EPA-600/1-76-030
                                          September 1976
     DIETARY SUBACUTE TOX1CITY OF
  ETHYLENEBISISOTHIOCYANATE  SULFIDE
         IN THE LABORATORY RAT
                By

          Ralph I. Freudenthal
              BATTELLE
         Columbus Laboratories
           505 King Avenue
         Columbus, Ohio  43201
        Contract No. 68-02-1717
          Project Officer

          Ronald L. Baron
 Environmental Toxicology Division
 Health  Effects Research Laboratory
 Research  Triangle Park, N.C.  27711
         TBAUY
         ' ""  . .V.tiW
         . :   .},  0881*.
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.
                                  11

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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.

     Studies of the metabolic fate of toxic chemicals give the Agency further
insight into the significance of these agents in the environment.  The
metabolism of toxicants generally results  in formation of chemicals of
unknown toxicological properties.  Chemical identification and toxicological
evaluation of these chemicals and their metabolites continues to be an
integral part of the environmental assessment necessary for continued safe
use of  chemicals.
                                          John H. Knelson, M.D.
                                               Director
                                   Health Effects Research Laboratory
                                  111

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                             CONTENTS

                                                                  Page
INTRODUCTION	    1

EXPERIMENTAL	    2
     Materials	    2
     EBIS Structure Confirmation and
       Purity Analysis	    2
     Thyroid Hormone Function 	    3
     Evaluation of Thyroid Function
       Test Kits	    3
     EBIS Study	    4

RESULTS 	    7
     Preliminary Study	    7
     EBIS Toxicity Evaluation 	    8

SUMMARY	,   26

REFERENCES	27
                                 IV

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                                INTRODUCTION
     The zinc and manganese salts of ethylenebisdithiocarbamic acid (EBDC)
are extensively used as crop fungicides.  Studies investigating the degrada-
tion of the EBDC fungicides have demonstrated the presence of ethylene thiourea
(ETU), ethylenediamine, ethylenebisisothiocyanate sulfide (EBIS, DITT [5, 6-
dehydro-3H-imidazo-l, 2, 4-dithiozole-3-thione], formerly called ETM), carbon
disulfide and inorganic metallic salts  (1, 2, 3, 4, 5).  Early studies evalu-
ating the toxicity of the EBDC fungicides reported a major effect to be thyroid
hyperplasia and neoplasia (6, 7).  The toxicity of ETU has also been studied
and found to be essentially similar to that of the parent fungicide with the
exception that ETU produces thyroid neoplasia in rats  (8, 9) and liver tumors
in mice (10).  The toxicity of EBIS, which has been demonstrated to occur as
an autooxidation product of EBDC's and may exist as a residue in and on food
crops, has not previously been evaluated.

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                                EXPERIMENTAL
                                  Materials

     Sprague Dawley derived rats were purchased from Charles River.   EBIS was
obtained from Trans World Chemicals Inc.  (Silver Spring, Md.), and methimazole
and amitrole from Sigma Chemical Co. (St.  Louis, Mo.).   Measurement of serum
triiodothyroxine (T-3) and thyroxine (T-4) was performed with the aid of
clinical kits purchased from Nuclear Medical Corp.   (Dallas, Texas).
used in iodine uptake studies was obtained from New England Nuclear (Boston,
Mass.).  125j_ was used in place of 131j because of its longer half life rela-
tive to the more commonly used isotope ^  I.
               EBIS Structure Confirmation and Purity Analysis

     EBIS was analyzed by mass spectroscopy and the spectra were compared to
an EBIS reference standard supplied by the Environmental Protection Agency
(Health Effects Research Laboratory, Research Triangle Park, N.C.).  Using
direct probe high resolution mass spectrometry, a strong parent ion was
detected at m/e 176 and fragmenta at m/e 86, m/e 72, m/e 64, and m/e 60.
These fragments were rationalized based on the currently accepted structure
for EBIS and are shown below.
                                                 m/e-86
                                                           (+N=C—S— S)
              m/e 176

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     The purity, based on analysis using high pressure liquid chromatography
and thin layer chromatography, was considered to be greater than 98 percent
based on the analytical standard.
                          Thyroid Hormone Function

     Thyroid hormone is biosynthesized in the thyroid gland by the iodination
of tyrosine .   lodotyrosines are coupled to form T-3 and T-4.  These active
hormones are released into the bloodstream and are distributed throughout the
body where they regulate metabolic processes.

     An increased body burden of thyroid hormone (either T-3 or T-4; both have
essentially identical hormone action) causes an increase in cellular oxygen
consumption.  There is an increase in the rate of metabolism of carbohydrates,
fats and proteins, a rise in cardiac output and an increased irritability of
the nervous system.  A decrease in thyroid hormone levels results in decreased
cellular metabolism.

     The thyroid and its feedback control system are designed to provide a
constant supply of thyroid hormone to peripheral tissues.  Chemical substrates
that: impair the synthesis or release of thyroid hormone from the gland cause
thyroid hyperplasia.  Continued exposure to antithyroid chemicals such as
propylthiouracil or methimazole results in the malignant transformation of the
benign hyperplastic thyroid tissue.  It is now well documented that chemicals
affecting thyroid function can initiate thyroid neoplasia (8, 9).

     The clinical test kits used in this study measure the amount of total
T-4 in the serum and the amount T-3 bound to thyroid binding globulin, the
carrier protein for thyroid hormone.  We also report the Free Thyroxine Index
(FTI) for each test group.  The FTI is a measurement of free serum T-4.  While
the T-4 level, measured by the T-4 radioimmunosassy kit, is influenced by
thyroid binding globulin  (TBG) concentrations, the FTI is a measurement of the
amount of T-4 free in serum, independent of the serum TBG concentration.
                   Evaluation of Thyroid Function Test Kits

     To show that chemically induced thyroid function alterations in rats
could be measured with the available techniques, two chemicals (methimazole
and amitrole) known to affect thyroid were administered to rats.   Groups of
six male and six female rats each received 0.6 mg/kg methimazole twice daily
for four days or amitrole daily at a dose of 4 g/kg, for four days.  Both
chemicals were administered by oral intubation, dissolved in distilled water.
Control rats received distilled water.  These dosage levels have been previ-
ously shown to cause measureable thyroid alterations (11, 12).

