7  The following
relationship is then suggested:
     LC5Q x respired fraction of LC^Q x body weight (kg)
          x percentage of deposition = LD50 , or
     LC50 (mg/m3) x 4.8 liters/1000 liters x 200 g/1000 g
          x 0.20 = LD50 in mg/kg .
                                   25

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     Calculation of the LD5g for each pesticide based on the above
relationship would indicate that, with the exception of parathion in
male rats, the inhalation route of pesticide administration was
7 to 24 times more toxic than intravenous dosing, as suggested by
the data presented in Table 10.
     There are two possible reasons for the greater inhalation toxicity.
First, material entering the body via the lung enters the left heart
and then is distributed throughout the tissues before it reaches the
liver, where the major detoxifying systems are located.  Therefore,
primary target organs and/or tissues might receive a relatively greater
portion of a given dose of pesticide by inhalation than by the oral
or intravenous routes, by which the dose would reach the liver first.
Second, the toxicity of organothiophosphates is greatly increased
when the organothiophosphates are converted to their oxygen analogs.
Neal et al.7»8 have shown that rabbit lung, in vitro, can convert
parathion to paraoxon, its oxygen analog; rabbit lung also can slowly
detoxify paraoxon to _p_-nitrophenol, although the lung contains only
about 3% of the enzyme activity found in the liver.
     This entire metabolic process can be altered (induced) by a
number of compounds including DDT, barbiturates, and steroid hormones
(particularly testosterone), which probably accounts for the differences
in tolerance between sexes seen with parathion and Thimet.
Tissue Distribution
     The attempt to recover unchanged parathion, methyl parathion,
and Thimet from plasma or liver after dosing by various routes of
administration was unsuccessful.  Two possible reasons for this are:
(1) Because the compounds tested are rapidly converted to their oxygen
analogs by plasma and particularly liver enzymes, they would not be
detected; and (2) these compounds are firmly bound not only to
acetylcholinesterases but also to other nonspecific esterases.  They
probably could not be removed from their bound esterases without
taking measures that would cause further hydrolysis of the pesticide
molecule.  Perhaps the only way to measure plasma half-life or liver

                                   26

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                              Table 10

             A COMPARISON OF THE ACUTE INTRAVENOUS LD50
    AND THE ACUTE INHALATION LC50 OF PARATHION, METHYL PARATHION,
        THIMET, GUTHION, AND AZODRIN IN MALE AND FEMALE RATS
Compound
          Intravenous
Sex       LD50(mg/kg)
            Calculated
            Inhalation
            LD50(mg/kg)
           Intravenous LD5Q/
           Inhalation
Parathion
Methyl
  parathion

Thimet
Guthion
Azodrin
Male
Female

Male
Female

Male
Female

Male
Female

Male
Female
 6.4
 4.5

 9.0
14.5

 2.2
 1.2

 7.5
 7.5

11.9
 9.2
5.14
0.66

1.23
1.38

0.29
0.05

0.33
0.38

0.78
0.85
 1.2
 6.8

 7.3
10.5

 7.6
24

23
20

15
10.8
                                  27

-------
content with these compounds is to use radioactive material such as a
double label with 35S and 32P isotopes or an uniformly labeled 11+C
compound.
Cholinesterase Inhibition
     Clearly death of animals treated with methyl parathion, Guthion,
or Azodrin was not correlated to the degree of cholinesterase inhibition
in the blood.  Although a moderate to great cholinesterase inhibition
occurred in rats treated with lethal doses of parathion and Azodrin,
very little inhibition was seen in the Guthion-treated animals except
by inhalation.  However, this may reflect the fact that the compounds
have different mechanisms of producing mortality as well as a similar
action of cholinesterase inhibition.  Because whole blood contains
both true and pseudo cholinesterases, a better correlation might have
been found if one or the other had been measured alone.  Another
possibility is that the pesticides might all produce a significant
cholinesterase inhibition in a critical location, the central nervous
system for example, to produce mortality by a common mode of action.
Pathology
     No significant gross or histopathological lesions attributable
to pesticide treatment were found in any of the animals examined.
These observations, therefore, support the position that the primary
hazard from treatment with these five pesticides is a biochemical
lesion.
                                   28

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                                SUMMARY

