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EPA-650/1-74-012
STUDY OF THE ECOLOGY
OF PESTICIDES
by
W. B . Deichmann
University of Miami
School of Medicine
P.O. Box 8216
Coral Gables, Florida 33124
Grant No. R801470
ROAP No. 21AFM, Task No. 54
Program Element No. 1E1078
EPA Project Officer: Dr. William F. Durham
Pesticides and Toxic Substances Effects Laboratory
National Environmental Research Center
Research Triangle Park, N.C. 27711
Prepared for
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
October 1974
-------
This report has been reviewed by the Environmental Protection Agency
»-, i :
and approved for publication. -^Approval does not signify that the
contents necessarily reflect the views and policies of the Agency,
nor does mention of trade names or commercial products constitute
endorsement or recommendation for use.
-------
TABLE OF CONTENTS
Part 1 Carcinogenicity: Effect in Mice Following Prolonged
Feeding of Dieldrin
Part 2 Effects of Feeding Aldrin and Dieldrin on the Tumor
Incidence in Albino Rats ............... 17
Part 3 Mouse Reproduction Study: Seven-Generation Study:
Retention of Dieldrin and DDT in the Tissues of Mice
Fed Aldrin and DDT 37
iii
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Part 1
CARCINOGENICITY: EFFECT IN MICE FOLLOWING
PROLONGED FEEDING OF DIELDRIN
INTRODUCTION
The members of the Aldrin-Dieldrin Advisory Committee* (1963-1965) found
available data on rat and mouse studies "...inconclusive as far as carcinogenesis
is concerned." Believing that the use of aldrin and dieldrin might continue
indefinitely for certain purposes, they suggested that additional studies be
initiated. The mouse studies reported here were conducted for the primary
purpose of providing information on the tumorigenicity of carcinogenicity of
dieldrin.
EXPERIMENTAL
A total of 684 weanling Swiss-Webster mice were divided into three groups.
Group 1 (125 males and 100 females) were fed the control diet of ground Purina
Laboratory Chow; Group 2 (129 males and 100 females) were fed the laboratory
chow supplemented with 3 ppm of dieldrin; and Group 3 (130 males and 100 females)
were fed the chow diet supplemented with 10 ppm of dieldrin. During the first
two months, the mice were fed half of these concentrations of dieldrin in the
diet. The animals were housed 10 to a cage and maintained in a room air-
conditioned by a single unit (thus avoiding exposure to chemicals that might
find their way into a general laboratory airconditioning system).
All animals were observed daily; those that were ill (particularly males,
because of fighting) were sacrificed by decapitation and subjected to necropsy.
All livers, all tumors, and all organs showing gross anatomical changes were
preserved for histological study. The weights of the livers were recorded.
RESULTS AND SUMMARY
Table 1 presents the ratio of liver-to-body weight of the male and female
mice fed 0, 3, and 10 ppm of dieldrin. Analysis of the data by the student "t"
test indicates that the differences noted, particularly the increase in the
liver-to-body weight ratio in the animals fed dieldrin 10 ppm, are statistically
not significant (p>0.05).
* Appointed in 1963 at the recommendation of the Secretary of HEW, Committee
comprised of: Hans Popper, M.D., Chairman; Alfred M. Boyce, Ph.D.; Wm. B.
Deichmann, Ph.D.; Harold C. Hodge, Ph.D.; Henry F. Kraybill, Ph.D.; James A.
Miller, M.D.; and Albert Tannenbaum, M.D. Subcommittee on Carcinogenesis:
Drs. Miller, Popper, and Tannenbaum.
-------
Table 1. RATIO OF LIVER TO BODY WEIGHT OF MICE FED DIELDRIN
Ratio of
Body Liver Liver-to-Body
Sex Weight Weight Weight
(g) (g)
Controls
M
F
25.2+8.5
32.0±7.6
3.1+2.0
2.8+2.1
0.088
0.088
Dieldrin 3 ppm
M 30.1+7.3 2.8+1.4 0.093
F 31.2+6.8 2.6+1.3 0.083
Dieldrin 10 ppm
M 31.1+5.8 3.4+1.2 0.109
F 30.9+6.6 3.9+1.7 0.125
-------
The relation of tumor deaths (mice dying with one or more tumors) to total
fatalities among the mice fed 0, 3, and 10 ppm of dieldrin, is presented in
Table 2. While the mean periods of survival of the control and experimental
male mice showed no significant differences (Table 3), the incidence of deaths
with tumors decreased from 23 per cent in the controls to 14 and 11 per cent,
respectively, in the groups fed 3 and 10 ppm of dieldrin. The relatively short
life span of the male mice was due to fighting and subsequent infections.
The mean life span of the female control mice was 18.6 months. While not
statistically significant, the mean life span of the experimental female mice
was more prolonged than that of the controls; in the females fed dieldrin 3 ppm,
it was 20.7 months, and in the dieldrin 10 ppm group it was 21.8 months. The
incidence of deaths with tumors as related to the total number of deaths was
63 per cent of the female control group, slightly higher, namely 70 per cent,
for the group of mice fed dieldrin 3 ppm, and considerably lower 47 per cent
for the female mice fed dieldrin 10 ppm.
Considering the number of mice per group examined histologically, which
ranged, for the males, from 81 to 93, and for the females from 70 to 78, and
considering that the life spans of the experimental male and female mice
compared well with those of their respective control groups, it must be
concluded that the reduction in the number of tumor deaths in all groups of
experimental mice, except in the females fed dieldrin 3 ppm is rather
noteworthy.
Table 3 presents the survival periods and the incidence of pathological
lesions (except in the liver) diagnosed microscopically. For each of the three
feeding groups (0, 3, and 10 ppm), the incidence of micropathological changes
was as follows:
Kidney;
Males - benign lesions, 0, 0, and 1 (adenoma), respectively;
Females - benign lesions, 0, 0, and 0, respectively.
Mammary and Subcutaneous:
Males - primary malignant lesions, 1, 1, and 0, respectively;
Females - primary malignant lesions, 11, 15, and 3, respectively
(carcinoma and adenocarcinoma).
Males - benign lesions, 0, 0, and 1 (sebaceous adenoma);
Females - benign lesions, 0, 0, and 1 (hemangioma).
Uterus:
Females - adenomatous hyperplasia, 12, 15, and 13.
- other benign lesions, 2, 1, and 0 (control: leiomyoma and
hemangioma; 3 ppm: fibroma).
-------
Table 2
Relation of Tumor Deaths to Total Fatalities in Mice Fed Dieldrin in the Diet
(as Confirmed
Monthly Tumor Deaths
Monthly Fatalities
Monthly Tumor Deaths
Monthly Fatalities
Monthly Tumor Deaths
Monthly Fatalities
Monthly Tumor Deaths
Monthly Fatalities
Monthly Tumor Deaths
Monthly Fatalities
Monthly Tumor Deaths
Monthly Fatalities
1234567
2
2252
1 15
121 44
1 1
1 1
1
2
1
8
1
7
1
5
2
7
1
1
2
3
0
9
1
4
7
1
5
2
2
2
2
1
n t h s
10 11 12
323
17 10 8
6
6 12 16
2
4 5 14
1 3
323
3 1
3 1
1
1
o f
13
2
5
5
1
6
1
1
1
1
2
by
Micropathological
F e e d i
14
1
2
3
13
1
9
2
3
i
1
3
15 16 17
Hal
^__B^_
Control
1
321
Dieldrin
4 2
Dieldrin
1
516
F e m a
Control
323
625
Dieldrin
3 1
333
Dieldrin
112
Examination)
n g E x p
18 19 20
e s
Diet
2
444
3 ppm
10 ppm
1
3 3
1 e s
Diet
451
752
3 ppm
462
682
10 ppm
1 3
2 7
21
1
4
1
1
2
2
1
2
e r i
22 23
1
3 2
1
6
5
7
6 4
7 5
1 2
4 9
m e :
24
1
1
4
9
2
4
2
2
n t -
25
1
2
2
3
5
2
4
26 27 28 29 30 31 32
1
1
1
2
4
1 13
2 15
432 4
541215
94 321
951523
Total
21
93
11
81
10
91
45
70
55
78
33
70
Per Cent
Tumor Deaths
23
14
70
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Table 3. MICROPATIIOLOGICAL DIAGNOSIS OF TUMORS (EXCEPT I.IVKR TUMORS) IN SWISS-WtBSTER MICE FED DIELDRIN FOR A LIFETIME
,..,
M
F
M
F
Mo'iin" |" No. of
Survival Mice
(i«,nth») per
Ho. of MJ.ce
Examined
Ilisto-
loeicallv
LHo. of Mice
Bearing
Tumor8
.lien iMalien ant
Kidney
-Controls-
12.7+3.7
18.6+4.2
12.0+3.9
20.7+4.8
125/115
100/78
129/101
100/91
« 93
70
81
78
11
15
8
21
10
30
3
34
Sarcoma (2") 1
Leukemic infiltration 5
Leukemic infiltration 2
Arayioidosis I
Lung 1 Lymplioid Tissues
|
Subcutaneous
and
Mammary Tissues
Uterus
or
Prostate
Gonads
Gastroenterlc
Tract
Other
-Controls-
Ade noma 2
Leukemlr infiltration 6
Amyloldosis 1
Adenoma 6
Adenuciircinoma 8
Adenoma 2
Leukemic infiltration 1
Adenoma 3
Adenocarc Lnomn 5
Leukemic infiltration 7
Lymphoma 5
Lymphoma 6
Lymphoma 1
Lymphoma 1 2
Carcinoma (2°) 2
Sq. carcinoma 1
Carcinoma 11
Adenocarcinoma 1
Carcinoma 15
Sarcoma (2") I
-Dlflltirln 10 ppm -
M
t
13.3±4.5
a.«.t
130/110
100/81
91
70
6
21
4
12
Adenoma I
Hyalinization 1
Leukemic Infiltration I
Adenoma 3
Leukemic infiltration 1
Adenocarcinoma I
Adenoma 3
Leukemic infiltration 2
Ade nocnrc 1 namn 2
Lymphoma 1
Lymphoma 1
Lymphusa rcona 3
Sarcoma (2°) J
PL b rosarcoma ( 2 " ) 1
Sebaceous adenoma 1
[lemangioma 1
Adenoc ;i re i no BUI 3
0
Leionyoma 1
Hemangiona 1
Hyperplasia 12
0
tiyperplasia 15
Fibroma 1
0
Cystic 4
0
Leukemic infil-
tration 1
Cystic 13
Hyperplasia
(polypoid) 6
Adenocarcinoma 1
Hyperplasia
(polypoid) 5
Sarcoma 1
Lymphoma 1
Hyperplasia
(polypoid) 4
Hyperplasia
(polypoid) 7
Lymphoma 2
Carcinoma 1
- -Dieldrln 10 ppm -
0
Hyperplasia 13
0
Adenoca re inomv 1
Cystic 13
Cyst adenoma 4
Hemangioma I
Hyperplasia
(polypoid) 1
Hyperplasia
(polypoid) 2
Adrenal , any loi dos is 1
Skin, lipoma 1
Adrenal, cortical adenoma 1
Adrenal anyloidosis 1
Spleen, leukemic infiltrate 2
Skin carcinoma 5
Skin, papilloma 2
Pancreas, leukemic infiltrate i
Pancreas, anaplaetlc carclnoma(2° ) i
Adrenal, anyloidosis 1
Spleen, anyloidosis 2
Pancreas, leukemic infilrrate 5
Spleen, leukemic infiltrate 5
Muscle, carcinoma (2*) 3
Lipoma
Spleen, carcinoma (2")
Islet cell hyperplasia
Adrenal, leukemic Infiltrate
Peritoneum, carcinoma (2°)
Peritoneum, sarcoma (2") 1
Skin , carcinoma 1
Pancreas, leukemic infiltrate 1
Spleen, hyalinization 1
Pancreas, sarcoma (2*) 1
Peritoneum, sarcoma i
Spleen, leukemic Infiltrate 1
* Number of nice started over number of nice autopsied.
