oEPA
              United States
              Environmental Protection
              Agency
              Environmental Monitoring
              Systems Laboratory
              P.O. Box 15027
              Las Vegas NV 89114
EPA-600 3-80-031
February 1980
              Research and Development
Selected  Toxicological
Studies of Dimilin in
Weanling  Male Rats

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This document is available to the public through the National Technical Information
Service, Springfield, Virginia  22161

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                                                  EPA-600/3-80-031
                                                  February 1980
SELECTED TOXICOLOGICAL STUDIES OF DIMILIN IN WEANLING MALE RATS
          Yogendra M. Patel  and John A. Santolucito
                  Exposure Assessment Division
         Environmental  Monitoring Systems Laboratory
                   Las  Vegas, Nevada  89114
             U.S.  ENVIRONMENTAL PROTECTION AGENCY
              OFFICE OF RESEARCH AND DEVELOPMENT
         ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
                   LAS VEGAS,  NEVADA  89114

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                                 DISCLAIMER
    This report has been reviewed by the Environmental  Monitoring  Systems
Laboratory, U.S. Environmental Protection Agency, and approved for publi-
cation.  Mention of trade names or commercial  products  does  not constitute
endorsement or recommendation for use.

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                                  FOREWORD
    Protection of the environment requires effective regulatory actions based
on sound technical  and scientific data.  The data must include the
quantitative description and linking of pollutant sources,  transport
mechanisms, interactions, and resulting effects  on man and  his environment.
Because of the complexities involved, assessment of exposure to specific
pollutants in the environment requires a total  systems approach that
transcends the media of air, water,  and land.  The Environmental  Monitoring
Systems Laboratory at Las Vegas contributes  to  the formation and  enhancement
of a sound monitoring-data base for  exposure assessment through programs
designed to:

        •  develop and optimize systems and  strategies for  moni-
           toring pollutants and their impact on the environment

        •  demonstrate new monitoring systems and technologies
           by applying them to fulfill special  monitoring needs
           of the Agency's operating programs

    This report provides significant new data on the toxicological  response
of weanling male rats to the pesticide, Dimilin.  This study specifically
deals with the effects of Dimilin on the circulating testosterone and  the
development of the reproductive organs.  For further information, the  reader
should contact the Exposure Assessment Division.
                                                George B.  Morga
                                                    D*i rector
                                  Environmental  Monitoring Systems  Laboratory
                                                   Las Vegas
                                    iii

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                                  ABSTRACT


    Effects of subacute doses of Dimilin [l(4-chlorophenyl)-3-(2,6-
difluorobenzyl)urea] on the reproductive system of weanling male rats were
examined over a period of 96 days.  The parameters evaluated were:
testosterone level in plasma, growth of reproductive organs (testes,
prostate, seminal vesicles) and adrenal glands, and histological  examination
of tissues for pathological changes associated with the administration of
Dimilin.  By intragastric intubation, groups of male rats,  25 days  old,  were
given 0, 15, 150, or 300 milligrams of Dimilin suspended in vegetable oil  per
kilogram per day for a period of 0, 14, 28, 42, and 96 days.

    The data indicate that Dimilin had no adverse effects on body weight or
organ weights of weanling rats, but decreased the levels of testosterone in
the plasma of animals of prepubertal age.  However, this effect of  Dimilin
began to disappear with the onset of puberty.  Histological examination of
the test animals having lower levels of testosterone in plasma failed to
reveal any Dimilin-induced changes in interstitial or germinal cells.  On the
basis of these observations, it is concluded that Dimilin,  at dose  levels of
15, 150, and 300 milligrams per kilogram per day, transiently depresses the
testosterone in plasma during the prepubertal period, yet has no delaying
effects on the development of the reproductive organs of male rats.