     A method was developed for generating pesticide aerosols 'in the
respirable particle size range of 0.3 to 3.0 vim.  Micrqanalytical
techniques also were developed for determining pesticide concentrations
of parathion, methyl parathion, Thimet, Guthion, and Azodrin by gas
chromatography.  Methods were developed for extracting these five
pesticides from rat blood and liver.
     A unique inhalation exposure chamber system was constructed that
permits the simultaneous exposure of up to four groups of animals to
four different predetermined concentrations of an aerosol from a
single generator source.
     In male and female rats, the acute oral, dermal, and intravenous
LD5Q and the acute inhalation LC50 were determined and compared for
parathion, methyl parathion, Thimet, Guthion, and Azodrin.  The
inhalation route of administration of all five pesticides appeared to
be the most toxic, followed by the intravenous and oral routes.  Thimet
was the most toxic of the five compounds by all routes of administration.
Females were more susceptible to Thimet and parathion than males.
There was no correlation between the tissue levels of pesticide and
the dose or the route of administration, nor was there correlation
between the inhibition of cholinesterase activity in blood and mortality.
     No significant pathological lesions that could be attributed to
treatment developed in the animals treated with any of the five
pesticides.
                                   29

-------
             FETOTOXICITY STUDIES - RESULTS AND DISCUSSION

Chloroform
     All the animals exposed to 20.1 ± 1.2 mg/1 (the highest level)' fell
asleep within a few minutes after initiation of exposure and continued
to sleep throughout the exposure period.  Some of the animals exposed to
10.9 ± 1.0 mg/1 (the mod-dose) of chloroform occasionally appeared to be
asleep.  All animals in the low-level exposure chamber and the control
chamber responded readily to light tapping on the chamber wall or to a
bright light stimulus.  One animal receiving the highest level of chloro-
form died during the night of the twelfth day of gestation.  At autopsy,
autolysis precluded any determination of resorptions at the nine
implantation sites.
     During the exposure period, the average body weights of the animals
receiving the high dose of chloroform decreased, whereas the weights of
those receiving the low dose did not change.  During the same period, the
control rats gained an average of 43 grams.  Table 11 summarizes the
body weight data.
     Food consumption was reduced in all the treated groups during the
chloroform exposure, and this response seemed to be dose-related.  Food
consumption returned to normal on the day chloroform exposures were
completed.  These data are summarized in Table 12.
     At autopsy, no gross pathologic lesions were noted that could be
attributed to the treatment.  Table 13 lists the litter sizes, number of
live and dead/resorbed fetuses, fetal weights, and the total number of
pregnancies per treatment group.  The number of resorptions increased in
the highest treatment group and in the restricted-food controls.  The
average fetal weights were somewhat less in the treated groups compared
with the air controls.  The overall fetal development of the restricted-
food controls were significantly less than those of the air controls.
No treatment-related changes in fetal ossification or occurrence of super-
numerary ribs were noted.  No gross defects were seen in any dose group.
                                  30

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                               Table 11

      AVERAGE BODY WEIGHTS OF PREGNANT RATS EXPOSED TO CHLOROFORM
  ATMOSPHERES FOR 1 HOUR DAILY DURING DAYS 7 THROUGH 14 OF GESTATION
                     Average Body Weightst During Days of Gestation
Treatment Group*         1           6           15           20

20.1 ± 1.2 mg/1       202 ±8     221 ± 7     192 ± 25*    252 ± 41
10.9 ± 1.7 mg/1       208 ±6     233 ± 9     213 ± 16     280 ± 25
4.6 ± 1.0 mg/1        203 ±7     223 ± 6     224 ± 12     280 ± 32
Air controls          209 ±7     231 ± 8     275 ± 12     335 ± 13
Restricted food
  controls            208 ±5     230 ± 6     195 ± 13     269 ± 32
* Ten animals per treatment group.
t In grams ± the standard error.
* Nine animals per group.
                               Table 12

 AVERAGE DAILY FOOD CONSUMPTION OF PREGNANT RATS EXPOSED TO CHLOROFORM
  ATMOSPHERES FOR 1 HOUR DAILY DURING DAYS 7 THROUGH 14 OF GESTATION

                        Average Daily Food Consumption (g/rat)
                               During Days of Gestation

Treatment Group*            1-7           8-14         15-20

20.1 ± 1.2 mg/1            16.0            5.8t         21.Ot

10.9 ± 1.7 mg/1            16.6            9.2          21.0

4.6 ± 1.0 mg/1             15.4           11.6          24.6

Air controls               16.4           19.8          24.2

Restricted food
  controls                 16.0            5.8          21.0
* Ten animals per group.
t Nine animals per group
                                   31

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                                                  Table 13

                          LITTER SIZE,  RESORPTIONS,  LIVE FETUSES,  AND FETAL WEIGHTS

                  AT DAY 20 OF GESTATION IN PREGNANT RATS EXPOSED  TO CHLOROFORM ATMOSPHERES

                           FOR 1 HOUR DAILY DURING DAYS 7 THROUGH  14 OF GESTATION




                                 Control   4.6 ± 1.0 mg/m5   10.9  ± 1.7 mg/m5   20.1 ± 1.2 mg/m5     RFCb
.w
K>
No. pregnant (term)
Av. implants
Av. mortality (%)
Av. weight (g)
Av. no. sternal ossification
centers
Av. no. caudal ossification
centers
Supernumerary ribs (%)
10
10.6
10
4.0