-------
Ovaries:
Females - primary malignant lesion, 0,0, and 1 (adenocarcinoma);
- benign lesions, 0, 0, and 5 (cystadenoma, hemangioma);
- cystic changes, 4, 13, and 13.
Lymphoid Tissue;
Males - malignant lesions, 5, 1, and 1 (malignant lymphoma);
Females - malignant lesions, 6, 12, and 4 (malignant lymphoma).
Gastroenteric Tract:
Males - primary malignant lesions, 0, 0, and 0;
Females - primary malignant lesions, 2, 1, and 0 (controls: adeno-
carcinoma and sarcoma; 3 ppm: carcinoma)
Males - polypoid hyperplasia, 6, 4, and 1;
Females - polypoid hyperplasia, 5, 7, and 2.
Lung;
Males - primary malignant lesions, 4, 0, and 1 (adenocarcinoma);
Females - primary malignant lesions, 8, 5, and 2 (adenocarcinoma).
Males - benign lesions, 2, 2, and 3 (adenomas);
Females - benign lesions, 6, 3, and 3 (adenomas).
Pancreas:
Males - benign lesions, 0, 0, and 0;
Females - benign lesions, 0, 1, and 0 (islet cell hyperplasia).
Skin:
Males - primary malignant lesions, 0, 0, and 0;
Females - primary malignant lesions, 5, 1, and 0 (carcinoma).
-------
Skin (continued)
Males - benign lesions, 1, 0, and 0 (lipoma);
Females - benign lesions, 2, 0, and 0 (papilloma).
Tables 2 and 3 and the above histopathological summary indicate
that:
a) The mean survival periods of the control and experimental male
mice (12.7, 12.0, and 13.3 months for the animals fed 0, 3, and
10 ppm of dieldrin) were considerably below those of the female
mice (18.6, 20.7, and 21.8 months, respectively, for the three
feeding groups).
b) Undoubtedly because of the reduced life span of the male mice,
the total number of deaths with benign or malignant tumors was
considerably below the total number of tumor deaths noted in
the female mice. For the 0, 3, and 10 ppm feeding groups, the
percentage of tumor deaths among the males was 23, 14, and 11;
for the females it was 63, 70, and 47 per cent.
c) While benign and malignant tumors were found in some mice of
all groups, clear-cut differences between experimental and
control mice were noted only as follows: among the female mice
fed dieldrin 10 ppm there was one mouse showing an adenocarcinoma
and five mice with cystadenoma and hemangioma of the ovaries.*
Cystic changes in the ovaries were noted in 4, 13, and 13 mice
fed 0, 3, and 10 ppm of dieldrin, respectively. Malignant
lymphoma and the associated leukemic infiltrations of many
tissues (lung, spleen, pancreas, kidney, liver, etc.) were noted
particularly in the females fed dieldrin 3 ppm. These changes
were noted in 6, 12, and 4 female mice of the groups fed 0, 3,
and 10 ppm of dieldrin, respectively. The female mice fed
dieldrin 3 or 10 ppm, when compared with the female controls,
showed a lower incidence of benign neoplasia of the uterus, benign
and malignant neoplasia of the lung, polypoid hyperplasia and
malignant neoplasia of the gastroenteric tract, and benign and
malignant neoplasia of the skin. Most of the decreases were dose-
related. The male mice fed dieldrin 3 or 10 ppm, when compared
with the male control group, showed a lower incidence of malignant
neoplasia of the lung and lymphoreticular system and polypoid
hyperplasia of the gastroenteric tract.
LIVER; Since organochlorine pesticides are known to produce liver
damage in certain strains of mice when fed in toxic concentrations for
a lifetime, the micropathological diagnoses of liver lesions are presented
in detail in Table 4.
Of the 15 types of non-neoplastic lesions of the liver listed in the
table, marked differences from group to group were noted only in regard to
dilatation of the sinusoids with associated protein precipitation and
hepatic cellular swelling and atrophic changes.
* There were no neoplastic lesions in the controls and those fed 3 ppm.
7
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Table 4. MICROPATHOLOGICAL DIAGNOSIS OF LIVER LESIONS
IN SWISS-WEBSTER MICE FED DIELDRIN FOR A LIFETIME
oo
Sex
Number
of Mice
Examined
Histo-
logically
I
NON-NEOPLASTIC LESIONS
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r-t
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cu
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CO
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fe
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00
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NEOPLASTIC LESIONS
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-------
All groups of male and female mice, but particularly the males and
especially those fed dieldrin 3 ppm and the control males, showed dilatation
of the sinusoids and protein precipitate in the sinusoids (a condition
resembling amyloidosis). In the male mice fed 0, 3, and 10 ppm of dieldrin,
dilatation of the sinusoids was noted in 17 of 93 control mice, in 28 of 81
mice fed 3 ppm, and in 7 of 91 mice fed 10 ppm. Protein precipitate was noted
in 7, 21, and 6 male mice fed dieldrin 0, 3, and 10 ppm, respectively. The
incidence of these changes in the control and experimental female mice was not
able.
There was a dose-related increase in cellular swelling and in the incidence
and degree of severity of atrophic hepatic changes in 2 mice (cellular swelling)
and 13 mice (atrophic changes) of 93 control males, in 10 mice and 18 mice,
respectively, of 81 males fed dieldrin 3 ppm, and in 11 mice and 44 mice of
91 males fed dieldrin 10 ppm. In the female mice, the changes were noted in
2 mice (cellular swelling) and one mouse (atrophic changes) of the 70 control
mice, in 3 mice and 20 mice, respectively, of 78 mice fed dieldrin 3 ppm, and
in 18 mice and 20 mice of 70 mice fed dieldrin 10 ppm.
Neoplastic lesions of the liver were noted only in a few animals. Among
the male control mice there was one animal with hemangioma and six mice with
leukemic infiltration. One of the male mice fed dieldrin 3 ppm and two of
those fed dieldrin 10 ppm developed leukemic infiltration. The only primary
malignant hepatocellular lesion in the entire experiment was noted in a control
female mouse (diagnosed as a hepatoma with fibrous component, D-2928).
Four female control mice and nine females fed dieldrin 3 ppm showed
leukemic infiltration. Of the females fed dieldrin 10 ppm, one animal exhibited
leukemic infiltration and one showed duct adenomas.
CONCLUSIONS
Dieldrin was fed as a dietary supplement to 259 male and 200 female
weanling Swiss-Webster mice. The control group included 125 male and 100
female weanling Swiss-Webster mice. No signs of intoxication were observed.
Survival; The mean survival of the three groups of male mice fed
dieldrin 0, 3, and 10 ppm, ranged from 12.0 to 13.3 months. (The cause of
early deaths was excessive fighting.) The mean survival of the three groups
of female mice ranged from 18.6 to 21.8 months. While the experimental male
and female mice except the males fed dieldrin 3 ppm survived the control
mice, the differences were statistically not significant.
Body and Liver Weights; All animals gained at a normal rate. At death
or sacrifice, the body weights of the control and experimental male and female
mice did not differ significantly. While there was an increase in liver
weights, and an increase in the ratio of liver-to-body weight of the male and
female mice fed dieldrin 10 ppm, the differences were not statistically
significant.
-------
Micropathology: A total of 163 control mice, 159 mice fed dieldrin
3 ppm, and 161 mice fed dieldrin 10 ppm, were examined histologically.
The number of male and female mice showing benign tumors was essentially
the same in the three groups a total of 26, 29, and 27 in the groups
fed dieldrin 0, 3, and 10 ppm, respectively.
When compared with the controls, the number of male and female mice
showing malignant tumors decreased as the feeding level of dieldrin
increased from 40 control mice with malignant tumors to 37 mice fed
dieldrin 3 ppm, and to 16 mice fed dieldrin 10 ppm.
Male Mice; The feeding of dieldrin in concentrations of 0, 3, and
10 ppm to male mice resulted in a dose-related increased incidence and
degree of severity in cellular swelling and atrophic changes in the liver,
but in a dose-related decrease in the incidence of leukemic infiltration.
There was also a dose-related decrease in malignant lesions of the lympho-
reticular system and in polypoid hyperplasia of the gastroenteric tract.
Finally, there was a decrease not dose-related in the incidence of
neoplastic lesions of the lung.
Female Mice; The feeding of dieldrin in concentrations of 0, 3, and
10 ppm to female mice resulted in a marked increase in the incidence and
severity of atrophic changes of the liver. But there was an essentially
dose-related decrease in benign neoplasia of the uterus, skin, and lungs.
Malignant neoplasia of the gastroenteric tract and skin in the animals fed
0, 3, and 10 ppm, showed a dose-related decrease.
Compared to the controls, the female mice fed dieldrin 3 ppm showed
an increase in polypoid hyperplasia of the gastroenteric tract, but of the
three groups, the females fed dieldrin 10 ppm showed the lowest incidence
of these lesions. This decrease occurred in spite of the fact that the
females fed dieldrin 10 ppm demonstrated the longest mean life span.
In the females fed 10 ppm, the ovaries and the mammary and subcutaneous
tissues showed an increased incidence of neoplastic lesions. These lesions
were absent in both the control females and those fed dieldrin 3 ppm.
10
-------
The primary contributors to this investigation include:
William E. MacDonald, Ph.D., Co-Principal Investigator
William B. Deichmann, Ph.D., M.D.(Hon.), Principal Investigator
Edward B. Blum, M.D., Pathologist
Michael Bevilacqua, M.D., Pathologist
W. A. D. Anderson, M.D., Pathologist
Joseph Freal, M.S., Chemist
Mary D. Balkus, Technician
M. D. Reuber, M.D., contributed to the pathological review
described in the Appendix.
11
-------
APPENDIX
Project No. 1
REVIEW BY SEVERAL PATHOLOGISTS OF SELECTED
LIVER SLIDES OF MICE FED DIELDRIN 0, 3, AND 10 PPM
Following the preliminary examination of all liver sections in our
laboratory by W. E. MacDonald, Ph.D., 25 slides were selected for review
by several pathologists. These included eight slides from control mice,
seven slides from mice fed dieldrin 3 ppm, and 10 slides from mice fed
dieldrin 10 ppm (Table 5). All sections were cut from tissues fixed in
buffered formalin solution and stained with hemotoxylin-eosin.