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                                  CONTENTS
Disclaimer	    ii
Foreword	   iii
Abstract	    iv
Tables	    vi
    1.   Introduction 	     1
    2.   Conclusion	     1
    3.   Experimental Procedures	     2
    4.   Results	     3
    5.   Discussion	     6
References	     7

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                                   TABLES


Number                                                                  Page


  1.    Experimental Design	   2

  2.    Effect of Dimilin on Body Weight of Male Rats
         Following Daily Administration of Dimilin 	   4

  3.    Effect of Dimilin on Organ Weights of Male Rats
         Following Daily Administration of Dimilin 	   5

  4.    Effect of Dimilin on Plasma Testosterone Level  in Male
         Rats Following Daily Administration of Dimilin	   6

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                                INTRODUCTION


    Insect growth regulators are a new group of compounds that disrupt growth
and normal development of the insect in various immature stages (1).   One of
these compounds, the insecticide Dimilin (Diflubenzuron; TH 6040;  1(4-
chlorophenyl )-3-(2,6-difIuorobenzyl)urea),  has emerged as one of the
promising urea-based larvicides.  This compound prevents the incorporation of
glucose into endocuticle through the inhibition of chitin synthesis  (2).
Dimilin has been successfully used in controlling a variety of arthropod
pests injurious to plants and animals (3,4).  This compound, when  spread
directly onto cattle, effectively reduces the egg hatching of stable  flies
and horn flies.  Secretion of Dimilin into  the egg damages the developing
embryo which accounts for the "sterility" observed in these insects.   This
compound has also shown excellent potential  for controlling the larval  stages
of mosquitos (5,6), Diptera (7,8), certain  Lepidoptera (9), and Coleoptera
(10).

    Dimilin's biotransformation in lactating cows, castrated sheep, and rats
has been reported.  It appears to be absorbed, metabolized, and excreted  in
the urine.  The major metabolites of Dimilin excreted by the cow,  sheep,  and
rats are the result of the hydroxylation of difluorobenzyol and chlorophenyl
rings and the cleavage between the carbonyl  and amide groups.  The mutagenic
potential  of the diflubenzuron metabolites  has also been investigated using
the Salmonella mutagenicity test (11).

    On account of Dimilin's high toxicity toward many destructive  insects and
its extremely low toxicity in rats (Oral LDso >10 g/kg)  and mice
>4.64 g/kg), Dimilin has been proposed for  use as an insecticide on
commercial crops for control  of such pests  as cornborers.  However, it has
been reported that when Dimilin was given in feed to baby chicks for  13
weeks, the males failed to mature—the combs, wattles, feathers, and  voice
remained undeveloped.  These adverse effects with Dimilin were accompanied by
decreases in plasma testosterone proportional to Dimilin dosage (12).

    Thus, in view of these adverse findings, and the lack of information
concerning other possible effects of Dimilin on the reproductive system
of weanling male rats (in terms of plasma testosterone,  development of
reproductive organs), the present study was undertaken.


                                 CONCLUSION
    When weanling male rats were given daily doses of 15,  150,  and  300
mg/kg body weight of Dimilin suspended in oil  over a  period  of  14,  28,  42,
and 96 days, Dimilin produced a transient depression  in plasma  levels of

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testosterone in the prepubertal period without causing any noticeable
delaying effect on the development of the reproductive organs.
                           EXPERIMENTAL PROCEDURES
ANIMALS AND DOSAGE

    Male Long-Evans rats were obtained as weanlings from Blue Spruce Farms,
Inc., Altamont, New York 12009.  They were acclimated to a controlled animal
room environment with a light cycle of 14 hours of light per 10 hours of
darkness for a period of 4 days.  The rats were approximately 25 days old
(55 g mean body wt) at the initiation of the study.  Dimilin (U.S. EPA Lot
no. PP312; 99% purity) was administered daily by intragastric intubation as a
suspension in vegetable oil to groups of rats throughout the duration of the
experiment.  The control animals were given only vegetable oil.  The dosage
levels of Dimilin were 0, 15, 150, and 300 milligrams of Dimilin per kilogram
of body weight.