6.0

4.7
19
+ 0.3
± 8
± 0.1

± 0.1

± 0.1
± 10
9
9.9 ±
3 ±
3.6 ±

5.9 ±

4.3 ±
10 ±
10
0.8
2
0.2

0.1

0.4
6
10.6
16
3.9

6.0

5.0
14
± 0.5
± 6
± 0.1

± 0.1

± 0.2
± 8
8
10.0
45
3.7

5.9

4.5
17
+
+
+

+

+
+
0.8
21a
o.ia

0.1 '

0.2
12
10
12.5
32
3.0

5^.5

3.3
5
± 0.6
+ 14
± O.la

± 0.2

± 0.2a
± 3
    ap<0.05

    ^Restricted Food Controls

-------
Ethylene Thiourea
     None of the animals exposed to ethylene thiourea exhibited any
toxic signs during the daily inhalation exposure periods.  However, as
shown in Table 14, those animals exposed to 120.4 mg/m3 (the highest
dose) appeared to have a slight decrease in weight gain, as compared
with the other dosage groups.  Statistically, they were not different
from the controls.  Food consumption also was less in the highest
group, as shown in Table 15.  However, one animal was not pregnant, and
another had the total litter resorbed, and this may account for the
observed difference.
     At autopsy, no lesions were observed that could be attributed to
the treatment.
     Table 16 lists the litter sizes, number of live and dead fetuses,
fetal weights, and the total number of pregnancies per treatment group.
The number of resorptions was highest in the group receiving 120 mg/m3
(the highest treatment group) of ethylene thiourea.  The number of
resorptions decreased as the dose decreased and was least in the air
controls.  There was a significant (p<0.01) dose-related reduction in
fetal weight and degree of ossification in the treated groups.  The
fetal weights  of the restricted food controls was significantly less
than the air controls—3.4 ± 0.2 g versus 4.1 ± 0.2 g.  No terata were
observed in any of the treatment groups.

Thimet
     The animals exposed to 1.94 mg/m3 (the highest concentration) of
Thimet exhibited toxic signs and mortality during the eight daily
exposures.  All the animals exhibited tremors, lacrimation, and
exophthalmos.   A total of five animals died--one after the third,
fourth, sixth, seventh, and eighth exposures, respectively.  Two rats
that died had bloody material in their intestines and bladder.  All
the animals that died were pregnant, but the one that died after the
eighth exposure appeared to be resorbing her entire litter.
                                  f3

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                               Table 14

  AVERAGE BODY WEIGHTS OF PREGNANT RATS EXPOSED TO ETHYLENE THIOUREA
   AEROSOLS FOR 3 HOURS DAILY DURING DAYS 7 THROUGH 14 OF GESTATION.


   1                        Average Body Weightst During
                                 Days of Gestation
Treatment
120'.
55.5
27.2
Air
4 + 8.
+ 5.5
± 3.1
Group*
0 mg/m3t
mg/m
mg/m
3
3
controls
Restricted
food

1
224
226
225
224

± 6
± 8
± 8
± 7

6
246
250
248
248

± 8
± 1
± 8
± 9

15
278
289
285
283

± 9
± 18
± 8
± 15

20
336
349
347
349

± 17
± 17
± 17
± 23

  controls          218 ±6     240 ± 12    262 ± 14§   323 ± 17 §
* Ten animals per group unless otherwise indicated.
t In grams ± the standard error.
$ Eight animals per group.
§ Nine animals per group.
                               Table 15

            AVERAGE DAILY FOOD CONSUMPTION OF PREGNANT RATS
              EXPOSED TO ETHYLENE THIOUREA AEROSOLS FOR
          3 HOURS DAILY DURING DAYS 7 THROUGH 14 OF GESTATION


                         Average Daily Food Consumption (g/rat)
                                During Days of Gestation
Treatment Group*          1 to 7        8 to 14        15 to 20

120.4 ± 8.0 mg/m3          16.8          17.5            20.9
55.5 ± 5.5 mg/m3           18.8          20.8            24.4
27.2 ± 3.1 mg/m3           17.7          19.2            25.2
Air controls               18.3          19.3            24.3
Restricted food
  controls                 16.8          17.5            20.9
* Ten animals per group.
                                   34

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                                                  Table 16

                         LITTER SIZE, RESORPTION,S LIVE FETUSES, AND FETAL WEIGHTS
               AT DAY 20 OF GESTATION IN PREGNANT RATS EXPOSED TO ETHYLENE THIOUREA AEROSOLS
                          FOR 3 HOURS DAILY DURING DAYS 7 THROUGH 14 OF GESTATION
    No. term pregnant