     After four treatment days, blood was taken by heart puncture from one-
half of each treatment group, to be used for T-3 and T-4 evaluations.  The
remaining rats were used for    I uptake studies.

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     The T-4 diagnostic kit quantitatively determines total serum thyroxine
by radioimmunoassay.  Ten microliters of serum is mixed with 200 microliters
of an acidic reagent, which completely inactivates endogenous thyroxine bind-
ing proteins.  Radioactive T-4-   I is then added to the test tube and
thoroughly mixed with the endogenous unbound T-4.  Anti-T-4 serum is added and
mixed well.  The mixture is incubated for 45 minutes at room temperature to
allow formation of antibody complexes.  The endogenous T-4 competes with the
isotopically labeled T-4 for the binding sites on the antibody.  The labeled
and unlabeled T-4 are each bound in proportion to their relative concentration
in the mixture.  Since the antibody will bind both T-4-   I and serum T-4
equally well, the amount of radioactive T-4 recovered will reflect the con-
centration of T-4 in the serum sample.

     After the 45 minute incubation, the antibody-bound T-4 is precipitated
by the addition of an ammonium sulfate solution to each tube.  Following
centrifugation, the free fraction of T-4 in the supernatant fluid is discarded
by decantation and the antibody-bound fraction is counted.  Serum T-4 levels
are then determined from a standard curve which is prepared from a series of
four serum standards assayed simultaneously with the experimental serum
samples.

     The T-3 kit used in this study actually measured the unsaturated TBG
binding capacity.  Radiolabeled T-3, added to a serum sample, competes with
endogenous T-3 for TBG binding sites.  Serum is added to the T-3-^  I reagent
and upon mixing, the labeled T-3 binds to serum TBG.  Separation of the free
fraction is accomplished by adding a silicate tablet which is allowed to stand
for 10 minutes prior to centrifugation.  After centrifugation, the supernatant
containing the bound fraction of T-3 is decanted.  The radioactivity on the
silicate is then compared to a standard curve which was assayed simultaneously
with the test samples.
                                 EBIS Study

     EBIS dissolved in corn oil was incorporated into powdered Purina rodent
feed, with a final corn oil concentration of 1 percent.  The test diets were
prepared fresh weekly.  The dietary levels of EBIS were 1000, 100, 10, 1, and
0 ppm.  The control group  received powdered diet containing 1 percent corn oil.
Five dosage groups each containing 60 male and 60 female rats, and a control
group of 30 male and 30 female rats, were placed on study as outlined in
Table 1.  At 30-day intervals, ten rats of each sex (five of each sex of the
control group) were used for the T-3 and T-4 thyroid function tests, for
hematology and for necropsy.  The remaining rats were used for thyroid
I25j uptake studies.  Food consumption and body weight were determined weekly
for each test animal.  Each rat was individually caged throughout the study.

     The 40 tissues removed at necropsy for histologic evaluation are listed
in Table 2.  Those tissues with asterick were also weighed.

     Tissues taken at necropsy were fixed in 10 percent buffered formalin,
sectioned at 5 micron and  stained with Hematoxylin-Eosin.  Blocks containing
tongue and skin from the animals on the 100 ppm/30 days feeding study and

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             TABLE 1.  EBIS PROTOCOL DESIGN
                         Number of Rats/Treatment Period
EBIS (ppm)
1000
1000
100
100
10
10
1
1
0
0
Sex
M
F
M
F
M
F
M
F
M
F
30-Days
20
20
20
20
20
20
20
20
10
10
60-Days
20
20
20
20
20
20
20
20
10
10
90-Days
20
20
20
20
20
20
20
20
10
10
       TABLE 2.  TISSUES REMOVED FOR HISTOLOGIC EVALUATION
Mammary Gland
Trachea
Lung
Abdominal Aorta
Heart*
Mesenteric Lymph Node
Mandibular Lymph Node
Tracheobronchial Lymph Node
Spleen*
Thymus
Kidneys*
Ureter
Urinary Bladder
Ovarv*
Uterus
Testicles*
Epididymus
Prostate
Seminal Vesicle
Salivary Gland
Tongue
Esophagus
Stomach
Duodenum
Jejunum
Ileum
Colon
Pancreas
Liver*
Thyroid*
Parathyroid*
Adrenal*
Eye
Ear
Sciatic Nerve
Rib
Femur
Diaphragm
Pituitary*
Brain*
Spinal Cord

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Several selected control blocks were stained  with toluidine  blue metachromatic
stain for mast cells.   Brain and cord sections from the animals on a diet  level
of 1000 ppm, which exhibited posterior paralysis, were stained with luxol  fast
blue.

     Hematology analysis was carried out from blood smears prepared by tail tip
cuts on the day prior  to sacrifice.   Polymorphonuclear leukocytes, eosinophils,
lymphocytes and monocytes were counted.

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                                  RESULTS
                             Preliminary Study

     Since it has been previously demonstrated that both amitrole and mpthima-
zole treatment for 4 days results in a substantial alteration of thyroid
function, the rats receiving these: chemicals were sacrificed after four daily
treatments and T-3, T-4 and 125I were determined.  The results of the thyroid
function assays are shown in Table 3.
               TABLE  3.  RAT THYROID FUNCTION  EVALUATION AFTER
                         PRETREATMENT WITH AMITROLE AND METHIMAZOLE
         Group
T-3(% Uptake)
T-4 (ug %)
                                                         125,
Uptake)
Control
Amitrole
Methimazole
68.9 + 1.50
68.8 + 1.22
65.1 + 2.07
6.65 + 0.74
4.28 + 1.03
1.95 + 0.84
2.84 + 0.52
0.73 + 0.14
9.79 + 0.89
     Neither amitrcle nor methimazole have an effect on thyroxine binding
globulin (TBG),  as indicated by the T-3 values.  In contrast, a significant
difference is observed in the total serum thyroxine (T-4) and iodide uptake.
It is interesting to note that while both amitrole and methimazole cause marked
thyroid hyperplasia and a decrease in free thyroxine, their effect on iodide
uptake differs.   Amitrole exerts an inhibitory effect on thyroid iodide uptake
while methimazole markedly stimulates iodide uptake.