EXPERIMENTAL DESIGN AND CONDUCT

    On the day before the start of pesticide administration, weanling male
rats were weighed and randomly assigned to control and treatment groups.
Those  rats in the lower and upper  ranges of body weights were first
eliminated.  The control group contained 15 animals and each test group
consisted of 8 animals to provide  optimal observations and contrast for
statistical evaluation.  The experimental design is presented in Table 1.
                        TABLE  1.   EXPERIMENTAL DESIGN
           No.  of
 Group    Controls    No.  of  Low  No.  of Medium   No. of High   Day of Sampling
         (oil only)    Dose Rats    Dose Rats	Dose Rats    and/or Sacrifice
I
II
III
IV
V
15*
15
15
15
15
-
8
8
8
8
-
8
8
8
8
-
8
8
8
8
1
14
28
42
96

* Controls  for  Group  I  received no treatment; controls for other groups
  received  vegetable  oil, without Dimilin, by intragastric intubation.

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All animals were housed individually in galvanized metal cages with food and
water supplied ad lib.  Body weights were recorded weekly and the dosage was
adjusted according to the mean body weight to attain the specified dosage
levels.

    On the day treatment began, blood samples from 15 non-treated animals
(Group I, Table 1) were collected for testosterone analysis.  The animals
were sacrificed; organ weights of testes, prostate, seminal  vesicle, and
adrenal glands were obtained and the tissues preserved in 10 percent neutral
buffered formalin.  The remaining groups of animals were sacrificed at the
same time of day throughout the experiment to minimize variation due to
diurnal rhythms of circulating testosterone.  Handling, ambient environment,
food, water, etc., were consistent for all animals.  Wet weights of the above
mentioned organs were recorded and the organs were fixed in  10 percent
neutral buffered formalin.

    The testosterone concentrations in plasma for the control animals and the
test groups were determined by a radioimmunoassay procedure  and the analyses
were carried out by Smith Kline Clinical Laboratories, Inc., Burbank,
California.  Testes from all  groups (control  and treatment groups) and the
adrenal glands from animals of Group III (Table 1) were submittted for
histopathological  evaluation.

    All histology and pathology examinations were performed  on 5-micron
sections stained with hemotoxylin and eosin by Dr. B. D. Ward, Veterinary
Pathologist, Mississippi State University, Starkville, Mississippi.

    The statistical analysis was performed on arithmetic means from log-
transformed data and employed analysis of variance procedures.
                                   RESULTS
    The mean body weights of the control and treatment groups following daily
administration of Dimilin are given in Table 2.  The results show that after
14, 28, 42, and 96 days of Dimilin treatment there are no significant
differences in body weights between the control and DimiTin-treated rats.

    Organ weights for the various treatment groups are given in Table 3.
One-way analysis of variance did not reveal any dose-related changes in the
various organ weights among the different groups.  There  are some minor
differences observed, however.  The only consistent observation is a
significant decrease in the relative adrenal  gland weights of all  animals on
Dimilin for 28 days.

    The values for the plasma testosterone levels are given in Table 4.  The
animals in Group II, which had received Dimilin for 14 days revealed a
decrease in plasma testosterone at all three dosage levels.  Lower levels of
testosterone were also observed in animals receiving Dimilin for 28 days at

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           TABLE 2.  EFFECT OF DIMILIN ON BODY WEIGHT OF MALE RATS
                  FOLLOWING DAILY ADMINISTRATION OF DIMILIN
        	Weights (g) are expressed as mean ± S.D.	
Group   Control9  15 mg/kg/dayb   150 nig/kg/day15   300 mg/kg/dayb  Remarks
  I   54.3 ± 4.7


 II  123.8 ± 10.5
124.3 ± 10.4   125.5 ± 8.7
III  212.3 ± 19.1   214.1 ± 23.6   221.5 ± 16.5
 IV  298.1 ± 35.2   291.8 ± 26.5   307.3 ± 25.9
  V  481.3 ± 34.3   488.6 ± 28.5   479.6 ± 21.0
              25 days of
              age

122.8 ± 14.5  14 days on
              Dimilin
              39 days of
              age

209.0 ± 14.4  28 days on
              Dimilin
              53 days of
              age

301.1 ± 33.3  42 days on
              Dimilin
              67 days of
              age

465.1 ± 35.2  96 days on
              Dimilin
              121 days of
              age
a = 15 animals each
b = 8 animals each
150 mg/kg/day, and at 300 mg/kg/day, and in animals receiving Dimilin for 42
days at 150 mg/kg/day.  However, it is interesting to note that by day 96,
the levels of plasma.testosterone in the treated animals had increased.  At
day 96, the plasma testosterone values of Dimilin treated animals were not
significantly different from the plasma testosterone values of the controls.