    Av. no.  implants

    Av. mortality (%)

    Av. weight (g)

    Av. no.  sternal ossification
^     centers
in
    Av. no.  caudal ossification
      centers

    Av. supernumerary ribs (%)
Control
10
11
2

4
6
5

.5
.8

.1
.0
.4
20
± 0.3
± 2.0

± 0.1
± 0.1
± 0.2
± 8
27.2 ± 3.
10
11.1 ±
4.5 ±

4.2 ±
6.0 ±
4.8 ±
13 ±
, 1 mg/m3

0.5
2.3

0.1
0.1
0.2a
4
55.5 ± 5.
10
11.5 ±
7.6 ±

4.0 ±
6.0 ±
5.0 ±
29 ±
,5 mg/m

0.4
3.2

0.1
0.0
0.1
11
120.4 ±8.0 mg/m3 RFCd

10.8
19.1

3.7
5.8
3.9
16
9 9
± 0.6 12.3 ± 0.4
± 10. 5a 6.1 ± 2.8
Vi >>
± 0.1° 3.4 ± 0.1°
± 0.1
± 0.1C
±5
    ap<0.05
    bp<0.01
    cp<0.001
    ^Restricted Food Controls

-------
     Table 17 presents the average body weights of pregnant rats exposed
to Thimet aerosols.  No differences in weight gain were noted that could
not be accounted for on the basis of the number of pregnancies in each
group, except for an unexplained weight loss in the restricted-food
controls on the fifteenth day of gestation.
     Food consumption, presented in Table 18, was not different among
any of the groups, considering the number of pregnancies in each group.
     Table 19 lists the litter size, number of live and dead/resorbed
fetuses, fetal weights, and number of pregnancies in each group.  The
highest dose of Thimet produced the highest fetal mortality as well as
maternal mortality.  Also, the average fetal weight at the highest dose
seemed to be slightly greater than the other groups.  No other fetal
effects were seen.  These observations were not the result of restricted
food intake or solvent (xylene) toxicity.

Bromacil
     No toxic signs were noted in any of the animals exposed to Bromacil
or to the DMSO solvent during the 8 days of treatment.   Table 20 presents
the body weights of all the animals in this study.   There is no difference
in the weight gain of any of the groups that cannot be explained on the
basis of nonpregnant animals in the group.
     The food consumption, presented in Table 21, reflects the weight
gains, and there is no difference among any of the groups.  No
restricted-food controls were included in the Bromacil study.
     Table 22 lists the litter size, number of live and dead/resorbed
fetuses, average fetal weight, and the number of pregnancies in each
group.  The group receiving 165 mg/m3 (highest dose) of Bromacil
appeared to have a slightly higher percentage of resorptions, although
the air controls and DMSO controls have a slightly higher resorption
rate than some of our previous control groups.  Significant (p<0.01)
dose-related reductions in fetal weight and caudal ossification were seen
in treated groups.   No gross pathology was noted in any of the groups.
                                  36

-------
                               Table 17
        AVERAGE BODY WEIGHTS OF PREGNANT RATS EXPOSED TO THIMET
    AEROSOLS FOR 1 HOUR DAILY DURING DAYS 7 THROUGH 14 OF GESTATION
                     Average Body Weightst During Days of Gestation
Treatment Group*          1           6          15          20
1.94 ± 0.48 mg/m3      214 ±5     240 ± 5     278 ± 12t   311 ± 19+
0.40 ± 0.15 mg/m3      216 ± 5     23.7 ±6     268 ± 7     337 ± 13
0.15 ± 0.04 mg/m3      214 ±5     235 ± 6     271 ± 7     333 ± 12
Restricted food
  controls             227 ±2     245 ± 3     236 ± 6     302 ± 10
Air controls           210 ±5     231 ± 5     257 ± 11    311 ± 18
Xylene controls           	      219 ± 3     254 ± 6     300 ± 16
* Ten animals per group.
t In grams ± the standard error.
t Five animals died during the inhalation exposure.
                               Table 18
   AVERAGE DAILY FOOD CONSUMPTION OF PREGNANT RATS EXPOSED TO THIMET
    AEROSOLS FOR 1 HOUR DAILY DURING DAYS 7 THROUGH 14 OF GESTATION
                          Average Daily Food Consumption (g/rat)
                                 During Days of Gestation
1-7
19.0
19.2
18.2
17.3
17.3
18.4
8-14
22t
20.1
19.7
17.5
18.1
19.2
15-20
26. 4t
24.4
23.1
—
20.8
22.1
Treatment Group*
1.94 ± 0.48 mg/m3
0.40 ± 0.15 mg/m3
0.15 ± 0.04 mg/m3
Restricted food
  controls
Air controls
Xylene controls
* Ten animals per group unless otherwise indicated,
t Five animals per group.
                                   37