     This preliminary study shows that the commercially available T-3 and T-4
kits can be used to measure chemically induced alterations in thyroid function
in the laboratory rat.

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                          EBIS Toxicity Evaluation

     Many of the rats receiving 1000 ppm EBIS showed marked toxicity by the enc
of the first week.  Partial to complete paralysis of the hind legs and lower
back, shaggy hair coat, extreme loss of weight and unresponsive disposition
were observed.  A substantial number of rats receiving the highest dose died
after having developed a posterior paralysis.  EBIS ingestion ranged from 1.4
to 11.3 mg/day.  A high percentage of the female rats died 7 to 10 days after
initiating the experiment, whereas the majority of the male rats died 11 to 14
days after the initial feeding, suggesting that the female rat may be more
susceptible than the male rat to the acute toxicity of EBIS.

     Daily observation of the rats receiving diets of 100, 10, or 1 ppm EBIS
or control diets showed no observable toxic effects.

     Because of the acute toxicity of EBIS at 1000 ppm, several rats were used
for thyroid function assays 7 days after being placed on the 1000 ppm test
diet.  The results from these assays are shown in Table 4.
             TABLE 4.  RAT THYROID FUNCTION AFTER  7-DAY INGEST EON
                       QF  1000  PPM EBIS  IN THE  DIET
Group (n)
Control (10)
EBIS (10)
T-3
59.
60.
(% Uptake)
9 + 0.76
8 + 1.13
T-4
4.88
1.13
+
+
>
•3
0
0
%)
.67
.68

3
2
125K
.73 +
.13 +
%
1.
I.
; )
17
16
FT1
2.74
0.73
     The  results of  the T-3 assay suggest that EBIS pretreatment at 1000 ppm
 for  7 days has no effect on thyroxine binding globulin.  The total capacity
 for  thyroid hormone  binding is unchanged.  A significant difference is seen in
 the  total serum T-4.  EBIS pretreatment resulted in a very significant
 decrease  in serum T-4 and the FTI, which measures free serum T-4.  EBIS pre-
 treatment at  1000 ppm also substantially decreased iodide uptake.

     In contrast to  the limited data for rats at the 1000 ppm dietary level,
 EBIS ingestion at  100,  10 or 1 ppm for 30, 60 or 90 days, had no measurable
 effect on thyroid function.  The T-3, T-4, 125j ancj pTj measurements for the
 rats receiving 100,  10 or 1 ppm EBIS were not significantly different from the
 values obtained with control rats.  These results are presented in Table 5.

     The  amount of  test diet consumed per week is presented in Table 6.  With
 the  exception of those rats receiving 1000 ppm EBIS, there was no significant
 difference in feed  consumption in any other group of rats.  A reduced food
 consumption was observed at 1000 ppm.

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     TABLE 5.  RAT THYROID FUNCTION - EBIS DIETARY STUDY
                                                        a,b
EBIS
(ppra)
100
100
100
100
100
100
10
10
JO
10
10
10
1
1
1
1
1
1
0
0
0
0
0
0
Days on
Study
30
30
60
60
90
90
30
30
60
60
90
90
30
30
60
60
90
90
30
30
60
60
90
90
Sex
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
T-3
(percent
uptake)
61,9 + 1.1
65.3 + 1.4
63.4 + 1.5
65.1 + 1.2
62.4 + 1.1
65.3 + 1.1
65.2 + 1.5
67.6 + 1.9
59.4 + 1.6
62.5 + 0.9
57.6 + 2.3
59.8 + 1.2
61.4 + 1.8
64.3 + 1.0
69.0 + 1.2
70.1 + 1.2
60.3 + 1.2
62.5 + 0.9
62.3 + 1.4
64.6 + 1.2
65.2 + 1.9
68.4 + 0.3
60.8 + 1.6
63.7 + 0.9
T-4
(Pg
percent)
3.8 + 0.7
5.1 + 0.6
4.2 + 0.8
6.2 + 0.8
3.2 + 0.5
4.9 + 0.9
3.2 + 0.6
5.6 + 2.0
3.8 + 0.7
5.7 + 0.8
3.6 + 0.4
5.2 + 0.9
4.1 + 0.8
4.9 + 1.1
2.3 + 0.9
3.4 + 0.7
3.2 + 1.5
5.4 + 2.1
4.5 + 0.2
5.2 + 0.6
3.9 + 1.5
4.2 H- 0.6
4.3 + 0.6
5.1 + 0.8
125,
(percent
uptake)
3.4 + 1.7
2.7 + 0.6
3.4 + 1.6
2.8 + 0.7
2.7 + 1.1
2.8 + 0.8
3.8 + 1.4
3.6 + 0.9
2.8 + 1.1
3.2 + 1.2
2.4 + 0.6
2.7 + 0.5
3.1 + 0.9
3.3 + 0.6
2.3 + 0.6
2.0 + 0.9
2.4 + 0.4
2.9 + 0.7
3.5 + 1.6
2.8 + 0.2
1.9 + 0.2
2.0 + 0.3
2.5 + 0.3
2.5 + 0.5
FTIC
2.34
3.32
2.69
4.02
2.61
3.21
2.11
3.77
2.28
3.56
2.11
3.10
2.52
3.20
1.43
2.46
1.89
3.38
2.89
3.37
2.57
3.26
2.57
3.86

 Expressed  as  the mean +_  S.D.

 Student's  t  test was  used to make  comparisons  between  the control
 and  treated animals.  No  significant  differences  were  found  between
 the  control and  corresponding  experimental  group.

:See  Table  3.

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     Table 7 and Figure 1 depict the amount of EBIS consumed.  In the 1000 ppm
group ingestion of EBIS ranged from 1.4 to 11.3 mg per day for the first week
of study while the rats receiving 100 ppm EBIS consumed from 0.6 mg to 3.2 mg
per day during the same period.

     The rats given 1000 ppm of EBIS in their diet for 4 days began eleciting
kyphosis (humped backs) and progressive weakness of the posterior limbs that
culminated in posterior paralysis.  The most severely affected animals showed
loss of weight due to malnutrition and dehydration.  One spontaneous death
occurred on day 6, three on day 7, and one on day 11 of the feeding experiment.
Groups of four animals were euthanized on days 9, 10 and 16, having exhibited
weight loss and posterior paralysis.