The pathologists* who diagnosed the hepatic lesions included our
laboratory consultants, E. Blum, M.D. and M. Bevilacqua, M.D.,
W.A.D. Anderson, M.D., and M.D. Reuber, M.D.
The slides were selected for review with the intent of presenting
a range of micropathological changes from essentially normal -to- various
degrees of inflammatory and degenerative changes -to- nodulation and
neoplastic lesions.
The information presented in Table 5 demonstrates that the pathologists
disagreed markedly in their diagnoses of the lesions as being either
non-neoplastic or neoplastic. This is a serious problem, the existence
of which has been recognized by pathologists and toxicologists for some
time, since it prevents an investigator who is truly seeking the facts
from concluding that a chemical is either a hepatic carcinogen or a
noncarcinogenic compound.
This dilemma was discussed at the working conference on liver cancer
in the summer of 1969, organized by the WHO International Agency for
Research on Cancer**- This group recommended that the term "hepatoma" be
abandoned because of the difference in meaning to clinical and experimental
pathologists. It was also recognized at this conference that "restorative
hyperplasia" is a better pathological term than "regeneration," and
"nodular hyperplasia" is preferable to "regeneration nodule."
* E. Blum, M.D. and M. Bevilacqua, Board-certified Pathologists,
Department of Pathology, Variety Children's Hospital, Miami, Florida;
W.A.D. Anderson, M.D., Professor of Pathology, University of Miami,
School of Medicine, Miami, Florida; and M. D. Reuber, M.D., Associate
Professor, Department of Pathology, University of Maryland, School of
Medicine, and pathological consultant to the U.S. Environmental
Protection Agency.
** World Health Organization, International Agency for Research on
Cancer, Liver Cancer, Proceedings of a Working Conference, Lyon, 1972;
Brit. Med. J., (5795), 261-262, 1972.
12
-------
Table 5. The Tunorigeniclty of Dieldrln in the Swiss-Webster Mouse
Examination of Liver Slides
Code
No.
1.
5.
6.
22.
11.
15.
17.
24.
3.
7.
13.
20.
I.D.
No. of
Mouse
2835
2856
4236
2849
2955
2928
2908
2916
2998
2987
3006
4297
Sex
M
M
M
M
F
F
F
F
M
M
M
M
Diet
Fed
Control
11
n
it
"
D-3ppm
n
tt
Ap;e
at
Death
(mos)
L 10
21
11
14
16
18
9
14
11
8
15
14
Diagnosis by
W.E.MacDonald, Ph.D.
U. of Miami
(1971)
Leukemic infiltration
Negative
Hemangioma & duct
proliferation
Amyloid
Hepatoma 4+
Hepatoma with fibrous
component 4+
Duct adenoma
Question of neoplasia;
question of malignancy.
Slight hypertrophy &
cloudy swelling
Amyloid
Some hypertrophy
Amyloid
Diagnosis by
W. A. D.Anderson, M.D.
U. of Miami
(Sept. 1971)
No neoplasia
No neoplasia
Hemangioma
Hepatoma
Not fibrosarcoma
No neoplasia
No neoplasia, amyloid
No neoplasia
Diagnosis by
Drs.Blum & Bevilacqua
Miami, Fla.
(1971)*
Negative
Minute inflammatory
infiltrates
Area of old infarction;
focal chronic inflamma-
tion; cavernous hemangioma
Minute inflammatory infil-
trates jmarked dilatation i
protein precipitate;
fibrosis.
Large area of cystic
dilatation of sinusoids
& veins with hemorrhage;
small area of atrophy.
Hepatoma with fibrous
component.
Negative
Negative
Negative
Amyloid
Diffuse inflammation
Focal atrophic with
amyloid
Diagnosis by
W. A. D.Anderson, M.D.
U. of Miami
(Sept. 1973)
No carcinoma
No carcinoma
Hemangioma
Amyloid
Hepatoma, questionable
carcinoma
Malignant tumor, type
uncertain
No carcinoma
Nodular hyperplasia
No carcinoma
Amyloid
Nodular hyperplasia,
inflammation
Amyloid
Diagnosis by
M.D. Reuber, M.D.
Representing EPA
(Mar. 1973)
No carcinoma
No carcinoma
Hemangioendo theli oma
Amyloid
Hepatocellular carcinoma
Kupffer cell sarcoma
No carcinoma
Hepatocellular carcinoma
No carcinoma
Amyloid
Hepatocellular carcinoma
Amyloid
* Most of the diagnoses by Drs. Blum and Bevilacqua were added to this table August 27, 1974.
..2
-------
The Tumorlgcnlclty of Dieldrln in the Swiss-Webster Mouse
Examination of Liver Slides
(continued)
Code
No.
9.
12.
16.
4.
19.
23.
2.
8.
10.
14.
18.
21.
25.
I.D.
No. of
Mouse
3102
2991
3071
3214
3224
3174
3293
3300
3230
3270
3285
3287
3254
Sex
F
M
F
M
M
M
F
F
F
F
F
F
F
Diet
Fed
D-3ppm
u
D-lOppi
ii
ti
it
ti
u
it
u
ARC
at
Death
(mos)
27
9
16
13
23
16
23
26
15
25
14
18
23
Diagnosis by
W.E.MacDonald, Ph.D.
U. of Miami
(1971)
Hyperplasia, hypertrophy,
modulation
Some cloudy swelling
Some cloudy swelling
Hypertrophy, hyperplasia,
nodulatlon
Hyperplasia, hypertrophy,
nodulation
Hypertrophy, hyperplasia,
nodulation
Hpperplasia, hypertrophy,
nodulation
Hyperplasia, hypertrophy,
nodulation, & leukemic
infiltration
Hyperplasia, hypertrophy,
nodulation
Hypertrophy, hyperplasia,
nodulation
Hydropic degeneration,
duct proliferation
Hypertrophy, nodulation,
and duct adenoma
Hypertrophy , nodulation
& secondary sarcoma
Diagnosis by
W. A. D. Anderson, M.D.
U. of Miami
(Sept. 1971)
No neoplasia
No neoplasia
No neoplasia
No neoplasia
No neoplasia
No neoplasia
No neoplasia
Diagnosis by
Drs.Blum & Bevllacqua
Miami, Fla.
(1971)*
Focal chronic inflammation
Nuclei swollen, minute
infiltrates
Small focal infiltrates
Congestion, early atrophy,
focal infiltrates
Atrophic changes with
hyperplasia
Small focal cellular
infiltrates
Focal infiltrates, one
areawith ducts & marked
inflammation
Focal infiltrates,
cellular swelling
Extensive necrosis,
probably an infarct
Liver cells swollen
Severe hydropic degen-
eration
Bile duct adenomata
Marked atrohhic changes
with large areas of fibroin
(cirrhosis)
Diagnosis by
W. A. D.Anderson, M.D.
U. of Miami
(Sept. 19 73)
Nodular hyperplasia
Degeneration
No carcinoma
Modularity ,degeneratior
questionable hepatoma
Nodular hyperplasia,
questionable hepatoma
Hepatoma, questionable
hepatooarcinoma
No carcinoma
Neoplasm, probable
carcinoma
Nodular hyperplasia,
questionable hepatoma
Hepatoma, questionable
carcinoma
Degeneration, no
carcinoma
No carcinoma
Hepatocarcinoma
Diagnosis by
M.D. Beuber, M.D.
Representing EPA
(Mar. 1973)
Hepatocellular carcinoma
No carcinoma
No carcinoma
..Hepatocellular carcinoma
Hepatocellular carcinoma
Hepatocellular carcinoma
No carcinoma
Hepatocellular carcinoma
Hepatocellular carcinoma
Hepatocellular carcinoma
Amyloid; focal large bile
duct proliferation
No carcinoma; bile duct
hyperplasia
Hepatocellular carcinoma
*Most of the diagnoses of Drs. Blum and Bevilacqua were added to this table August 27, 1974.
-------
Dr. M. D. Reuber recognized that there does exist a "gray area"
in the diagnosis of pathological lesions in carcinogenic testing, and
states that ..."The area that causes the most problems are the lesions
in which there are focal atypical or malignant cells," and he asks ...
"Should this be considered as a malignant tumor?" * In spite of these
reservations, Dr. Reuber, in contrast to the diagnoses of the four other
pathologists involved in this investigation, most frequently diagnosed
a h^j atic lesion as malignant.
The dilemma of the diagnosis of the pathological liver lesion
remains unsolved, yet far-reaching decisions are being made by the
Environmental Protection Agency and other agencies and laboratories
regarding the carcinogenicity or noncarcinogenicity of a compound.
* Report: Discussion Group #8, Criteria for Tumor Diagnosis and
Classification of Malignancy, M. D. Reuber, Leader. Conference on
Carcinogenesis Testing in the Development of New Drugs, National
Academy of Sciences, Washington, D.C., May 23-25, 1973.
15
-------
Part 2
EFFECTS OF FEEDING ALDRIN AND DIELDRIN ON THE TUMOR
INCIDENCE IN ALBINO RATS
INTRODUCTION
This report presents results with two rat feeding experiments.
I. Feeding Experiment No. 1: Reference is made to the report published in
1970 by Deichmann et_ a±.* which dealt with the feeding of aldrin and
dieldrin to weanling Osborne-Mendel rats in concentrations of 0, 20, 30, and
50 ppm. In this publication it was stated that the overall tumor incidence
(benign and malignant) in both male and female rats fed aldrin or dieldrin
was significantly lower than the incidence of these tumors in male and female
control rats. The paper concluded that "when fed for a lifetime to albino rats,
aldrin and dieldrin (and endrin) are neither tumorigenic nor carcinogenic."
In the 1970 report, tumor deaths were related to the total number of rats
with which the feeding experiment was initiated. (Consideration was not given
to the reduced periods of survival of some of the groups of rats.) We now
realize that it would have been more realistic to relate tumor incidence to
the number of male and female rats that were actually at tumor risk. This
eliminates those rats that, for various reasons, died during the early months
of the experiment, namely, a total of six males and 46 females from a total
number of 800 rats, or about 6 per cent. Thus, we considered that the number
of rats at tumor risk were those that were alive at the beginning of the llth
month. The one tumor death before the llth month, a female rat fed dieldrin
50 ppm, was included in the analysis.
In order to conduct the age adjusted statistical analysis of the tumor
incidence, the statistician (Dr. P. F. Hunt, Statistics Unit, Sittingbourne
Research Centre, England) was supplied with the following information for each
animal:
a) its period of survival,
b) its cause of death, and
c) tumors found or not found in the liver, mammary tissues,
lymphoreticular system, uterus, ovaries, and other organs
arid tissues, as based on gross observation and microscopic
examination and on the diagnosis of a tumor as either
benign or malignant.