-------
OJ
00
                                                   Table 19
                           LITTER SIZE, RESORPTIONS, LIVE FETUSES, AND FETAL WEIGHTS
                      AT DAY 20 OF GESTATION IN PREGNANT RATS EXPOSED TO THIMET AEROSOLS
                            FOR 1 HOUR DAILY DURING DAYS 7 THROUGH 14 OF GESTATION
No
Av
Av
Av
Av
Av
. pregnant (term)
. implants
. mortality
. weight (g)
10.
(%) 3.
3.
. no. sternal ossification
centers 6.
. no . caudal
centers
Supernumerary
ossification
4.
ribs (%) 9.
Control
15
1 ±0.7
8 ±1.6
8 ±0.1
0 ±0.0
4 ±0.1
9±5.7
0.15 ±

10.0
5.7
3.9
5.9
4.5
12.2
0.04 mg/m3 0.40 ±0.15 mg/m3 1.94 ±0.48 mg/m
10
+ 1.1
± 2,2
± 0.1
± 0.1
± 0.2
± 5.2
9
11.7 ±
3.6 ±
3.8 ±
5,9 ±
4.3 ±
5.9±

0.2
1.9
0.1
0.1
0.2
3.0

10.2
30.8
4.1
5.8
4.3
3.4
5e
± 2.1
±18.7
± 0.2a
± 0.2
± 0.4
± 7.6
3 RFCd
8
9.5 ±1.1
6.6±2.5
3.7 ±0.1
6.0 ±0.0
4.4 ±0.2
13.5 ±9.6
Xylene
7
11.6 + 1.1
7.4 ±3.2
3.9± 0.1
6.0 + 0.0
4.4 ±0.2
0
   ap  0.05
   bp  0.01
   Cp  0.001
   Restricted Food Controls
   eFive additional rats died during the exposures to Thimet.  All rats were pregnant, .but  they
    were not evaluated for live or dead fetuses.

-------
                               Table 20
       AVERAGE BODY WEIGHTS OF PREGNANT RATS EXPOSED TO BROMACIL
   AEROSOLS FOR 2 HOURS DAILY DURING DAYS 7 THROUGH 14 OF'GESTATION
                     Average Body Weightst During Days of Gestation
Treatment Group*          1           6          15          20
165 ± 6 mg/m3          195 ± 16    219 ± 17    251 ± 24    307 ± 41
78 ± 3 mg/m3           206 ± 15    227 ± 13    253 ± 19    305 ± 37
38 ± 2 mg/m3           200 ± 11    222 ± 10    258 ± 14    316 ± 19
DMSO controls          202 ± 13    227 ± 13   . 262 ± 15    318 ± 30
Air controls           198 ±7     227 ± 11    265 ± 13    326 ± 23
* Ten animals per group.
t In grams ± the standard error.
                               Table 21
  AVERAGE DAILY FOOD CONSUMPTION OF PREGNANT RATS EXPOSED TO BROMACIL
   AEROSOLS FOR 2 HOURS DAILY DURING DAYS 7 THROUGH 14 OF GESTATION

                        Average Daily Food Consumption (g/rat)
                               During Days of Gestation
Treatment Group*
165 ± 6 mg/m3
78 ± 3 mg/m3
38 ± 2 mg/m3
DMSO controls
Air controls
1-7
16.9
17.8
17.3
18.8
19.2
8-14
19.7
19.5
20.0
15.5
16.4
15-20
22.9
22.5
22.9
24.2
24.0
* Ten animals per group.
                                   39

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                                              Table 22
                      LITTER SIZE, RESORPTIONS, LIVE FETUSES, AND  FETAL WEIGHTS
                AT DAY 20 OF GESTATION IN PREGNANT RATS EXPOSED TO BROMACIL AEROSOLS
                       FOR 2 HOURS DAILY DURING DAYS 7 THROUGH 14  OF  GESTATION
No. pregnant (term)

Av. implants

Av. mortality (%)

Av. weight (g)

Av. no. sternal ossification
  centers

Av. no.  caudal ossification
  centers

Supernumerary ribs (%)
Control
38 ± 2 mg/m3
19
10.8
8.9
4.3
6.0
5.2
20.2
± 0.
± 3.
± 0.
± 0.
± 0.
± 4.
4
1
04
02
2
4
11.
4.
4.
6.
4.
2.
10
2 ±
7 ±
1 ±
0 ±
5 ±
0 ±