     To determine whether the posterior paralysis, observed in rats after feed-
ing EBIS at 1000 ppm for about 8 days, was reversible, a separate group of rats
were placed on the 1000 ppm EBIS test diet until they developed paralysis.  They
were then removed from the test diet and allowed access to control diet.  The
clinical signs of paralysis disappeared within four days.  When these animals
were placed back on diets containing 1000 ppm EBIS, paralysis again occurred
in about 8 days.  Again, removel from the test diet resulted in alleviation of
the clinical signs establishing that the paralysis is reversible.

     During the course of the experiment, one animal in the 1 ppm/60 days
dosage group and three animals in the 0 ppm/60 days dosage group developed
skin irritation of the neck and shoulders which resulted in self multillation
due to scratching.  This resulted in a denuded, ulcerated area that was
resolved by scab formation and overgrowth of new skin.  No EBIS related clin-
ical signs could be discerned within any of the remaining dosage groups
(0-100 ppm for 30-90 days).  All animals, except those 4 described above,
appeared normal and in good condition when presented for necropsy.

     Individual body weight data were recorded weekly throughout the study.
Table 8 summarizes the body weight data  (group mean + S.D.) for the 12-week
test period.  Only those rats which received the 1000 ppm EBIS diet lost
weight.  No difference in body weight gain was observed for those rats receiv-
ing 100, 10, or 1 ppm EBIS or the control diet.  This is shown in Figure 2,
the cumulative body weight gain for 12 weeks.

     Hematology values are shown in Table 9.  No significant differences were
measured for the rats receiving 100, 10, 1, or 0 ppm EBIS.

     At the conclusion of the study gross necropsy including organ weight data
and evaluation of thyroid function was performed.  The number of animals
examined is described in Table 10.  Table 11 lists the organ weights obtained
from the animals on study for 30, 60, and 90 days.  There was no significant
difference in organ weights between those rats on the EBIS diets and the
control rats.  Organ weight to body weight ratios for the test animals were
calculated and are presented in Tables 12 and 13.  No significant difference
exists between groups.  .Organ weight to brain weight ratios were calculated
and are presented in Tables 14 and 15.  There was no significant difference
in brain weight ratios between groups.
                                      11

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  100
   90
   80
   70
   60

   50

   40

   30
   20
M
           1,000 ppm
M


F
                   100 ppm
                     x	x
           ,	     «.--x	
          X"'
en  10
E   9
co   8
5   7
U   6
   1.0
   0.9
   0.2
   O.I
     0
                                  7    8
23456
                    Week
 FIGURE  1. EBIS CONSUMED PER  WEEK (mg)
10    II     12
                                13

-------
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    6001
    5501
    500 \
    450 [
    400 \
CD
    350 \
    300 f
    250}
    200}
    1501
  M Oppm
  M  I ppm
  M 10 ppm
  M 100 ppm
                                         O- —
                                         £*-—•
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                        —A  F I  ppm
                        — -•  F 10 ppm
                        —K  F 100 ppm
    lOOl
            I
1

          Start   I    2345    6789   10   II   12
                                    Week No.
                           FIGURE 2. BODY WEIGHT CHANGE
                                   15

-------
TALBE 9.  HEMATOLOGY VALUES
EBIS
(ppm)
1000
1000
100
100
100
100
100
100
10
10
10
10
10
10
1
1
1
1
1
1
0
0
0
0
0
0
Days on
Study Sex
13
13
30
30
60
60
90
90
30
30
60
60
90
90
30
30
60
60
90
90
30
30
60
60
90
90
F
M
F
M
F
M
F
M
F
M
F
M
F
M
•n
r
M
F
M
F
M
F
M
F
M
F
M
Polymorphonuclear
leukocytes Eosinophils
17.3 + 7.0
17.3 + 7.0
13.4 + 4.3
18.0 + 6.2
15.6 + 7.2
15.2 + 4.5
16.9 + 8.1
14.0+ 6.3
15.3 + 7.4
19. 3 + 8.4
20.1 + 4.8
19.0 + 11.6
15.6 + 6.8
18.9 + 7.2
17.4 + 7.9
20.4 + 5.2
20.1 + 8.2
21.2 + 9.3
19.1 + 6.5
15.7 + 6.3
18.2 + 5.6
12.8 + 3.4
13.0 + 6.8
18.0 + 10.8
18.7 + 5.7
18.3 + 9.7
~ • 	 • — . — . — 	
0.6 + 1.1
3.6 + 1.1
2.9 + 2.1
1.8 + 1.3
4.2 + 3.9
2.1 + 1.5
2.3 + 1.3
2.2 + 1.4
2.4 + 2.7
1.6 + 1.4
2.5 + 2.1
2.0 + 1.3
2.8 + 2.0
2.0 + 1.6
1.9 + 1.1
I- 5 + 1.1
3.1 + 5.1
2.2 + 1.2
2.8 + 1.2
1.8 + l.l
1.6 + 1.1
2,2 + 1.3
3.3 + 2.6
3.5 + 0.6
1.8 + 1.5
Lymphocytes

76.0 + 10.4
75.7 + 15.1
74.8 + 6.6
68.7 + 8.8
76.5 + 9.0
80.3 + 4,3
78.6 + 8.6
80.9 + 7.3
80.3 + 7.9
75.3 + 9.9
74.7 + 6.0
76.7 + 12.5
78.9 + 9.0
77.4 + 8.5
73.9 + 13.5
74.6 + 5.4
76.0 + 6.6
69.1 + 10.9
73.9 + 7.4
78.8 + 6.2
65.8 + 7.5
74.2 + 3.1
80.8 + 9.2
66.7 + H.6
75.0 + 6.1
77.8 + 9.1
— 	 — 	 . 	 .
Monocytcs

5.0 + 3.1
5.7 + 2.5
8.9 + 3.6
11.5 + 3.9
3.5 + 1.5
2.2 + 1.6
1.9 + 1.8
3.2 + 1.6
1.9 + 1.9
2.8 + 1.6
2.5 + 1.8
2.3 + 1.3
2.0 +~ 1.1
1.5 + 1.2
3.5 + 1.4
3.4 + 2.4
5.8 + 2.1
5.5 + 2.6
4.4 + 1.7
3.7 + 1.3
3.8 + 2.9
2.8 + 2.1
3.6 + 0.6
3.0 + 0.0
2.3 + 2.1
       16