Tables 1 and 2 summarize these data. Dr. P. F. Hunt's report is as
follows:
* Tumorigenicity of Aldrin, Dieldrin and Endrin in the Albino Rat. Ind. Med.
and Surg., 3£ (10), 426-434, 1970.
17 '
-------
Table 1. SUMMARY - MALE RATS
TUMORIGENICITY OF ALDRIN AND DIELDRIN IN MALE RATS
This summary pertains ONLY to those rats that were alive at 11 months and that lived up to 31 months.
Group
Control
Aldrin
20 ppm
Aldrin
30 ppm
Aldrin
50 ppm
No. of
Rats
Alive
at
LI Mos.
:
100
50
50
50
Dieldrin
20 ppmj 50
Dieldrin ,,
30 ppmj
Dieldrin
50 ppm| 48
Maximum
Survival
after
Start of
Feeding
Experiment*
(months )
28
28
29
31
29
29
29
Mean
Survival
after
Start of
Feeding
Experiment*
(months)
20.6±4.8
19.4±4.2
19.7±4.1
20.2±3.7
19.6+5.0
21.0±4.1
19.5+4.1
Rats with Tumors Dying Between: t
11 and 12 Mos.
No. of
Rats
* with
Tumors
0
0
0
0
1
0
0
Percent
of Rats
with
Tumors
0
0
0
0
2
0
0
13 and 18 Mos.
No. of
Rats
with
Tumors
6
2
2
2
2
1
1
Percent
of Rats
with
Tumors
6
4
4
4
4
2
2
19 and 24 Mos.
No. of
Rats
with
Tumors
6
2
3
1
2
4
0
Percent
of Rats
with
Tumors
6
4
6
2
4
9
0
25+ Months
No. of
Rats
with
Tumors
8
1
2
1
0
2
0
Percent
of Rats
with
Tumors
8
2
4
2
0
4 '
0
11 Mos. until
Term nation
No. of
Rats
with
Tumors
20
5
7
4
5
7
1
Percent
of Rats
with
Tumors
20
10
14
8
10
15
2
oo
* This includes all rats whether or not they developed tumors.
-------
Table 2. SUMMARY - FEMALE RATS
TUMORIGENICITY OF ALDRIN AND DIELDRIN IN FEMALE RATS
This summary pertains ONLY to those rats that were alive at 11 months and that lived up to 31 months.
Group
Control
Aldrin
20 ppm
Aldrin
30 ppm
Aldrin
50 ppm
No. of
Rats
Alive
at
LI Mos
100
49
45
28
Dieldrin
20 ppm| 50
Dieldrin
30 ppmj 41
Dieldrin
50 ppm Jy
Maximum
Survival
after
Start of
Feeding
Experiment*
(months)
28
26
27
25
29
29
28
Mean
Survival
after
Start. of
Feeding
Experiment1'
(months)
20.5±4.1
18.9+3.9
19.9±3.7
18.5+3.8
20.5+3.1
19.2+4.3
19.7±4.8
Rats with Tumors Dying Between:
11 and 12 Mos.
No. of
Rats
with
Tumors
0
2
1
1
2
2
1
Percent
of Rats
with
Tumors
0
4
2
4
4
5
3
13 and 18 Mos.
No. of
Rats
with
Tumors
16
5
4
4
4
2
1
Percent
of Rats
with
Tumors
16
10
9
14
8
5
3
19 and 24 Mos.
No. of
Rats
with
Tumors
31
10
17
6
15
11
12
Percent
of Rats
with
Tumors
31
20
38
21
30
25
31
25+ Months
No. of
Rats
with
Tumors
13
3
2
0
3
1
2
Percent
of Rats
with
Tumors
13
6
4
0
6
2
5
.
.
11 Mos. until
Termination
No. of
Rats
with
Tumors
60
20
24
11
24
16
16
Percent
of Rats
with
Tumors
60
41
53
39
48
39
41
* This includes all rats whether or not they developed tumors.
-------
Statistical Analysis of Tumour Incidence in the Deichmann
Aldrin/Dieldrin Rat Study No.l
(by Dr. P. F. Hunt)
A summary of the tumour incidence from this study is shown in
Tables 3 and 4 for male and female rats respectively.
If no account is taken of the survival rates for the different
treatment groups the tumour incidence can be considered solely in
relation to the initial group sizes. Thus in this study where controls
contained 100 rats and the other groups generally contained 50 rats and
under the assumption that each rat, regardless of group, has the same
chance to display a tumour, twice as many tumors would be expected in
control as in each treatment group.
As an example of this consider the male rats with at least one
tumour:-
£ A20 A30 A50 D20 D30 D50 Total
Group Size : 100 50 50 50 51 50 50 401
No. of animals
with tumour (0) : 20 3 8 3 5 6 1 46
Expected No. of
tumours based on
group size (E) : 11.5 5.7 5.7 5.7 5.9 5.7 5.7
0/E : 1.74 0.53 1.40 0.53 0.85 1.05 0.18
o
A X test would reject at the 5% level the assumption of equal tumour
incidence among the groups. However, this type of analysis makes no
allowance for the different survival times experienced by each treatment
group.
Regardless of treatment an older animal is more likely to have a
tumour than a younger animal. In consequence if the animals in one group
have survived longer than another similar group they are likely to display
more tumours.
The method of overcoming this problem is to analyse the study month
by month so that within any period the tumour risk can be considered
constant and the risk to each group will be proportional to the number of
survivors in those groups at the start of the period. The expectations
for each month are summed to produce the total number of expected tumours
for each group. In the case of our example the figures are :-
20
-------
DEICHMANN ALDRIN/DIELDRIN STUDY : TABLE 3 SUMMARY OF TUMOUR INCIDENCE IN MALE RATS
NUMBER OF ANIMALS WITH TUMOURS OF SPECIFIC TYPE
NUMBER OF ANIMALS WITH
'ANIMALS
,^.T,,VNI JLV EACH
: TREATMENT
CONTROL
ALDF.IN 20PPM
ALDRIN 30PPM
ALDRIN 50PPM
CIELDRINv20P?M
D-__::-?.LN 30PPM
DILLDRIN 50PPM
100
50
50
50
51
50
50
LIVER : MAMMARY
B
1
0
0
0
0
0
0
M
1
0
0
0
0
0
0
B
5
0
1
0
1
1
0
\i
1
0
0
0
0
0
0
LYMPHO-
RETICULAR
9
3
7
2
3
5
0
PROSTRATE
B
0
0
0
0
0
0
0
M
0
0 "
0
0
0
0
0
TESTES
B
1
0
0
0
0
0
0
M
0
0
0
0
0
0
0
OTHER
B
2
0
1
1
1
0
1
i
M
A
0
0
0
0
0
0
AT LEAST
ONE
TUMOUR
20
, 3
8
3
5
6
1
MORE THAN
ONE
TUMOUR
4
0
1
0
0
0
0
AT LEAST
ONE
MALIGNANT
TUMOUR
14
3
7
2
3
5
0
MORE THAT;
ONE
MALIGNANT
TUMOUR
1
0
0
0
0
0
0
B : Benign Tumour
M : Malignant Tumour
-------
DEICKMANN ALDRIN/DIELDRIN STUDY : TABLE 4 SUMMARY OF TUMOUR INCIDENCE IN FEMALE RATS
\SD1ALS
IN EACH
TP.EAIME.NT TREAP.i.VT
CONTROL
AIDSIX 20PPM
ALDKIX 30PPM
ALDRIN 50PP.M
DIHLDFvIX 20PPM
100
49
50
NUMBER OF ANIMALS WITH TUMOURS OF SPECIFIC TYPE
LIVER MAMMARY
B
0
0
0
50
50
0
0
i
UiL.i.-.-._, J^I-.-V.
:;_: .::.::: SOPPM
48 0
t
1
M
0
0
0
0
0
B
24
8
17
6
16
i
0 1 6
50 ,0 019
;
M
9
2
3
2
2
3
LYMPHO-
RETICULAR
10
1
1
2
2
1
i
l ; 2
'
UTERUS
B
2
4
5
2
0
4
M
3
0
0
0
1
0
i
3
0
!
OVARY
B
"
1
0
0
0
0
0
M
0
0
0
0
0
0
OTHER .
B
2
0
0
0
0
1
1 0
1 '
M
1
1
0
NUMBER OF AS DIALS WITH
AT LEAST
ONE
TUMOUR
44
16
23
I
2
0
1
0
10
21
13
15
MORE THAN
ONE
TUMOUR
7
1
3
2
0
3
1
AT LEAST
ONE
MALIGNANT
TUMOUR
20
4
4
4
5
5
4
MOKE THAN1
ONE
MALIGN AST
TUMOUR
3
0
0
1
0
0
O
B : Benign Tumour
M : Malignant Tumour
-------
£ A20 A30 A50 D20 D30 D50
Expected No. of
tumours based on
survival (E) : 13.6 4.7 5.5 5.3 5.9 6.4 4.7
0/E : 1.47 0.63 1.46 0.57 0.85 0.94 0.22
An alternative way of considering the effect of the differences in
survival would be to take the expectations above and calculate "adjusted
initial group size" assuming identical survivability. This would give
"Adjusted initial
size" : 118 41 48 46 51 56 41
From either consideration it can be seen that the differences in
actual tumour numbers have been affected by survivability and that
adjustment in this respect has reduced the differences between observed
and expected tumours.
o
The significance of the x is now below 10%.
The principle tumour categories from Tables 3 and 4 have been
analyzed by the above method and the results are given in Tables 5 and 6.
Results
Males: None of the variates gave significant results. The largest
discrepancies between observed and expected tumours, either expressed as
a difference or a ratio, generally occurred in control.
Females: None of the variates gave significant results.
Conclusion
There were no significant differences in tumour incidence between
the treatment groups in this study. (Ref: T357/3226)
Dr. Hunt's conclusions support the conclusions reported by Deichmann et al.
(1970) that, when fed for a lifetime to weanling male and female Osborne-Mendel
rats, aldrin and dieldrin, in dosages of 20, 30, and 50 ppm, are neither
tumorigenic nor carcinogenic.