    The histological evaluation of the testicular tissues of the control and
treated groups and the adrenal glands of Group III (Table 1) animals,
revealed no histopathological changes attributable to Dimilin.

    Eighteen plasma  samples  (12 experimental plasma samples and 6 samples
from pooled plasma of untreated animals) were submitted for an inter-
laboratory cross-check comparison to Endocrine Sciences, Tarzana, California.
The results of the entire 18 samples were subjected to the t-test for

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  TABLE 3.   EFFECT OF DIMILIN ON ORGAN WEIGHTS OF MALE RATS FOLLOWING DAILY ADMINISTRATION OF  DIMILIN
Orqan Weights (mg)
Group
I
Control a
II
Control3
15 mg/kg/dayb
150 mg/kg/dayb
300 mg/kg/dayb
HI
Control3
15 mg/kg/dayb
150 mg/kg/dayb
300 mg/kg/dayb
IV
Control3
15 mg/kg/dayb
150 mg/kg/dayb
300 mg/kg/dayb
V
Control3
15 mg/kg/dayb
150 mg/kg/dayb
300 mg/fcg/dayb
Testes
337.3
1156.3
1209.0
1252.0
1178.8
2341.4
2246.3
2283.0
2277.5

2965.5
2931.6
2915.3
2918.8

3448.1
3451.5
3510.6
3644.5*
± 42.3
± 257.5
± 150.2
± 118.4
± 211.9
± 197.0
± 298.4
± 270.6
± 308.4

± 361.5
± 239.9
± 204.3
± 257.2

± 239.5
± 131.1
± 232.9
± 200.1
Prostate
11.7 ±4.3
43.3 ± 9.2
47.9 ± 7.1
48.0 ± 6.0
44.6 ± 14.1
116.8 t 34.9
94.0 ± 26.5
102.4 ± 16.4
103.4 t 11.6

165.1 ± 51.2
155.6 ± 35.4
176.1 t 64.8
163.5 ± 52.4

345.7 ± 33.8
356.4 ± 43.5
344.1 t 59.5
347.6 ± 64.6

are expressed as mean ± S.D.
Seminal Vesicles
With Fluid
Intact Without Fluid
23.6 ± 7.0
40.3 ± 7.4
45.3 ±11.7
41.9 ± 8.5
44.6 ± 8.3
210.1 ± 54.4
193.3 ± 49.4
196.6 ± 38.4
211.3 ± 61.0

661.0 ±
607.6 ±
699.3 ±
632.8 ±

1244.5 ±
1264.1 ±
1259.9 ±
1336.5 ±

187.7
53.5
169.6
154.5

187.5
186.6
158.1
116.8
.
267.3
249.3
287.8
239.4

360.3
358.3
353.3
369.9

± 57.5
± 28.3
± 75.4
± 51.7

± 46.4
± 77.9
± 41.1
± 46.9
Adrenal
21.0
40.5
35.6
37.8
41.5
69.9
60.8*
59.8*
60.3*

53.3
47.4
47.9
46.1

58.3
61.8
58.6
56.3
±3.5
± 8.4
± 5.7
± 2.4
± 7.6
± 11.6
± 7.8
± 4.8
± 3.8

± 10.8
±9.9
± 4.3
t 4.2

± 13.0
± 15.3
± 10.4
± 8.8
Remarks
25 days
14 days
39 days
28 days
53 days

42 days
67 days




96 days
121 days

of age
on Dimilin
of age
on Dimilin
of age

on Dimil in
of age




on Dimilin
of age

a = 15 animals each
b = 8 animals each
* Significantly different from Control  P<.05 (one-way analysis of variance)