-------
TABLE 10. GROSS NECROPSIES AND HISTOLOGIC EVALUATIONS
EBIS
(ppm)
1000
100
100
100
10
10
10
1
1
1
0
0
0
Days on
Study
Varied
90
60
30
90
60
30
90
60
30
90
60
30
Number of
Animals Subjected
to Gross Necropsy
17
20
20
20
19
20
19
20
20
20
8
11
9
Number of
Animals Subjected
to Histologic Review
17
10
10
10
0
0
0
0
0
0
8
11
9
                   17

-------












iAN WEIGHTS OBTAINED AT NECROPSY
3
o
TABLE 1











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-------
                            TABLE 12.   ORGAN WEIGHT:BODY WEIGHT  RATIO
EBIS
(ppm)
100
100
10
10
1
1
0
0
100
100
10
10
1
1
0
Days
on
Study
90
90
90
90
90
90
90
90
60
60
60
60
60
60
60
Sex
F
M
F
M
F
M
F
M
F
M
F
M
F
H
F
Heart
(g)
3.31 x ID"3
2.89 x 10-3
2.99 x 10-3
3.06 x 10-3
3.51 x 10-3
3.03 x 10-3
3.13 x 10-3
2.81 x lO"3
3.46 x 10-3
2.92 x lO-3
3.32 x 10-3
3.11 x 10-3
3.44 x lO"3
3.19 x 10-3
3.14 x 10-3
Spleen
(g)
1.78 x ID"3
1.65 x 10-3
1.68 x 10-3
1.66 x 10-3
1.87 x 10~3
1.67 x 10~3
2.16 x HT3
1.71 x 1CT3
1.99 x 10-3
1.75 x 10-3
2.08 x ID"3
1.64 x 10-3
1.88 x 10~3
1.70 x 10-3
1.67 x 10-3
Rt. Kidney
(g)
3.46 x lO-3
3.27 x 10-3
3.15 x 10-3
2.23 x 10-3
3.47 x 10-3
3.14 x 10-3
3.43 x lO"3
3.18 x ID"3
3.46 x 10-3
3.27 x 10-3
3.39 x 10-3
3.05 x ID"3
3.47 x ID"3
3.54 x 10-3
3.15 x 10-3
Left Kidney Ovary
(g) (paired) (g)
3.31 x lO-3 3.94 x 10~4
3.22 x 10~3
3.08 x 10~3 3.73 x 10~4
3.10 x 10-3
3.31 x 10-3 3.68 x 10~4
3.12 x HT3
3.40 x ID"3 4.57 x 10~4
3.24 x 10-3
3.28 x ID"3 4_OQ x 10_4
3.20 x 10-3
3.28 x ID"3 3.36 x 10-4
2.95 x ID"3
3.38 x ID"3 5.23 x 10-4
3.48 x ID"3
3.06 x 10-3 3.68 x 10-4
Testicle
(paired) (g)

1.11 x 10-2

1.02 x 10-2

1.14 x 10-2

1.09 x lO-2

1.08 x ID"2

1.18 x 10"2

1.09 x ID"2

  0      60    M    2.73 x 10"3   1.52 x 10"3   3.04 x 10"3   3.08 x 1Q-3                1.05 x 10~2

100      30    F    4.06 x ID"3   2.13 x 10~3   3.86 x 10"3   3.83 x 10"3   5.18 x  10~4
100      30    M    3.22 x 10-3   x.gg x 1Q-3   3.79 x ID'3   3.79 x 10" 3                1.25 x 10-2
 10      30    F    3.37 x 1Q-3   2.07 x 10~3   3.79 x 10~3   3.71 x 10~3   5.69 x  lO"4
 10      30    M    3.32 x 10-3   2.00 x 10~3   3.63 x 10~3   3.56 x 10~3                1.25 x 10"2
  1      30    F    3.83 x lO-3   2.44 x 10~3   3.88 x 10~3   3.79 x 10"3   4.79 x  10~4
  1      30    M    3.38 x 10-3   1.79 x 10~3   3.52 x 10~3   3.47 x 10~3                1.14 x 10"2
  0      30    F    3.44 x 10~3   2.15 x 10~3   3.89 x 10~3   3.72 x 10~3   4.48 x  10"*
  0      30    M    3.21 x 10"3   2.08 x 10~3   3.51 x 10'3   3.55 x 10~3                1.17 x 10'2
                                            19