0.5
3.0
0.09a
0.0
0.2b
2.0
78 ± 3 mg/m3

10.
5.
3.
5.
4.
13.
8
8 ±
3 ±
9 ±
9 ±
8 ±
3 ±

0.8
2.2
0.07b
0.03
o.ia
6.5
165

10.
7.
4.
6.
4.
20.
± 6
9
9 ±
6 ±
0 ±
0 ±
7 ±
0 ±
DMSO Air
mg/m Control Control
9 10
0.7 10.9 10.7
3.7 9.2 9.4
0.09a 4.3 ± 0.2 4.3 ± 0.3
0.03
0.2a
8.8
ap<0.05
bp<0.01

-------
Simazine
     Animals exposed to the highest dose of Simazine usually did not
appear as active after each inhalation exposure as those in the other
groups.  However, they all appeared normal on the day after exposure
when they were being prepared for the next inhalation treatment.  The
average body weights, shown in Table 23, and the food consumption of
the animals, shown in Table 24, do not indicate any differences among
the treated and the control groups.  No gross pathology was noted at
the time of sacrifice.
     Table 25 lists the litter size, number of live and dead/resorbed
fetuses, average fetal weight, and number of pregnancies in each group.
There were no differences in the number of resorptions among the
groups.  The fetal weight and degree of caudal ossification were less in
the treated groups compared to the controls.  No treatment-relative
terata were noted in any group.
     SRI's chloroform study was initiated to reproduce the results of
Schwetz et al.1  Those investigators exposed animals to 30, 100, and
300 ppm chloroform for 7 hr/day, whereas our exposures were much
higher--950, 220, and 4100 ppm

                    /mg/m3   (24.450)
                    \-LUUU    ^ 211V/ J. W If J         I

However, our exposure time was reduced to 1 hour daily to reduce the
stress on the animals.  The results obtained with our highest dose of
4100 ppm for 1 hr/day are similar to those obtained by Schwetz et al.1
at 300 ppm for 7 hr/day.  The percentage of resorptions observed by
Schwetz et al. was 61%, and ours was 44%.  Their average live litter
size was 4.7, whereas ours was 5.6.  Thus, we essentially have repeated
their experimental results using different exposure times and
chloroform concentrations.
                                  41

-------
                               Table 23
       AVERAGE BODY WEIGHTS OF PREGNANT RATS EXPOSED TO SIMAZINE
   AEROSOLS FOR 2 HOURS DAILY, DURING DAYS 7 THROUGH 14 OF GESTATION
                       Average Body Weightst During Days of Gestation
Treatment Group*          1           6           15           20
317 ± 89 mg/m3         182 ± 7     211 ± 8     238 ± 13     309 ± 31
77 ± 56 mg/m3          183 ± 9     212 ±9     239 ± 11     316 ± 22
17 ± 12 mg/m3  .        186 ± 7     217 ±6     241 ± 14     316 ± 22
DMSO controls          202 ± 13    227 ± 13    262 ± 15     318 ± 30
Air controls           198 ±7     227 ± 11    265 ± 13     326 ± 23
* Ten animals per group.
t In grams ± the standard error.
                               Table 24
  AVERAGE DAILY FOOD CONSUMPTION OF PREGNANT RATS EXPOSED TO SIMAZINE
   AEROSOLS FOR 2 HOURS DAILY DURING DAYS 7 THROUGH 14 OF GESTATION
                           Average Daily Food Consumption (g/rat)
                                  During Days of Gestation
Treatment Group*
317 ± 89 mg/m3
77 ± 56 mg/m3
17 ± 12 mg/m3
DMSO controls
Air controls
1-7
12.4
12.5
12.5
18.8
19.2
8-14
19.2
19.1
19.2
15.5
16.4
15-20
24.5
24.8
23.9
24.2
24.0
* Ten animals per group.
                                   42

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                                              Table 25

                      LITTER SIZE, RESORFTIONS, LIVE FETUSES, AND FETAL WEIGHTS
                AT DAY 20 OF GESTATION IN PREGNANT RATS EXPOSED TO SIMAZINE AEROSOLS
                       FOR 2 HOURS DAILY DURING DAYS 7 THROUGH 14 OF GESTATION
No. pregnant (term)

Av. implants

Av. mortality (%)

Av. weight (g)

Av. no. sternal ossification
  centers

Av. no. caudal ossification
  centers

Supernumerary ribs
Control
19
10.8
8.9
4.3
n
6.0
5.2
20.2
± 0.
± 3.
± 0.
± 0.
± 0.
± 4.
4
1
04
02
2
4
17 ±
•z
.12 mg/m
77
10
10.6
15.3
3.8
5.9
4.5
4.1
± 1.
± 9.
± 0.
± 0.
± 0.
± 2.
0
7
06b
1
2a
7
10.
8.
4.
6.
4.
11.
± 56 mg/m3
10
8 ±
7 ±
0 ±
0 ±
8 ±
7 ±

0.
3,
0.
0.
0.
6.