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    TABLE 4.  EFFECT OF DIMILIN ON PLASMA TESTOSTERONE LEVEL IN  MALE  RATS
                   FOLLOWING DAILY ADMINISTRATION OF DIMILIN
                Plasma Testosterone Values are expressed as
        	Mean ± S.D. (ng/D)	

Group   Control9    15 mg/kg/dayb  150 mg/kg/dayb  300 mg/kg/dayb   Remarks


  I   13.3 ±8.6          -              -             -         25 days  of
                                                                 age

 II   74.7 ± 81.3    21.4 ± 57.2*   16.7 ± 34.2*    4.2 ± 4.3*   14 days  on
                                                                 Dimilin
                                                                 39 days  of
                                                                 age

III  213.3 ± 163.4  217.8 ± 96.9   115.5 ± 77.5*  113.6 ± 72.3*  28 days  on
                                                                 Dimilin
                                                                 53 days  of
                                                                 age

 IV  480.0 ± 264.2  319.8 ± 328.5  145.0 ± 120.2* 353.3 ± 200.2  42 days  on
                                                                 Dimilin
                                                                 67 days  of
                                                                 age

  V  177.9 ± 112.5  251.0 ± 194.8  269.4 ± 173.7  439.9 ± 297.0  96 days  on
                                                                 Dimilin
                                                                 121 days of
                                                                 age


a =  15  animals  each
b =  8 animals each
*Significantly  different from control  P<.05 (one-way analysis of variance)
differences; there was  no  statistically significant difference between the
testosterone values reported by the two clinical laboratories.


                                  DISCUSSION


    The data presented  in  this report indicate that Dimilin had no adverse
effects on body weight  or  organ weights of weanling rats; however, a decrease
in levels of testosterone  in plasma was noted.  Testosterone values were

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significantly lower in the prepubertal  period,  but this  effect  of Dimilin
began to disappear with the onset of puberty.   Histological  examination of
the testicular tissues of animals having lower  levels  of testosterone  in
plasma did not reveal  any Dimilin-induced changes  in interstitial or germinal
cells.  Similarly, no changes were observed in  the weights  of seminal
vesicles or prostates of these animals.

    In conclusion, the results of this  study show  that Dimilin  given to male
weanling rats at dosage levels of up to 300 milligrams per  kilogram per day
transiently depresses the testosterone  levels in plasma  during  the
prepubertal period, yet has no delaying effect  on  the  development of the
reproductive organs.


                                  REFERENCES
1.  Marx, J. L.  Chitin Synthesis  Inhibitors:   New Class of Insecticides.
    Science 197:1170,  1977.

2.  Post, L. C., B. J. Dejong,  and W.  R.  Vincent.  l-(2,6-disubstituted
    benzoyl)-3 Phenylurea Insecticides:   Inhibitors of Chitin Synthesis,
    Pestic. Biochem.  Physiol.  4:473,  1974.

3.  Ivie, G. W., and J. E. Wright.  Fate  of  Diflubenzuron in the Stable Fly
    and House Fly.  J. Agric. Food Chem.  26:90,  1978.

4.  Ivie, G. W.  Fate  of Diflubenzuron in Cattle and Sheep.  J. Agric. Food
    Chem. 26:81, 1978.

5.  Mulla, M. S., H. A. Darwazeh,  and  R.  L.  Norland.  Insect Growth
    Regulators:  Evaluation  Procedures and Activity Against Mosquitos.
    J. Econ. Entomol.  67:329, 1974.

6.  Schaefer, C. H., H. W. Wilder, and F. S. Mulligan.  A Practical
    Evaluation of TH 6040 as a  Mosquito Control Agent in California.  J.
    Econ. Entomol.  68:183,  1974.

7.  Miller, R. W.  TH  6040 as a Feed Additive for Control of the Face Fly
    and House Fly.  J. Econ. Entomol.  67:697, 1974.