-------
TABLE 13.   ORGAN WEIGHT:BODY WEIGHT RATIO
EBIS
(ppm)
100
100
10
10
1
1
0
0
100
100
10
10
1
1
0
0
100
100
10
10
1
1
0
0
Days
on
Study
90
90
90
90
90
90
90
90
60
60
60
60
60
60
60
60
30
30
30
30
30
30
30
30
Sex
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
Liver
(g)
3.51 x 10- 2
3.62 x KT2
3.21 x 10~2
3.43 x 10" 2
3.42 x 10~2
3.37 x ID"2
3.91 x ID"2
3.72 x 10" 2
3.79 x ID-2
3.83 x 10"2
3.70 x ID'2
3.20 x ID'2
3.71 x 10-2
3.71 x ID"2
3.45 x 10-2
3.21 x 10-2
4.58 x ID'2
4.30 x 10- 2
4.05 x ID-2
3.91 x 10-2
4.23 x 10-2
3. 77 x 10'2
4.47 x 10-2
4.;.5 x 10~ 2
Pituitary
(g)
6.32 x ID"5
2.66 x lO-5
6.72 x 10- 5
2.55 x 10"5
6.74 x 10-5
2.81 it 10-5
3.75 x 10-5
2.77 x 10-5
7.78 x 10-5
2.90 x ID'3
5.88 x 10-5
2.59 x 10-5
4.98 >. lO'5
2.68 x 10-5
5.26 * 10-5
2.18 x 10-5
5.58 x 10-5
4.92 x 10-5
6.32 x 10-5
3.25 x LO-5
6.75 x ID"5
2.84 x 10-5
5.89 x 10-5
3.61 x ;.0-5
Thyroid
(paired) (g)
8.58 x ID"5
6.57 x 10-5
9.77 x 10-5
7.08 x 10-5
8.9° x 10-5
6.57 x ID"5
9.10 x ID"5
6.13 x 10-5
8.89 x 10-5
8.63 x 10-5
7.50 x 10-5
7.35 x ID'5
7.58 x ID'5
6.73 x 10-5
9.30 x 10-5
7.42 x 10-5
6.92 x 10-5
7.56 x ID-5
8.38 x 10"5
7.96 x ID"5
9.46 x ID"5
7.19 * 10-5
1.14 x 10"4
7.12 x 10-5
Adrenal
(Paired) (mg)
2.22 x 10-4
1.01 x 10"4
2.33 x 10"4
1.05 x 10- 4
2.49 x 10" 4
1.03 x ID"4
2.57 x HT4
1.32 x 10-4
2.46 x 1Q-4
1.31 x 10-4
2.39 x 1Q-4
1.08 x 10'4
2,86 x 10-4
1.26 x 10-4
2.17 x 10-4
1.56 x 10-4
3.02 x 10-'*
1.78 x 10-''
2.45 x 10-4
1.43 x 10-4
3.08 x 10-4
1.36 x 10~4
3.52 x 10-"
1.56 x 10-4
Brain
W
7.02 x ID"3
4.38 x 10-3
6.36 x 10-3
4.08 x 10-3
6.70 x 10"3
4.27 x 10-3
6.63 x 10"3
4.47 x 10-3
6.75 x 10"3
4.49 x 10-3
6.81 x 10-3
4.81 x 10-3
6.63 x ID'3
4.32 x 10-3
6.60 x ID'3
4.07 x ID-3
7.26 x 10-3
5.34 x 10-3
7.79 x 10-3
4.97 x ID'3
7.88 x 10-3
5.04 x 10-3
8.03 x 10-3
5.04 x ID-3
             20

-------
TABLE 14.   ORGAN WEIGHT: BRAIN WEIGHT RATIO
EBIS
(ppm)
100
100
10
10
1
1
0
GI
100
100
10
10
1
1
0
0
100
100
10
10
1
1
0
0
Days on
Study
90
90
90
90
90
90
90
90
60
60
60
60
60
60
60
60
30
30
30
30
30
30
30
30
Sex
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
Heart
(g)
4.71
7.83
4.69
7.49
5.23
7.08
4.71
6.29
5.13
6.50
4.87
6.48
5.18
7.38
4.76
6.71
5.61
6.03
4.32
6.68
4.87
6.65
4.29
6.37
x 10"1
x 10"1
x 10~!
x 10"!
x 10"1
x 10"1
x 10~]
x lO'l
x 10"1
x ID-1
x lO"1
x 1Q-1
x 10"!
x 10"1
x 1Q-1
x 1Q-1
x 10"!
x 10"1
x 10-1
x 10"1
x 10"1
x 10-1
x 10^1
x 10-1
Spleen
(g)
2.54 x
3.78 x
2.63 x
4.06 x
2.79 x
3.91 x
3.26 x
3.83 x
2.95 x
3.96 x
3.05 x
3.41 x
2.84 x
3.94 x
2.53 x
3.73 x
2.93 x
3.52 x
2.66 x
4.02 x
3.10 x
3.56 x
2.68 x
4.13 x
lo-1
lo-1
10-1
ID"1
lo-1
10-1
lo-1
10-1
10-1
10"1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
Rt. Kidney
(g)
4.92 x
7.47 x
4.95 x
7.91 x
5.18 x
7.36 x
5.18 x
7.10 x
5.13 x
7.28 x
4.97 x
6.33 x
5.23 x
8.20 x
4.77 x
7.48 x
5.32 x
7.11 x
4.86 x
7.31 x
4.92 x
6.99 x
4.84 x
6.97 x
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
Left Kidney
(g)
4.71 x
7.37 x
4.85 x
7.58 x
4.94 x
7.31 x
5.13 x
7.24 x
4.87 x
7.14 x
4.81 x
6.14 x
5.10 x
8,06 x
4.63 x
7.57 x
5.27 x
7.11 x
4.76 x
7.16 x
4.81 x
6.89 x
4.64 x
7.04 x
10"!
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
10-1
Liver
(g)
4.99
8.28
5.10
8.40
5.10
7.89
5.89
8.31
5.62
8.53
5.42
6.65
5.60
8.59
5.22
7.89
6.30
8.06
5.20
7.86
5.37
7.49
5.56
8.83
            21

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TABLE 15.  ORGAN WEIGHT: BRAIN WEIGHT RATIO
EBIS
(ppm)
100
100
10
10
1
1
0
0
100
100
10
10
1
1
0
0
100
100
10
10
1
1
0
0
Days on
Study
90
90
90
90
90
90
90
90
60
60
60
60
60
60
60
60
30
30
30
30
30
30
30
30
Ovary Testicle
Sex (paired) (g) (paired) (g
F 5.61 x 10~2
M 2.54
F 5.88 x 10"2
M 2.49
F 5.49 x 10"2
M 2.66
F 6.88 x 10"2
M 2.44
F 5.93 x 10"2
M 2.42
F 4.93 x 10"1
M 2.45
F 7.88 x 10~2
M 2.53
F 5.59 x 10"2
M 2.59
F 7.13 x 10~2
M 2.33
F 7.30 x 10~2
M 2.52
F 6.08 x 10" 2
M 2.27
F 5.58 x 10~2
M 2.33
Pituitary
) (mg)
8.99 x 10~3
6.08 x 10~3
10.60 x 10"3
6.23 x 10~3
10.10 x 10"3
6.57 A 10~3
5.65 x 10~3
6.19 x 10~3
11.51 x 10" 3
6.46 x 10~3
8.62 x 10~3
5.38 x 10~3
7.51 x 10~3
6.21 x 10~3
7.98 x 10~3
5.38 x 10"3
8.09 x 10~3
9.25 x 10~3
8.11 x 10"3
6.55 x 10"3
8.57 x 10"3
5.63 x 10~3
7.34 x 10~3
7.16 x 10~3
Thyroid
(paired) (g)
1.22 x 10~2
1.50 x 10~2
1.54 x 10~2
1.73 x 10~2
1.34 x 10~2
L.54 x 10~2
1.37 x 10~2
1.37 x 10~2
1.32 x 10~2
1.92 x 10~2
1.10 x 10~2
1.53 x 10~2
1.14 x 10~2
1.56 x 10"2
1.41 x 10~2
1.82 x 10~2
0.95 x 10" 2
1.42 x 10~2
1.08 x 10~2
1.60 x 10~2
1.20 x 10"2
1.43 x 10~2
1.42 x 10~2
1.41 x 10~2
Adrenal
(paired) (mg)
3.16 x 10~2
2.31 x 10~2
3.66 x 10~2
2.58 x 10~2
3.72 x 10"2
2.41 x 10~2
3.88 x 10~2
2.95 x 10~2
3.65 x 10~2
2.91 x 10~2
3.51 x 10~2
2.25 x 10~2
4.32 x 10~2
2.91 x 10~2
3.29 x 10~2
2.35 x 10~2
4.15 x 10~2
3.34 x 10~2
3.14 x 10~2
2.88 x 10~2
3.91 x 10~2
2.71 x 10~2
4.38 x 10~2
3.09 x 10~2
             22