23
-------
TABLE 5
DEICHMANN ALDRIN'/DIELDRIN STUDY : OBSERVED AND EXPECTED Tl::OURS IK MALE RATS
TREATMENT
NUMBER 01- AN DIALS WITH
AT LEAST ONE TUMOUR
MORE THAN ONE TUMOUR
WITH AT LEAST ONE MALIGNANT TUMOUR
CONTROL
ALDRIN 20PPM
.C.Z7.IX 30PPM
.V.3RIX 50PPM
DIELDRIS 20PPM
DIELDRIN 30PPM
DIELDRD; SOPPM
X* +
0
20
3
8
3
5
6
1
E
13.6
4.7
5.5
5.3
5.9
6.4
4.7
0/E
1.47
0.63
1.46
0.57
0.85
0.94
0.22
9.3
0
4
0
1
0
0
0
0
E j 0/E
1.7
0.6
0.5
0.6
0.6
0.6
0.4
2.37
0.00
1.84
0.00
0.00
0.00
0.00
6.5
0
14
3
7
2
3
5
0
E
10.1
3.8
4.0
4.0
4.2
4.5
3.5
0/E
1.39
O.SO
1.75
0.50
0.72
1.11
0.00
9.1
.
o
0>
+ X is distributed as x^ with 6 degrees of freedom
0 : Observed nunber of tumours E : Expected number of tumours based on surviving treatment group sizes throughout study
0/E ; Ratio 0 to E
-------
TABLE 6
DEICHMANN ALDRIN/DIELDRIN STUDY : OBSERVED AND EXPECTED TUMOURS IN FEMALE RATS
TREATMEST
CCSTROL
U.DRD; 20??M
i-LORIN 2CPPM
--LORES 50PPM
3IELDRI:; :OP?M
3IELDRIS 30PPM
3IELDRIX 50PFM
x2
NUMBER OF ANIMALS WITH A MAMMARY TUMOUR
BENIGN
0
24
8
17
6
16
6
9
E
28.1
9.0
10.5
4.7
0/E
0.85
0.89
1.62
1.27
14.5 , 1.1C
8.9 ' 0.67
10.2 0.88
6.7
MALIGNANT
0
9
2
3
2
2
E
7.8
2.1
2.7
1.1
3.7
i
3 | 2.1
1 2.7
0/E
1.15
0.96
1.13
1.90
0.55
1.44
0.38
3.3
TOTAL
0
33
10
20
8
18
9
E
35.9
11.1
13.1
5.8
18.2
11.0
10 : 12.9
'
0/E
0.92
0.90
1.52
1.39
0.99
0.82
0.78 j
6.3 :
i
NUMBER OF ANIMALS WITH
AT LEAST ONE TUMOUR
0
44
16
23
10
21
13
15
E
46.6
14.8
17.3
7.8
23.9
14.7
17.0
0/E
0.94
1.08
1.33
1.29
0.88
0.89
0.89
3.9
MORE THAN ONE TUMOUR
0 1 E
7
1
3
2
0
5.8
1;7
2.1
0.8
2.8
3 ! 1.7
1
1 ! 2.1
1
0/E
1.22
0.59
1.45
2.43
0.00
1.74
0.47
7.1
AT LEAST ONE
MALIGNANT TUXO'JX
0
20
4
4
.4
5
5
4
E
15.2
4.9
5.7
2.7
7.5
' 4.8
5.4
0/E
1.32
0.81
0.71
1.51
0.67
1.05
0.7*
4.2
2 2
X is distributed as y with 6 degrees of. freedom
a A
oo 0 : Observed puri>er of tumours
E : Expected nu:.._,er of rumours based on surviving treatment group sizes throughout study
v> 0/E : Ratio of 0 to E
-------
II. Feeding Experiment No. 2; A second rat feeding experiment with aldrin*
was started on May 20, 1971, with two strains of female rats: Osborne-
Mendel and Sprague-Dawley. These studies were initiated with the approval and
support of NIH (BS 00052-07-S). Financial support for the completion of the
feeding portion of this experiment and initiation of the mammary tumor histology
was provided by EPA (R 801470), supplemented by the Shell Chemical Company.
The primary reason for the use of female rats was that in our first aldrin-
dieldrin feeding experiment (Feeding Experiment No. 1; Deichmann et al., 1970),
the tumors that developed most frequently were found primarily in females, i.e.,
mammary tumors. The decision to use females in Feeding Experiment No. 2 was made
because signs of acute toxicity (tremors and convulsions in the animals suffering
marked loss of body weight) reduced life span, and a marked increase in the
ratio of liver-to-body weight were all noted primarily in the female Osborne-
Mendel rats of Feeding Experiment No. 1. Finally, it was decided to employ two
strains of rats since the susceptibility to tumors varies in different strains
of the same species.
Osborne-Mendel Rats;
Group A - 50 weanlings (age 28-30 days), fed the control diet.
Group B - 50 weanlings, fed the control diet supplemented with
20 ppm aldrin.
Group C - 50 weanlings, fed the control diet supplemented with
50 ppm aldrin.
Sprague-Dawley Rats;
Group D - 50 weanlings (age 28 days), fed the control diet.
Group E - 50 weanlings, fed the control diet supplemented with
20 ppm aldrin.
Group F - 50 weanlings, fed the control diet supplemented with
50 ppm aldrin.
The Osborne-Mendel strain rats, like the rats in Feeding Experiment No.l,
were raised in our laboratory from a colony obtained several years ago from the
Food and Drug Administration in Washington, D.C. The Sprague-Dawley rats were
purchased from the Sprague-Dawley Farm in Madison, Wisconsin. The protocol and
experimental details were essentially like those of Feeding Experiment No.l, in
that the animals were housed two or three in a hanging wire cage in quarters
maintained at 76 + 3° F. They were observed daily and were weighed twice a
month, more often when indicated, i.e., when weight increased rapidly because
of the growth of a tumor or when body weight was lost rapidly due to advanced
age. Appearance and growth, as well as regression of tumors, were observed
by visual inspection and recorded. Palpation of a tumor was avoided.
The experimental diets, prepared once a week, were analyzed periodically
by gas-liquid chromatography to assure the desired concentrations and homogeneous
distribution of aldrin. Pesticide concentrations in the control diet were:
aldrin <0.1 ppm, p,p'-DDT <0.3 ppm, p,p'-DDE <0.1 ppm, and p,p'-DDD <0.2 ppm.
A more extensive analysis carried out on one sample of control diet revealed
the following concentrations: dieldrin <0.0004 ppm, and DDT plus DDE <0.0005 ppm.
* Formerly referred to as "Effect of aldrin as an inhibitor in the production
of mammary tumors in rats..." (EPA Grant No. R801470, 1973-74).
26
-------
Moribund animals were anesthetized by diethyl ether and sacrificed by
exsanguination. Those that were found to lose body weight were removed from
the racks and placed in cages on a table to permit closer observation. Every
effort was made to avoid loss of tissues resulting from the death of an animal
during the night or over the weekend.
At autopsy, all mammary tumors were taken and measured; in addition, samples
of mammary tissue of all rats, as well as sections of organs showing gross
changes, were taken and preserved for microscopic examination. Sections were
also taken and preserved of the liver, lung, kidney, spleen, pancreas, heart,
adrenal, ovary, uterus, mesenteric and mediastinal lymph nodes, stomach, and
intestines of all animals (in the event that it should become desirable to
study them histologically).
Results
Body Weights; Figures 1 and 2 present the body weights of the two strains
of rats. The mean body weights of the Osborne-Mendel rats fed aldrin 20 and 50 ppm
reached a plateau (360 g) of approximately 30 g below that of the control rats.
The Sprague-Dawley rats fed aldrin 50 ppm gained weight more rapidly than the
controls and reached a level of approximately 350 g, while those fed aldrin 20 ppm
levelled off when their weights reached 315-320 g. The control animals reached
a mean body weight of approximately 280 g. Almost every Osborne-Mendel and
Sprague-Dawley rat began to lose body weight during the last months of the
feeding experiment.
The significance of the body weight changes noted is not clear. It is
noteworthy, however, that this is not the first time that investigators have
noted that certain strains of rats fed organochlorine pesticides gained more
rapidly than controls. We (Deichmann, ejt al_. , 1972) reported this for weanling
Osborne-Mendel rats fed aldrin 7.5 ppm, DDT 50 ppm, or DDT 50 ppm plus aldrin
7.5 ppm.
Survival: Of the 50 animals initiated per group, all animals of both
strains were alive at age 120 days except for one Sprague-Dawley rat fed the
control diet. The feeding of aldrin at 20 and 50 ppm to both strains of rats
was accompanied by a dose-related reduced period of survival: the mean period
of survival of the Osborne-Mendel control rats was 689 days; for the experimental
rats mean survival was 640 and 570 days, respectively. The same observations
were made for the Sprague-Dawley rats, except that their period of survival was
shorter than that of the Osborne-Mendel rats: the mean period of survival of
the Sprague-Dawley controls was 567 days, and that of the experimentals 540 and
409 days, respectively. The primary cause of death of these animals was
pneumonitis, usually associated with advanced age and/or tumors of the mammary
and uterine tissues. (Table 7)
Table 7 also presents the incidence of all tumors noted at necropsy in
these animals. In agreement with our aldrin-dieldrin Feeding Experiment No.l,
the Osborne-Mendel rats again demonstrated a high incidence of mammary tumors.
Tumors of the uterus and ovaries followed in frequency. A few animals in all
groups showed tumors in the lymph nodes, lungs, kidneys, adrenals, and spleen.
A single control or experimental rat showed a tumor in either the heart,
intestine, bladder, bone, or fat. No tumors were found in the liver of the
control or experimental rats.
27
-------
Body Weight
in grama
400
360
'120
280
"240
ZOO
160
120
80
0T control
X aldrin 20 ppm
O aldrin 50 ppm
Figure 1
Body weights of female Osborne-Mendel rats
10
20
25
30
-------
Body Weights
in grams
360
control
X ftldrin 20'ppm
nldrin 50 ppm
lita of female Sprague-Dwley rats
Months:
-------
Table 7. INCIDENCE OF TUMORS (AT NECROPSY) IN RATS FED ALDRIN
(second aldrin rat feeding experiment)
Group
A
" B
C
D
E
F
Diet Fed
Control
Aldrin
20 ppm
Aldrin
50 ppm
Control
Aldrin
20 ppm
Aldrin
50 ppm
No. of
Rats at
Age of
120 Days
50
50
50
49
50
50
Mean
Survival
of all
Rats
(days )
689
640
570
567
540
409
No. of
Rats
with
Tumors
47
46
33
26
23
18
h
CO
|
S
§
0)
5
co
a)
H
M
CO
0
00
§
Osborne-Mendel Rats
34
29
13
Sprj
5
3
2
15
14
16
igue-Dc
18
15
14
17
15
8
iwley ]
5
8
2
3
5
5
lats
Q)
H
i-J
J3 CO
>* O
h-I 53
7
12
6
1
1
oT
c
H
3
2
1
rH
CO
0)
<
I
2
1
t-l
cfl
-------
In the Sprague-Dawley rats (Table 7) the tumor incidence was lower than
in the Osborne-Mendel rats. In this strain, uterine tumors were most frequent,
followed by ovarian and mammary tumors. Occasionally, a single animal showed
a tumor in the lymph nodes, adrenal, heart, or bladder. No tumors were found
in the liver, kidneys, lungs, intestine, spleen, bone, or fat.
Since this feeding experiment was initiated largely for the purpose of
studying the effect of aldrin on mammary tumors, tables were prepared for the
statistician (Dr. P. F. Hunt), presenting the following information and data
for a risk analysis of mammary tumor development:
a) number of rats alive at the first of each month,
b) number of rats showing first evidence of a mammary tumor during
the month,
c) number of deaths during each month,
d) number of rats dying with mammary tumor(s) during the month,
e) and number of rats "at risk" at the first of each month.