8.  Miller, R. W., C.  Corley, and  K. R. Hill.   Feeding TH 6040 to Chickens:
    Effect on Larval House Flies in Manure and  Determination of Residues in
    Eggs.  J. Econ. Entomol. 68:181, 1975.

9.  Granett, J. and D. M. Dunbar.   TH  6040:  Laboratory and Field Trials for
    Control of Gypsy Moths.  J. Econ.  Entomol.  68:99, 1975.

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10. Moore, R. F. and H. M. Taft.   Boll  Weevils:   Chemosterilization of Both
    Sexes with Busulfan plus Thompson-Hayward  TH  6040.  J. Econ. Entomol.
    68:96, 1975.

11. Seuferer, S. L., H. D. Braymer, and J.  J.  Dunn.   Metabolism of
    diflubenzuron by Soil  Microorganisms and Mutagenicity of the
    Metabolities.   Pestic. Biochem. Physio!.  10:174, 1979.

12. Smalley, H. E.  Comparative Toxicology  of  Some  Insect Growth Regulators.
    Clin. Toxicol. 9:27, 1976.

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                                   TECHNICAL REPORT DATA
                            (Please read Instructions on the reverse before completing)
1. REPORT NO.

  EPA-600/3-80-031
                             2.
                                                           3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
SELECTED  TOXICOLOGICAL STUDIES OF DIMILIN
IN WEANLING MALE  RATS
             5. REPORT DATE
               February  1980
             6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Yogendra M.  Patel  and John A. Santolucito
                                                           8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Environmental  Monitoring Systems Laboratory
Office  of  Research and Development
U.S. Environmental  Protection Agency
Las Vegas, Nevada 89114
             10. PROGRAM ELEMENT NO.
              A2AL1D
             11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
U.S. Environmental  Protection Agency—Las Vegas,  NV
Office of  Research  and Development
Environmental  Monitoring Systems Laboratory
Las Vegas, Nevada  89114
             13. TYPE OF REPORT AND PERIOD COVERED
              Final	
             14. SPONSORING AGENCY CODE
              EPA/600/07
15. SUPPLEMENTARY NOTES
This work  was  performed for EPA's Office of Toxic  Substances
Washington,  D.C.   20460
16. ABSTRACT
The effects of the  subacute doses of Dimilin [l(4-chlorophenyl)-3-(2,6-
difluoropbenzyl)urea]  on  the reproductive system of weanling  male rats were examined
over a period of 96 days.   The parameters evaluated were:   plasma testosterone level,
growth of reproductive organs (testes, prostate, seminal  vesicles)  and adrenal glands,
and histological examination of tissues for pathological  changes  associated with the
administration of Dimilin.   The animals, 25 days old, were  given  0, 15, 150, and 300
milligrams/kilogram/day of  Dimilin suspension in vegetable  oil  by intragastric
intubation for a period of  0, 14, 28, 42, and 96 days.  The data  indicate that Dimilin
lad no adverse effects on body weight or organ weights of weanling  rats, but a
decrease in circulating testosterone in the plasma of animals  of  prepubertal age was
noted.  However, this  effect of Dimilin began to disappear  with the onset of puberty.
The histological examination of the test animals with lower circulating testosterone
in plasma failed to reveal  any Dimilin-induced changes in interstitial  or germinal
cells.  On the basis of these observations, it is concluded that  Dimilin, at 15, 150,
and 300 mg/kg/day dosage  levels,  transiently depresses the  testosterone in plasma in
the prepubertal period, yet  has no delaying effects on the  development  of the
reproductive organs.
17.
                               KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
                                              b.lDENTIFIERS/OPEN ENDED TERMS
                             COSATI Field/Group
urea
boxicology
 ats
Dimilin
 99A
 57S
 57Y
18. DISTRIBUTION STATEMENT

RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)

IINH ASSTFTFH	
21. NO. OF PAGES

	16
                                              20. SECURITY CLASS (Thispage)
                                              UNCLASSIFIED
                                                                         22. PRICE
EPA Form 2220-1 (Rev. 4-77)   PREVIOUS EDITION is OBSOLETE

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