7
6
09a
01
2
6
317

11
8
3
5
4
25
DMSO Air
± 89 mg/m3 Control Control
9
.3 ±
.3 ±
.7 ±
.9 ±
.4 ±
.6 ±
9 10
0.5 10.9 10.7
3,4 9.2 9.4
0.06b 4.3 ± 0.2 4.3 ± 0.3
0.03
0.2b
8.8
ap<0.01

bp<0.001

-------
     Ethylene thiourea exposures were established to reproduce the
results of Khera,2 who orally dosed rats and rabbits with 5, 10, 20,
4.0, and 80 mg/kg, respectively.  To compare Khera1 s data with those
from our inhalation studies, we converted our data to an mg/kg basis.
Assuming that a 200-g rat has a respiratory volume of 80 ml/minute,11
then in 60 minutes the rat will have breathed 4800 ml, or 4.8 liters,
of air.  If we assume 100% absorption/ deposition of material in the
respiratory tract, we can then make the following estimate of the
daily dose:

     Aerosol concentration (mg/m3) 80 nl/min x exposure time (min)


                          =   daily dose in
                 t   g)

Table 26 shows the conversion of all the aerosol concentrations to
mg/kg.  On this basis, our highest possible dose of ethylene thiourea
was 8.67 mg/kg.  Khera found only a decrease in mean fetal weight in
the 80-mg/kg treatment group, whereas we found a slight decrease in
fetal weight plus a slight increase in fetal mortality in our highest
dose group (120.4 mg/m , or 8.67 mg/kg).  This is not surprising, since
inhalation administration usually is much more efficient, comparing
closely to intravenous administration.
     Thimet was the most toxic of the compounds tested for terato-
genicity.  The major prenatal observations were a slight increase in
fetal mortality in the high-dose group and possibly a slight increase
in fetal weight.  Maternal mortality precluded exposure to higher
doses.
     Bromacil or Simazine did not produce any prenatal changes of weight
or fetal mortality in any of the treated groups.  This was not
unexpected, since both these compounds are relatively insoluble.
                                  44

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                               Table 26

                    CONVERSION OF DAILY MEAN AEROSOL
                   CONCENTRATIONS TO DOSAGES IN mg/kg*
    Compound
Chloroform
Ethylene Thiourea
Thimet
Bromacil
Sima2:ine
  Daily                                  Daily
Exposure                                 Dose
Time (hr)     Aerosol Concentration     (mg/kg)

    1          20.1 mg/1 (4100 ppm)     480
    1          10.9 mg/1 (2200 ppm)     260
    1           4.6 mg/1 (950 ppm)      110

    3              120.4 mg/m3            8.67
    3               55.5 mg/m3            4.00
    3               27.2 mg/m3            1.96

    1              1.94 mg/m3             0.047
    1              0.40 mg/m3             0.047
    1              0.15 mg/m3             0.004

    2              165 mg/m3              7.92
    2               78 mg/m3              3.75
    2               38 mg/m3              1.83

    2              317 mg/m3             15.22
    2               77 mg/m3              3.70
    2               17 mg/m3              0.82
* Assuming a respiratory volume of 80 ml/min for a 200-g rat and
  100% deposition and absorption of the aerosol.
                                   45

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                                SUMMARY

     Pregnant rats were exposed daily to five different chemicals by
the inhalation route from day 7 through day 14 of gestation.
     Chloroform, ethylene thiourea, and Thimet caused some prenatal
changes of increased fetal mortality and possibly some fetal weight
loss.
     Bromacil and Simazine had no prenatal effect on rats under .these
experimental conditions.
     No compounds produced treatment-related teratogenic effects.
     None of the dams exhibited gross pathological changes that could
be attributed to the inhalation exposures.
                                   46

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                            RECOMMENDATIONS

     These studies suggest that inhalation is perhaps the most sensitive
route of administering pesticides.  Furthermore,  other routes of
administration do not necessarily predict the toxicity of a chemical
administered by inhalation.  Since inhalation is  probably the most
common route of exposure, inhalation toxicity studies should be
performed on all pesticides that are volatile or  prepared/administered
as a dust or aerosol.
     Although no teratology was observed in these studies, further
work should continue in this area because inhalation is the route of
exposure most often encountered.
     These studies should be extended to include  inhalation mutagenesis
with compounds that are suspected of producing mutations.  Compounds
tested by oral or percutaneous administration may be poorly absorbed
or destroyed (in the gut).  Inhalation more nearly represents an
intravenous dose, and many materials are readily  transported across
the alveolar membranes into the circulation.
                                   47

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                               REFERENCES
 1.  B. A. Schwetz, B.K.J. Leong, and P. J. Gehring.  Embryo and
     fetotoxicity of inhaled chloroform in rats.  Toxicol. Appl.
     Pharmacol. 28, 442-451 (1974).