These data are summarized in Table 8 under the following headings:
1) number of rats per group,
2) number of rats with a single mammary tumor,
3) number of rats with multiple mammary tumors,
4) total number of rats with mammary tumors,
5) mean survival of all rats per group,
6) mean survival of rats that showed mammary tumors,
7) mean survival of the rats without mammary tumors,
8) mean age of the rats at the first appearance of a mammary tumor.
Osborne-Mendel Rats: The mammary tumor incidence was highest among
control rats (34 versus 29 and 13 in the 20 and 50 ppm groups) (Table 8).
The feeding of aldrin 20 ppm to Osborne-Mendel strain rats (when compared
with the controls) did not markedly influence the mean latent period until
the first tumor appeared (597 versus 590 days), nor did it markedly influence
the mean survival of the rats (705 versus 696 days) that developed mammary
tumors.
The Osborne-Mendel rats fed aldrin 50 ppm showed again (as in Feeding
Experiment No.l) a marked reduction in survival. There was also a reduction
in the mean latent period in the appearance of the first tumor.
Sprague-Dawley Rats; The most notable difference between the two strains
of rats was the markedly lower number of Sprague-Dawley rats that developed
single and/or multiple mammary tumors. While a total of 34 Osborne-Mendel
control rats developed mammary tumors, only five Sprague-Dawley controls
developed such tumors. The mean survival of the Sprague-Dawley control rats
with tumors was 574 days, while the mean survival of the Osborne-Mendel
controls was 705 days.
In the Sprague-Dawley rats fed aldrin 20 ppm, the mean age of an animal
at the appearance of the first tumor was 668 days, while in the Osborne-
Mendel rats fed aldrin 20 ppm, it was 590 days.
31
-------
Table 8. EFFECT OF FEEDING ALDRIN ON MAMMARY TUMORS
IN FEMALE OSBORNE-MENDEL AND SPRAGUE-DAWLEY RATS
(second aldrin rat feeding experiment)
Group
No.
A
B
C
D
E
F
Diet Fed
Negative Control
Control Diet
Aldrin 20 ppm
Aldrin 50 ppm
Negative Control
Control Diet
Aldrin 20 ppm
Aldrin 50 ppm
No. of Rats
at
Weaning
(28 days)
50
50
50
50
50
50
5 Started
at
Age of
120 Days
50
50
50
49
50
50
No. of I
Single
Tumors
Osborne
21
23
9
Sprague
2
3
2
lats with
Multiple
Tumors
- Mendel
13
6
4
- Dawley
3
0
0
Total No.
of Rats
with Tumors
Rats
34
29
13
Rats
5
3
2
Mej
All
Rats
(days)
689
640
570
567
540
409
in Survd
Rats
with
Tumors
(days)
705
696
671
574
699
534
_val of
Rats
without
Tumors
(days )
655
563
534
566
530
404
Mean Age of Rat
at Appearance
of First Tumor
(days)
597
590
537
536
668
394
u>
to
-------
The mean survival of the Sprague-Dawley rats fed 50 ppm, when compared
with those fed aldrin 20 ppm, was markedly reduced (Table 8).
Age Adjusted Risk Analyses; The risk analyses based on the data in
Tables 7 and 8 were again conducted by Dr. P. F. Hunt*. These analyses
(Table 9) were carried out in two ways: by considering deaths with a tumor
in relation to rats alive at the start of each month, and by considering the
number of rats first showing evidence of a tumor in relation to those which
had shown no evidence of a tumor at the start of the month. One would
expect that these two types of analysis would be in good agreement, which in
fact was the case. The results in the two strains of rats were very similar.
The risk analysis studies showed that there were no significant
differences between the respective controls and the aldrin-treated Osborne-
Mendel and Sprague-Dawley rats when considered separately, although the
lower survival in the aldrin 50 ppm Osborne-Mendel and Sprague-Dawley rats
was a complication. The survival of the aldrin 20 ppm groups was only
slightly less than the respective controls.
Analyses of the data presented showed no indication of an increase in
mammary tumor incidence related to the feeding of 20 or 50 ppm of aldrin.
Summary
Based primarily on the incidence of all tumors (Table 7) found at necropsy
in the female Osborne-Mendel and Sprague-Dawley rats fed aldrin in the diet
at 0, 20, and 50 ppm, and on the age adjusted risk analyses (Tables 8 and 9)
carried out on the number of rats that developed mammary tumors (predominantly
in the Osborne-Mendel rats) it is concluded that the lifetime feeding of
aldrin in concentrations of 20 and 50 ppm started at weaning age to female
Osborne-Mendel and weanling female Sprague-Dawley rats neither increased nor
decreased the incidence of tumors in these animals above that noted in the
respective control groups.
NOTE; While this report with Osborne-Mendel and Sprague-Dawley female rats
deals primarily with the number of tumors and the number of rats that
developed tumors, we await the completion of the microscopic studies to
determine whether the feeding of aldrin altered the distribution of benign
and malignant tumors.
* Statistics Unit, Sittingbourne Research Centre, England.
33
-------
Table 9. OBSERVED AND EXPECTED NUMBERS OF MAMMARY
TUMORS IN ANIMALS RECEIVING 20 OR 50 PPM ALDRIN
Control
20 ppm
50 ppm
X2 (2df)
Control
20 ppm
50 ppm
X2(2 df)
Analysis of death with tumor Analysis
0 E 0
0/E
Osborne-Mendel Rats
: 34 36 34
0.95
: 33 27.8 33
1.19
: 15 18.3 15
1.03
: 2.0
Sprague-Dawley Rats
: 5 Q>94 5.3 5
: 3 3.7 3
0.82
: 2 1.0 2
1.94
: 1.1
of first evidence
of tumor
E
0/E
33.1
1.03
27.1
1.22
21.9
0.69
4.1
1.11 4-5
4.0
0.75
1.5
1.36
0.5
34
-------
This investigation was conducted primarily by:
William B. Deichmann, PhD, MD(hon)., Professor of Pharmacology
and Director, Research and Teaching Center of Toxicology -
Principal Investigator.
William E. MacDonald, Ph.D., Assistant Professor of Pharmacology,
and Associate Director, Research and Teaching Center of
Toxicology - Co-Principal Investigator.
Dr. P. F. Hunt, Statistician, Statistics Unit, Sittingbourne
Research Centre, England.
Dewey A. Cubit, B.S., Research Associate.
A. Gerald Beanley, M.S., Research Associate.
Mary A. Balkus, Laboratory Technician.
35
-------
REFERENCES
Deichmann, Win. B. , MacDonald, W.E., Blum, E. , Bevilacqua, M. ,
Radomski, J., Keplinger, M. , and Balkus, M. Tumorigenicity of Aldrin,
Dieldrin, and Endrin in the Albino Rat. Ind. Med. Surg., 39_: 426-434, 1970.
Hunt, P. F. (Statistics Unit, Sittingbourne Research Centre, Sittingbourne,
Kent, England). Personal communication, 1974.
Deichmann, Wm.B., MacDonald, W.E., Cubit, D.A., and Beasley, A.G.
Effects of Starvation in Rats with Elevated DDT and Dieldrin Tissue Levels.
Int. Arch. Occ. Hlth (Int. Arch. Arbeitsmed.), 29; 1-20, 1972; Pesticides
and the Environment: A Continuing Controversy (published by Intercontinental
Medical Book Corp., New York, 1973), p. 11-12.
36
-------
Part 3
Mouse Reproduction Study
Seven-Generation Study; Retention of Dieldrin
and DDT in the Tissues of Mice Fed Aldrin and DDT
In 1970, M. L. Keplinger, Wm. B. Deichmann and F. Sala published
a report on the "Effects of Combinations of Pesticides on Reproduction
in Mice." Even though the concentrations of dieldrin and DDT in
abdominal body fat were determined in these mice, the data were not
included in the report. There was a definite indication that
succeeding generations fed aldrin 3 and 5 ppm retained higher
concentrations of this pesticide in body fat.* This observation,
without confirmation, was considered alarming and it was largely for
this reason that this part of the investigation was repeated. All
mice of the 1970 six-generation study were kept frozen at -5°C for
varying periods of time (1 to 15 months) prior to analysis for
organochlorine pesticides. It was also considered, at that time, that
increasing concentrations of dieldrin and DDT in abdominal fat do not
necessarily reflect an increasing total body load of pesticide.
In the investigation reported here, both the concentration and the
total retention of pesticides were determined in abdominal fat and
in the total carcass.
Experimental
Diets Fed: The parent (P) generations of Swiss-Webster mice
were selected randomly from our laboratory stock at weaning age
(3-4 weeks; 9-12 g body weight). The animals were divided into three
experimental groups of 10 females and six males each and were fed
ground Purina Laboratory Chow supplemented as follows:
Group 1 - aldrin** 5 ppm; Group 2 - aldrin 10 ppm; Group 3 -
DDT***100 ppm. Group 4 - 20 females and 12 males, which
served as controls, were fed the uncontaminated control diet
for seven generations.
The P, F^ to Fg and F^ to Fy generations of Groups 1 to 3 were fed
the respective experimental diets; all F^ generations of Groups 1, 2,
and 3 were fed the control diet from weaning to sacrifice.
* Reported to NIH in October 1968 (NIH Grant ES00052-06).
** p,p-DDT 99+% [l,l-bis-(p-chlorophenyl)-2,2,2-trichloroethane],
Aldrich Chemical Co., Inc.
*** Technical aldrin 95% (hexachlorohexahydro-endo, exo-dimethanonaphthalene),
Shell Chemical Co.
37
-------
Analysis of Tissues for Dieldrin, p,p'-DDT, p^'-DDE, and p,pf-DDD;
The animals were sacrificed by cervical dislocation, weighed, depilated
with a commercial thioglycolate preparation, then washed with water and
dried with absorbant paper. After a portion (approx. 0.2 g) of abdominal
fat was removed from each mouse, the whole animal was minced with
scissors, then macerated in a cooled 200 ml Sorvell Omnimixer with an
equal volume of 0.73% saline until a slurry of uniform consistency was
obtained. Total lipids and their pesticide contents were extracted (by
the method of Folch et^ al., 1957) from a 2.0 g aliquot of the slurry,
representing 1.0 g of mouse. Pesticide residues were determined in the
total lipid fraction and in samples of abdominal fat without additional
further clean-up (by the method of Radomski and Fiserova-Bergerova, 1965).
The pesticide residues were analyzed by gas-liquid chromatography, using
Micro Tek 2000 R gas-chromatographs equipped with tritium foil electron
capture detectors. Columns were of boro-silicate glass 6' x 1/4" OD
packed with 10% DC 200/5% QF-1 coated on chromosorb W (HP) and 1.50%
OV-17/1.95% QF-1 coated on chromosorb W (HP) and 1.50% OV-17/1.95% QF 1
on chromosorb W (HP).
Results
Body Weights; The mean body weights of the control mice and the
experimental mice fed either aldrin 5 or 10 ppm, or DDT 100 ppm, at
sacrifice (age 260±15 days),are presented in Table 1. While there were
some statistically significant differences (p <0.05) in the body weights
of some of the groups of these animals over seven generations, there was
no significant pattern.