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     Tissues were taken for histologic examination from the animals receiving
EBIS at 1000 and 100 ppm and also from control rats.   Histopathologic observa-
tions for all experimental groups presented for evaluation are described in
Table 16.  Tissues examined from the 1000 ppm animals that died spontaneously
primarily exhibited mild congestion of the pulmonary aveolar capillaries and
pulmonary veins coupled with mild to moderate nonsuppurative interstitial pneu-
monitis, and bronchitis.  Mild congestion was also observed in liver sinusoids
and in the pyramidal zone of the kidney.   One of these animals that died on
day 6 had a marked suppurative interstitial nephritis and another, which died
on day 11, had dissecting hemorrhage in skeletal muscles of the rear legs,
abdominal wall and vertebral area.  The remaining 12 animals which were sacri-
ficed exhibited similar pulmonary, hepatic and nephritic lesions, but in a
smaller percentage than the five animals that died spontaneously.

     The only histopathologically observable changes in the animals feed
100 ppm was the presence of mast cell granules in the connective tissue of the
tongue and elsewhere.  This occurred only in the animals feed 100 ppm EBIS for
30 days.  This observation was extremely prominent when tissues were stained
with H & E but was not apparent with toluidine blue stained sections.  The only
unique non EBIS-related lesion was the suppurative folliculitis and ulcerative
dermatitis that appeared in three control animals and one rat feed 1 ppm EBIS
for 60 days.  Histopathologic changes such as inflammatory cell infiltration
and congestion of the lungs, heart, kidneys and liver, and random lesions of
the lymph nodes,adrenals, stomach, brain, and prostate appeared with similar
frequency among both control and exposed groups.

     The pulmonary lesions combined with otitis media observed in several of
the rats which received 1000 ppm EBIS are suggestive of lesions noced with
chronic murine pneumonia and are apparently not related to EBIS.  The advanced
nephritic syndrome or hemorrhagic conditions which were found respective1? in
two animals which died spontaneously were not encountered in an" -'-"^r animals
of this group.  No histologic lesion could be identified i- either H & E or
luxol fast blue-stained sections of brain, spinal -°rd> or peripheral nerves
from paralyzed animals.

     The etiology of the dermatitis, observed in a small number of 0 to 100 ppm
diet fed animals, remains undetermined after repeated dry mount preparations,
skin scrappings, culturing, and histologic examination of numerous sections
of affected skin and surrounding tissue.  One might speculate that the small
number of animals involved may indicate an individual variation in sensitivity
to either a cleaning agent or non EBIS diet component associated with the feed
cup, as the lesions were all at the shoulder level and cephalad.

     The nature of the changes was in general very mild and found in all
dosage levels and in controls.  The incidence and degree of pulmonary involve-
ment exhibited in rats which received 1000 ppm EBIS may be relatable to non
lethal viral infections that are often seen in laboratory rats, but no attempt
was made to verify their presence.  The similarity of occurrence, or low
incidence of other changes noted are interpreted as being of a non EBIS-induced
nature.  The only deviation from this interpretation is the 90 percent
                                      23

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incidence of an increase in affinity for the hematoxylin eosin stain,  of mast
cell granules in the 100 ppm/30 days diet group.   Comparative sections from
this and other groups stained with toluidine blue stain for metachromasia
exhibited no discernable change in granule size,  color, or number.   No varia-
tion in the handling of these tissues throughout  their processing has  been
uncovered.

     The data presented show no significant measurable effects of dietary EBIS
at dosage levels of 100 ppm and below.   Severe toxic responses were observed
at 1000 ppm within one week of feeding.
                                       25

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                                  SUMMARY
     Ethylenebisisothiocyanate sulfide (EBIS) was fed to groups of rats at 0,
1, 10, 100, and 1000 ppm for up to 90 days,.  Only those rats receiving EBIS at
1000 ppm demonstrated a toxic respoiise to the test chemical reflected as a
reversible paralysis of the hind legs noteci within 8 to 14 days.  If left on
the 1000 ppm diet, the animals soon died.  When removed from the diet, the
animals recovered, only to become ataxic on further dietary exposure at the
high level.  No histologic lesion could be identified in either H t* E or luxol
fast blue stained sections of brain, spinal cord, or peripheral nerves from
the paralyzed animals.  The ability to reverse the paralysis by removing the
animals from the test diet coupled with the lack of histologicaliy observable
lesions adds credence to a purposed biochemical lesion.

     Ingestion of 1000 ppm EBIS for 7 days also resulted in measurable changes
in thyroid function.  Total serum thyroxirie levels were markedly decreased as
was iodide uptake by the thyroid.