 2.  K. S. Khera.  Ethylenethiourea:  Teratogenicity study in rats and
     rabbits.  Teratology .1, 243-252 (1973).

 3.  R. A. Vukovich, A. J. Triolo, and J. M. Coon.  Rapid method for
     detection of parathion by electron capture gas chromatography
     without prior cleanup.  J. Agr. Chem. 17, 1190-91 (1969).

 4.  S. Hestrin.  The reaction of acetylcholine and other carboxylic
     acid derivatives with hydroxylamine and its analytical application.
     J. Biol. Chem. 180. 249-61 (1949).

 5.  J. H. Fleisher and E. J. Pope.  Colorimetric method for determination
     of red blood cell cholinesterase activity in whole blood.  Arch.
     Ind. Hyg. .9, 323-34 (1954).

 6.  J. V. Dilley and J. Doull.  The acute inhalation toxicity of
     methyl parathion in rats and mice.  Department of Pharmacology,
     University of Chicago Toxicity Laboratory, unpublished data.

 7.  Task Group on Lung Dynamics.  Deposition and retection models
     for internal dosimetry of the human respiratory tract.  Health
     Physics 12_, 173-207 (1966).

 8.  R. A. Neal.  Studies of the enzymatic mechanism of the metabolism
     of diethyl 4-nitrophenyl phosphorothionate (parathion) by rat
     liver microsomes.  Biochem. J. 105, 289-97 (1967).

 9.  R. A. Neal.  A comparison of the in vitro metabolism of parathion
     in the lung and liver of the rabbit.  Toxicol. Appl. Pharmacol.
     23, 123-30 (1972).

10.  L. I. Kleinman and E. P. Radford, Jr.  Ventilation standards for
     small animals.  Journal of Applied Physiology 19, 360-363 (1964).
                                   48

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                                   TECHNICAL REPORT DATA
                            (Please read Instructions on the reverse before completing)
1. REPORT NO.
   EPA-600/1-78-003
                             2.
                                                           3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
  Teratology  and  Acute Toxicology of Selected Chemical
  Pesticides  Administered by Inhalation
                                                           5. REPORT DATE
                                                              January 1978
                6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
  Gordon W. Newell and James V. Dilley
                                                          8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS

  Stanford   Research Institute
  Menlo  Park,  California 94025
                10. PROGRAM ELEMENT NO.
                  1EA615
                11. CONTRACT/GRANT NO.

                    68-02-1751
 12. SPONSORING AGENCY NAME AND ADDRESS
   Health Effects Research Laboratory
   Office of  Research and Peyelopment
   U.S.  Environmental Protection Agency
   Research Triangle Park. N.C. 27711
                13. TYPE OF REPORT AND PERIOD COVERED
HERL-RTP.NC
                14. SPONSORING AGENCY CODE
                    EPA-600/11
 15. SUPPLEMENTARY NOTES
 16. ABSTRACT
   A method was developed for generating pesticide aerosols within the  respirable
   particle size range of 0,3 to 3.0ym.  Analytical methods were established for determi-
   ning pesticide concentrations in  chamber air samples and in tissues.   A unique chambe
   exposure system was developed that  permitted the simultaneous exposure of four
   different groups of rats to  four  different concentrations of pesticide from.a single
   generation source. Parathion, methyl  parathion, Thimet, Guthion,  and Azodrin were
   administered to rats by the  oral, dermal, intravenous or inhalation  routes, and the
   LD5Q.S or LCsos were compared.   Inhalation was the most toxic route of administration,
   followed by the intravenous, oral,  and then dermal routes.  Females  were more sensiti
   than males to parathion and  Thimet  by all routes of administration.  Azodrin was more
   toxic to females by the intravenous and oral routes, and Guthion  was more toxic to
   females by dermal application.  No  correlation was found between  mortality and choli-
   nesterase inhibition or blood or  liver pesticide content.  No gross  or histopathologi
   cal lesions were identified  that  could be attributed to pesticide treatment. Timed-
   pregnant rats were exposed  to vapors/aerosols of chloroform, ethylene thiourea, Thimet,
   Bromacil, and Simazine for  1 to 3 hours daily on days 7 through 14 of gestation. No
   dose-related terata were found  in any of the studies.
                                                i/e
17.
                               KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
                                             b.lDENTIFIERS/OPEN ENDED TERMS
                              c. COSATI Field/Group
   congenital abnormalities
   toxicity
   respiration
   pesticides
                                06  T
18. DISTRIBUTION STATEMENT

   RELEASE  TO PUBLIC
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     UNCLASSIFIED
21. NO. OF PAGES
   61
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EPA Form 2220-1 <9-73)
                                            49

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