Total Lipids; Comparison of the F^ with the P generations of all
groups of mice showed an increase in total body lipids (g/mouse). The
increases were statistically significant (p <0.05) in all groups except
in the female control mice. The increase in total lipids was dramatic
in male mice fed aldrin 10 ppm. In subsequent generations, total body
lipids showed no specific pattern except in the male and female mice fed
aldrin 10 ppm, in which total body lipids remained consistently elevated
from the F-^ to Fg generations. (Table 2)
Concentrations of Pesticides in the Lipids of the Total Carcass
and Abdominal Fat; Since organochlorine pesticides may not be distributed
uniformly in all fatty tissues of the body, concentrations of dieldrin,
p,p'-DDT, p,p'-DDE, and p,p'-DDD were determined in the total lipids of
the entire carcass and in abdominal fat taken at necropsy (Table 3 and
Figures 1-3). The pesticide concentrations in total body lipids and in
abdominal fat were essentially the same. Statistically significant
differences were noted only in males and females of the P generation
mice fed DDT 100 ppm. The mean concentrations of total DDT in the abdominal
fat of male and female mice fed DDT 100 ppm were 980 and 950 ppm,
respectively (Figure 3), while in the lipids of the entire carcass, the
mean concentrations of total DDT were 514 and 423 ppm, respectively.
(Table 3).
39
-------
Table 1
Body Weights of Mice (g) at Time of Sacrifice
Mean and Standard Deviation per Generation
(Ages of Mice: 260 ± 15 Days)
f\ j
Fed P
Control M 35.3+2.0
(4)*
F 36.9+3.3
(19)
Aldrin M 40.0+2.8
5 ppm (5)
F 34.0+2.3
(10)
Aldrin M 33.1+2.7
10 ppm (13)
F 33.0+3.5
(8)
DDT M 34.6±1.4
100 ppm (4)
F 32.2+1.1
(9)
42.
36.
40.
37.
46.
38.
36.
37.
Fl
2+4.2
(11)
8+4.8
(19)
1+3.3
(6)
5+6.4
(10)
4+4.2
(6)
2+3.3
(8)
0+2.3
(5)
2+3.7
(8)
40
39
40
35
38
36
38
35
GT7 M
F2
.0+4.2
(12)
.2±4.8
(20)
.3+6.3
(6)
.8+3.4
(9)
.7+1.8
(6)
.8+3.4
(9)
.9+2.8
(5)
.7+3.8
(10)
E R
38
35
37
35
39
36
39
37
A T I (
F3
.9+2.7
(11)
.2+3.4
(18)
.2+7.1
(6)
.1+3.0
(9)
.5+3.9
(5)
.4+4.3
(9)
.0+3.8
(6)
.1+6.9
(10)
)M C ___.
F4
38.5±3.4
(11)
33.3±3.0
(15)
37.7+4.9
(6)
35.7+4.4
(7)
40.0+5.7
(6)
37.6+5.6
(9)
33.4±4.8
(6)
30.4±3.4
(8)
32.
36.
33.
33.
39.
38.
34.
39.
FS
2±2.6
(ID
8+4.2
(17)
2+2.5
(5)
2±4.9
(7)
4±2.9
(6)
6+1.4
(6)
7+4.2
(6)
1+3.4
(5)
36
36
42
35
36
37
41
41
F6
. 4±1 . 8
(9)
.2±2.2
(13)
.2±3.2
(6)
.2±3.,3
(6)
.5+2 ,,8
(7)
.4+4.0
(7)
.7+4.9
(5)
.0±2.0
(3)
* Number of mice per group
40
-------
Table 2
Total Lipids (g/mouse)
(Mean and Standard Deviation per Generation)
Compound GENERATIONS
SBX
Fed ?z± P F! F2 F3
Control M 2.92+0.46 6.31±1.32 5.60+1.14 6.38+1.63 4.57+1.14 2.50+1.09 6.85±2.70
F 4.02±0.69 5.06±1.29 5.38±2.60 4.17±1.14 3.64±1.32 2.94+0.40 5.20+2.61
Aldrin M 3.88+.0.80 6.77±3.08 7.90±2.74 7.18+2.56 7.68±3.95 2.97±3.95 8.65±2.21
5 ppm F 3.64±1.02 6.32±3.04 6.13±2.82 6.30+1.23 7.02+5.79 2.18±0.48 6.86+2.24
Aldrin M 2.12±0.53 10.90+.1.63 6.60+1.71 8.85+1.35 7.26+3.56 5.14+1.21 6.18+2.52
10 ppm F 3.04±1.38 6.90+1.52 5.94±3.98 7.79±1.89 7.01±4.85 5.20+1.91 7.71+3.39
DDT M 2.25+0.52 5.44+1.11 7.19+1.29 5.42+1.21 3.29+1.31 2.26+0.77 7.37+3.85
100 ppm F 2.86±1.12 6.28+2.08 5.61±1.57 6.63+2.10 2.60±0.59 3.87+0.59 5.90±1.19
41
-------
Parent (P) to Fo Generations Fed Dieldrin; In the second generation
mice (Pi) fed aldrin 5 ppm, the mean concentration of dieldrin in the lipids
of the entire carcass rose to a value of 50% above the concentration found
in the P generation (mean values: 34-51 ppm), (Table 3). Total retention
of dieldrin in the entire carcass of these mice rose approximately three
times above the quantities found in the P generations (mean values: 0.12
to 0.37 mg/mouse), (Figure 4). No further significant changes in concen-
tration and total retention of pesticides occurred in the F£ and Fo
generations.
In the second generation (F^) male and female mice, when compared to
the P generations fed aldrin 10 ppm, the increases in mean dieldrin
concentration in the lipids of the entire carcass approximately doubled
(mean values: 60 to 118 ppm), (Table 3). Total retention increased almost
eight-fold in the mice fed aldrin 10 ppm (mean values: 0.13 to 1.0 mg/mouse),
(Figure 5). No further statistically significant changes in the concen-
tration and total retention of dieldrin in male and female mice fed aldrin
10 ppm occurred in the F2 and F3 generations.
Parent (P) to FS Generations Fed DDT; In the mice fed DDT 100 ppm,
the concentration of total DDT in the lipids of the entire carcass
increased markedly (69%) from the P to the second (F^) generation.
Retention of total DDT in the total body also increased significantly,
approximately five-fold in the second generations (mean values: 1.1 to
5.2 mg/mouse). Comparison of the concentration and retention of total DDT
in the male and female mice of the F2 and Fg generations fed DDT 100 ppm
showed no statistically significant changes when compared with the F^
generation mice.
F^ Generations: The question was raised whether the increases in
pesticide concentration in the Fj_, F2, and F3 generation mice were
possibly related to pesticide-induced inherited characteristics. To test
this hypothesis, all groups of the F^ generation experimental mice were
fed the control diet from weaning to sacrifice, with the feeding of
pesticide diets resumed to the F5 and F6 generations.
At sacrifice o'f all experimental F4 generation mice (which had absorbed
pesticides in utero and via mother's milk, and ingested the pesticide-free
diet from weaning to death), it was found that concentrations of dieldrin,
p,p'-DDT, p,p'-DDE, and p,p'-DDD in the lipids of the total body and in
abdominal fat, as well as the retention of these pesticides in the total
carcass, were below the levels of sensitivity of the analytical^method.
Briefly summarizing: in the Swiss-Webster mouse, the retention of
dieldrin following the feeding of aldrin with the diet to four generations
(P, F]_, F2, and F3) led to a significant increase in the concentration of
dieldrin in the lipids of the total carcass and abdominal fat, and to a
significantly increased total retention of dieldrin in the total carcass
in the second generation (F^). There were further gradual, though not
statistically significant, increases in concentration in the total
retention of dieldrin in F2 and F^ generation mice. The fifth
42
-------
150
I 10°
a
Tl
e
o
-
g
o
a
o
50
Figure 1
Dieldrin (ppm) in Abdominal Fat of Mice Fed Aldrin 5 ppm
F2
F3
Generations
Male A-
Female O
IfL.
43 '
-------
Figure 2
Dieldrin (ppm) in Abdominal Fat of Mice Fed Aldrin 10 ppm
300
A
a
c
200
g,
a
M
8
100
Generations
Male A
Female O
44 V
-------
Figure 3
Total DDT (ppm) in Abdominal Fat of Mice Fed DDT 100 ppm
3000
4J
-------
Table 3
Dieldrin and Total DDT Concentrations (ppm)
in Total Lipids of Entire Carcasses
(Mean and Standard Deviation per Generation)
Compound
Fed SeA
P
F!
F2
-GENERATIONS
F3 F4 FS
F6
Control M <<<<<<<
F <<<<<<<
Aldrin
5 ppm M
F
Aldrin
10 ppm M
F
DDT
100 ppm M p,p'
P»P'
P,Pf
Total
F P,p'
P,P'
P»Pf
Total
-DDT
-DDE
-DDD
DDT
-DDT
-DDE
-DDD
DDT
Minimum detectable
31+
381
601
601
4681
35+
111
5141
3631
29+
31+
423+
levels :
10
10
12
16
51
12
2
63
49
9
15
74
50+ 7
521 7
911 12
145+ 40
810+181
66+ 10
34+ 6
9101193
585±127
48+ 32
31+ 10
664+160
dieldrin
P
P
P
,p'-DDT
,p'-DDE
,p'-DDD
56+ 5
661 6
89+ 8
1361 8
9911121
38+ 29
101 9
10391184
754+130
34+ 32
36+ 20
824+184
<1.0 ppm
<2.0 ppm
<1.0 ppm
<1.0 ppm
501
751
1191
1141
17
35
12
25
9541233
49+
361
36
13
10391275
865+179
26+
42+
24
14
933+194
' 88+
< 931
c 1151
' 1491
16
53
5
64
! 6491145
' 291
: 251
11
7
' 7031163
' 6021154
' 301
' 25+
16
15
= 567+185
56+
671
1211
1591
8151
48+
581
9211
5731
731
51+
7
75
30
19
26
31
42
44
99
44
20
697+159
46
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Figure 4
Dieldrin in Total Carcass of Mice Fed Aldrin 5 ppm (mg/mouae)
1.00
/"X
$
v^ 0. 75
0)
§
V
P.
n 0.50
TJ
O
to
4)
Pu
« 0.25
4J
O
H
/
r'l
^
p
J
f
j
_
^-/
/
/
Fl
-1
. *
,'
{
y
F2
i
\
4
* 1
V
<
\
\
. i
F3
\
\
V
v Ay
^y Jf
F4
y
f
.