     Dietary levels of 100, 10, 1 and 0 ppm EBIS for 90 days produced no
observable toxicity.  Growth as seen by body weight increases, diet consump-
tion and thyroid function were normal.  No EBIS-related lesions were detected
during the histopathologic evaluation of the 42 tissues taken from rats which
received 100 ppm EBIS.  A no effect level for this 90 day dietary study for
EBIS is 100 ppm in the diet, equivalent to an average intake ranging from
67 mg/kg body weight at week 1 to 31 nig/kg body weight at week 12.
                                      26

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                                  REFERENCES

 1.  Bontoyan, W. R. and Looker, J. B., Degradation of Commercial Ethylene
     Bisdithiocarbamate Formulations to Ethylenethiourea under Elevated
     Temperature and Humidity.  J. Agr. Food Chem 21, 338-341 (1973).

 2.  Watts, R. R., Storrher, R. W. and Onley, J. H., Effects of Cooking on
     Ethylenethiourea.  Bull. Environ. Contam. 12, 224-226 (1974).

 3.  Newsome, W. H. and Laver, G. W.,  Effects of Boiling on the Formation
     of Ethylenethiourea in Zieb-Treated Foods. Bull. Environ. Contam. 10,
     151-154 (1973).

 4.  Vontc, J. W., Chemical Decomposition of Bisdithiocarbamate Fungicides and
     Their Metabolism by Plants and Microorganisms.  Doctoral Thesis, (1975)
     Utrecht.

 5.  Thorn, G. D. and Ludwig, R. A., The Dithiocarbamates and Related
     Compounds.  Elsevier Publishing Co. Amsterdam (1962).

 6.  Smith, R. B. Finnegan, J. K., Larson, P. S., Sahyoun, P. F.,
     Dreyfuss, M. L. and Haag, H. B.,  Toxicologic Studies on Zinc and Disodium
     Ethylene Bisdithiocarbamates. J.  Pharmacol. Exp. Ther. 109, 159-166 (1953).

 7.  Ivanova-Chemishanska, L., Markov, D. V. and Dashev, G., Light and Electron
     Microscopic Observations on Rat Thyroid After Administration of Some
     Dithiocarbamates.  Environ. Res.  _4, 201-212  (1971).

 8.  Ulland, B. M. , Weisburger, J. H., Weisburger, E. K., Rice, J. M., and
     Cypher, R., Thyroid Cancer in Rats from Ethylene Throurea Intake.
     J. Natl Cancer Inst. 4j?, 583-584  (1972)

 9.  Grahan, S. L., Hansen, W. H., Davis, K. J., and Perry, C. H., Effects of
     One-Year Administration of Ethylenethiourea Upon the Thyroid of the
     Rat. J. Agr. Food Cehm. 2±, 324-329 (1973)

10.  Innes, J. R., Ulland, B. M. , Valerio, M. G., Bioassay of Pesticides and
     Industrial Chemicals for Tumorigenicity in Mice. J. Natl Cancer Inst.
     ^2, 11-1-1114 (1969).

11.  Gaines, T. B., Kembrough, R. D., and Linder, R. E., The Toxicity of
     Amitrole in the Rat.  Toxicol. Appl. Pharmacol. 2.6, 118-129 (1973).

12.  Pharmakiotis, A. D. and Alexander, W. D., Repeated Administration of
     35s-Methimazol-;:  Pattern of Accumualtion and Oxidation by the Rat
     Thyroid.  Endocrinology J34, 1508-1513 (1974).

                                        27

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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1 REPORT NO. 2.
EPA-600/1 -76-030
4. TITLE ANDSUBTITLE
Dietary Subacute Toxicity of Ethylenebisisothiocyanate
Sulfide in the Laboratory Rat
7 AUTHOR(S)
Ralph I. Freudenthal
9 PERFORMING ORGANIZATION NAME AND ADDRESS
Battelle
Columbus Laboratories
505 King Ave.
Columbus, OH 43201
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
3. RECIPIENT'S ACCESSION-NO.
5. REPORT DATE
September 1976
6. PERFORMING ORGANIZATION COD
8. PERFORMING ORGANIZATION REP
10. PROGRAM ELEMENT NO.
1 EA61 5
11. CONTRACT/GRANT NO.
68-02-1717
13 TYPE OF REPORT AND PERIOD CC
Interim
14. SPONSORING AGENCY CODE
15 SUPPLEMENTARY NOTES
is.ABSTRACT  Ethylenebis^soth^ocyanate suifide (EBIS)  was fed to groups of rats at C
  10,  100,  and  1000  ppm for  up  to  90 days.   Only those rats receiving EBIS at 100C
  demonstrated  a toxic  response to the test chemical reflected as a reversible pay
  of the  hind legs noted within 8  to 14 days.   If left on the 1000 ppm diet, the i
  soon died.  When removed  from the diet,  the  animals recovered, only to become a
  on further dietary exposure at the high  level.   No histologic lesion could be ic
  fied in either H & E  or luxol fast blue  stained sections of the brain, spinal cc
  or peripheral  nerves  from  the paralyzed  animals.   The ability to reverse the pay
  by removing the animals from  the test diet coupled with the lack of histologica"
  observable lesions adds credence to a proposed biochemical lesion.
        Ingestion of 1000 ppm  EBIS for 7 days  also resulted in measurable changes
  thyroid function.   Total  serum thyroxine levels were markedly decreased as was  •
  uptake  by the thyroid.   Dietary levels  of 100, 10, 1 and 0 ppm EBIS for 90 day:
  produced  no observable toxicity.  Growth as  seen by body weight increases, diet
  consumption and thyroid function were normal.  No EBIS related lesions were dete<
  during  the histopathologic evaluation of the 42 tissues taken from rats which r<
  100 ppm EBIS.  A  no effect level for this 90 day dietary study for EBIS is 100  |
  t^o. d^-t, =MJtvaieiiL to an average intake ranying from 67 mg/kg body weight at
  week 1  to 31  mg/kg body weight at week 12.
17. KEY WORDS AND DOCUMENT ANALYSIS
•a. DESCRIPTORS
Isothiocyanates
toxicity
toxic tolerances
rats
13. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
b.lDENTIFIERS/OPEN ENDED TERMS
EBIS
ethyl enebi si sothiocyana!
19. SECURITY CLASS (This Report)
UNCLASSIFIED
20. SECURITY CLASS (This page)
UNCLASSIFIED
c. COSATI Field/
06, T
e
21. NO. OF PAGES
32
22 PRICE
EPA Form 2220-1 (9-73)
                                           28

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