-
»
'/
(
\
\ F5
j
'r
I * y^*
/ ./
/
f
"
F6
Generations
Male A
Female O-
^ ^
47
-------
Figure 5
DieIdrin in Total Carcass of Mice Fed Aldrin 10 ppm (mg/mouse)
2.00
a
^ 1.50
01
1
M
-------
Figure 6
Total DDT in Total Carcass in Mice Fed DDT 100 ppm (me/mouse)
15.00
oo
3
M
O
O
O
Pesticides per
Tota
O
O
7'
F2
Generations
Male A
Female O-
F5
r
4
49
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generations of experimental mice, born of parents that carried a
considerable load of dieldrin, were fed from weaning the control diet,
leading to a complete depletion of this pesticide even though the animals
had absorbed dieldrin In utero and via lactation. Concentration and
retention of DDT followed the same general pattern as that of dieldrin.
Comparison of the Concentration and Total Body Retention of Pesticides
in P and Fg Generations: The P and F5 generations provide "comparable"
baseline data. They were essentially identical, differing only in that
(1) the parents of the P generations, in contrast to the parents (F^) of
the F5 generations, did not absorb pesticides in utero and in mothers'
milk, and that (2) the parents and grandparents of the P generation mice
had no (known) exposure to aldrin (dieldrin) or DDT and its metabolites,
while the parents (namely, F^) of the FS generations were the offspring of
parents, grandparents, and great grandparents that had ingested these
pesticides throughout their lifetime. Comparison of the pesticide concentra-
tion and total retention in P and 7$ generations reveals the following
(for both sexes combined) mean values:
Aldrin 5 ppm
Mean concentration of dieldrin in the lipids of the
entire carcass:
P generation 35 ppm
FIJ generation 90 ppm
Mean retention of dieldrin in total carcass:
P generation 0.13 mg
F^ generation 0.23 mg
Aldrin 10 ppm
Mean concentration of dieldrin in the lipids of the
entire carcass:
P generation 60 ppm
Fc; generation 132 ppm
Mean retention of dieldrin in total carcass:
P generation 0.15 mg
F^ generation 0.69 mg
DDT 100 ppm
Mean concentration of total DDT in the lipids of the entire
carcass:
P generation 468 ppm
Ftj generation 635 ppm
Mean retention of total DDT in total carcass:
P generation 1.34 mg
F5 generation 2.13 mg
50
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Conclusions
The concentration of dieldrin or p,p'-DDT and metabolites in the
entire body and the total retention of these pesticides in the entire
carcass were determined over seven generations of Swiss-Webster mice
fed diets supplemented with aldrin 5 or 10 ppm or DDT 100 ppm.
As the generations progressed, particularly from Fo on, conception
was frequently delayed for three to four weeks. When pregnancy did not
occur within one month, the male was replaced by one of proven fertility.
Other signs of toxicity, such as tremors and reduction in body weight,
were not observed.
When sacrificed at age 260 + 15 days, total body lipids in the F-^
generations of all groups showed statistically significant increases
when compared with the respective P generations. In subsequent generations,
total body lipids showed no specific trend with the exception of the mice
fed aldrin 10 ppm. In these animals, total body lipids remained elevated
from FI to Fg generations.
In all generations, the pesticide concentrations in abdominal fat
were essentially the same as the concentrations in the total body lipids
with one notable exception, namely, in the mice of the P generation fed
DDT 100 ppm. The mean concentrations of DDT and its metabolites in the
abdominal fat of these animals were approximately twice the concentrations
in the lipids of the total body.
Mice fed aldrin 5 or 10 ppm, or DDT 100 ppm, showed a significant
increase in both concentration and total retention of dieldrin or DDT and
metabolites in the total carcass of the second generation (F^) when compared
with the concentration and retention of these compounds in the P generation
mice. No further significant changes occurred in generations F£ and F3.
Feeding of the control (pesticide-free) diet to all groups of the F^
generation experimental mice from weaning to sacrifice resulted in the
complete excretion of pesticides absorbed by the animals while in utero
and with their mothers' milk.
Resumption of the feeding of aldrin 5 and 10 ppm, and DDT 100 ppm, to
generations F5 and Fg resulted, in the F5 generation, in markedly elevated
total body pesticide levels and in markedly increased concentrations of
pesticides in the total body lipids (when compared with the concentration
and retention in the P generation), and to further increases in concentra-
tion and total pesticide retention in the F/- generation.
The increase, in both pesticide concentration and retention, in the
F$ generation above that of the P generation suggests that pesticide-
induced inherited characteristics played a role in modifying the mechanism
underlying pesticide retention. It will require further analyses of these
data and additional studies to find the answer.
51
-------
Primary contributions to this investigation were made by
the following:
William B. Deichmann, Principal Investigator
William E. MacDonald, Co-Principal Investigator
Dewey A. Cubit, Chemist
A. Gerald Beasley, Chemist
Mary D. Balkus, Technician
52
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References
Keplinger, M.L., Deichmann, Wm.B., and Sala, F.
Effects of combinations of pesticides on reproduction in mice.
(Presented at the Sixth Inter-American Conference on Toxicology
and Occupational Medicine, August 1968); published in
Pesticides Symposia by Halos and Associates, Inc., Miami,
Florida, 1970 (p. 125-138).
Radomski, J.L. and Fiserova-Bergerova, V.
The determination of pesticides in tissues with the electron
capture detector without prior clean-up.
Ind. Med. Surg., _34: 934-939, 1965.
Deichmann, Wm. B.
Progress Report to National Institutes of Health (Grant No.
ES 00052-06), 1969.
Folch, J., Lees, M., Sloan, S.G.H.
A simple method for the isolation and purification of total
lipids from animal tissues.
J. Biol. Chem., 226; 497-509, 1957.
Deichmann, Wm.B:, MacDonald, W.E., and Cubit, D.A.
DDT tissue retention: sudden rise induced by the addition of
aldrin to a fixed DDT intake.
Science, 172: 275-276, 1971.
53
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO. 2.
EPA-650/1-74-012
4. TITLE AND SUBTITLE
Study of the Ecology of Pesticides
7. AUTHOR(S)
W. E. Deichmann
9. PERFORMING ORG\NIZATION NAME AND ADDRESS
University of Miami, School of Medicine
Research and Teaching Center of Toxicology
P.O. Box 8216
Coral Gables, Florida 33124
12. SPONSORING AGENCY NAME AND ADDRESS
( ' nvironrrEntal Protection Agency
1 ational Environmental Research Center
.Research Triangle Park, N.C. ?77ll
3. RECIPIENT'S ACCESSION-NO.
5. REPORT DATE
October 1974
6. PERFORMING ORGANIZATION CODE
8. PERFORMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
1E1078
11. CONTRACT/GRANT NO.
R801470
13. TYPE OF REPORT AND PERIOD COVERED
Final Report 1973-1974
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Mean survival of the groups of mule mice fed dieldrin 0. 3. and 10 ppm fur .1 lifetime, ranged from 12.0 to 13.3 months. Mean survival of the females
ranged from 18.6 to 21.8 months. While I here was an increase in liver weights, and an increase in the ratio of liver-to-body weight of the male and fe-
male mice fed dicldrin 10 ppm. the differences were not statistically significant. The number of male and female mice with benign tumors was essen-
tially the same in the three groups: a total of 26 of 163 control mice, 29 of 159 mice ted dieldrin 3 ppm. and 27 of 161 mice fed dieldrin 10 ppm.
When compared with the controls, the number of male and female mice showing malignant tumors decreased as the feeding levels of dicldrin increased:
from 40 control mice to 37 mice fed dicldrin 3 ppm, and to 16 mice fed dieldrin 10 ppm. Review of selected liver slides by several pathologists demon-
strated a difference in their diagnosis of the lesions as being cither non-neoplastic or neoplastic. Difficulties in reaching a unanimous decision have been
recognized lor some time and became the subject of discussion at the working conference on liver cancer in the summer of 1969. organi/ed by WHO
International Agency for Research on Cancer.
In the aldrin-dieldrin-endrin feeding study (Deichmann el a!., Ind. Mcd. Surg. 39:426-434, 1970), the number ol animals dying wild tumors was re-
lated to the total number of rats with which the feeding experiment was initialed. Recalculation of the data, but based on the number of male and fe-
male rats that were actually at tumor risk, confirmed the original conclusions that there were no statistically significant differences in the tumor inci-
dence between the control and experimental rats. A second rat-feeding experiment with aldrin was conducted with two strains of weanling female rats:
Osbornc-Mendel and Spraguc-Dawley (50 rats per dose level). The reasons for the use of female rals were I hat in llie 1970 aldrin-dieldrin feeding ex-
periment (1) tumors were noted most frequently in females, i.e., mammary tumors; (2) signs of acute loxicily, a reduced life span, and a marked in-
crease in the ratio of liver-to-body weight were noted primarily in the female rals. Two strains were used, since susceptibility to tumors varies. Based
on (1) the incidence of all tumors found at necropsy in the female Osborne-Mendcl and Spraguc-Dawley rats led aldrin with the diet at 0, 20, and 50
ppm, and on (2) an age-adjusted risk analysis carried out on the number of rats that developed mammary tumors, it is concluded that the lifetime feed-
ing of aldrin 20 and 50 ppm neither increased nor decreased the incidence of tumors in these two strains of female rats above that noted in the respec-
tive control groups. It will require completion of the microscopic studies to determine whether the feeding of aldrin altered the distribution of benign
and malignant tumors.
In the third study, Swiss-Webster mice were fed aldrin 5 and 10 ppm, and DDT 100 ppm, for seven generations. As the generations progressed, parti-
cularly from F3 on, conception was frequently delayed from three to four weeks. No other signs of toxicily were noted. When sacrificed at age 260
± 15 days, total body lipids in the ! l generations of all groups showed statistically significant increases when compared with the respective parent (P)
generations. In subsequent generations, total body lipids showed no specific trend with the exception of mice fed aldrin 10 ppm, in which total body
lipids remained elevated from generations !'[ to 1;6. In all generations, the pesticide concentrations in abdominal fat were essentially the same as the
pesticide concentrations in total body lipids, except in mice of the P generation fed DDT 100 ppm, in which the concentrations of total DDT in ab-
dominal tat were approximately twice the concentrations in the lipids of the total body. All experimental mice showed a significant increase in both
concentration and total retention of dieldrin or total DUT in the total carcass of the second generation (I-1) when compared with the P generation.
Feeding of the pesticide-free diet to all experimental mice of the T"4 generations resulted in the complete excretion of the pesticides absorbed by thesi;
animals while in utcro and with their mothers' milk. Resumption of the feeding of the experimental diets by generations I'5 and l;6 resulted in marked-
ly elevated body pesticide retention and in markedly increased concentrations of pesticides (dieldrin or DDT plus metabolites) in the total body lipic
and to some further increases in concentration or retention in the l;6 generations.
17.
KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS
Carcinogen icity
Mice
Aldrin
Reproduction
Retention
Dieldrin
Liver Tumors
DDT
Swiss -Webster
Seven Generation
Rats
18. DISTRIBUTION STATEMENT
Unlimited
b.lDENTIFIERS/OPEN ENDED TERMS
19. SECURITY CLASS (This Report)
unclassified
20. SECURITY CLASS (This page)
unclassified
c. COSATI Held/Group
21. NO. OF PAGES
22. PRICE
EPA Form 2220-1 (9-73)
54
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