United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Cincinnati OH 45268
EPA-600 1-78-069
December 1978
Research and Development
&EPA
Fate of Ingested
Chrysotile Asbestos
Fiber in the
Newborn Baboon
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series. This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals — but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/1-78-069
December 1978
FATE OF INGESTED CHRYSOTILE ASBESTOS
FIBER IN THE NEWBORN BABOON
by
William H. Hallenbeck
Kusum Patel-Mandlik
School of Public Health
University of Illinois
Chicago, Illinois 60680
Order No. CA-7-3159-J
Project Officer
James R. Millette
Exposure Evaluation Branch
Health Effects Research Laboratory
Cincinnati, Ohio 45268
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Health Effects Research Laboratory,
U. S. Environmental Protection Agency, and approved for publication. Approval
does not signify that the contents necessarily reflect the views and policies
of the U. S. Environmental Protection Agency, nor does mention of trade names
or commercial products constitute endorsement or recommendation for use.
ii
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FOREWORD
The U. S. Environmental Protection Agency was created in response to in-
creasing public concern about the dangers of pollution to the health and wel-
fare of the American people and their environment. The complexities of en-
vironmental problems originate in the deep interdependent relationships be-
tween the various physical and biological segments of man's natural and social
world. Solutions to these environmental problems require an integrated pro-
gram of research and development using input from a number of disciplines.
The Health Effects Research Laboratory was established to provide sound
health effects data in support of the regulatory activities of the EPA. Eval-
uating man's exposure to environmental health hazards is a key segment in
developing such a data bank. Studies of exposure require indentification,
characterization, and quantification of physical, chemical, and biological
agents found in the environment. In addition, exposure assessment involves
the determination of conditions that cause agents to be released into the
environment, the study of the routes and pathways to man, and research into
the body's ability to prevent the entrance of environmental hazards.
This report presents the results of a study to determine if ingested as-
bestos fibers can penetrate the gastrointestinal tract and be transported to
other tissues in the body. An understanding of how the body handles asbestos
is important in determining the potential health effects of asbestos in drink-
ing water.
R. J. Garner
Director
Health Effects Research Laboratory
iii
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ABSTRACT
The objective of this research was to determine if orally administered
chrysotile asbestos fibers can penetrate through the gastrointestinal tract
of the neonate baboon and be recovered in selected tissues. A neonate baboon
was given a cumulative dose of 3 x lO1^ chrysotile fibers per kilogram. At
the end of a 9-day feeding period test and control animals were sacrificed.
The following tissues were collected: kidney, liver, spleen, heart, lymph
nodes, urine, blood, and gastrointestinal tract.
Many methodological problems were encountered in preparing tissues for
evaluation by electron microscopy. After developing a satisfactory preparation
technique, tissue samples of kidney cortex, kidney medulla, spleen, and liver
were analyzed. Definitive data are presented which demonstrate the recovery
of chrysotile asbestos from kidney cortex tissue. Data concerning the kidney
medulla, spleen and liver tissues must be considered preliminary as more de-
finitive work is in progress concerning these and other tissues.
This report was submitted in fulfillment of Contract No. W00112 by the
School of Public Health, University of Illinois Medical Center under the spon-
sorship of the U. S. Environmental Protection Agency. This reports covers
the period September 8, 1977 to September 15, 1978 and work was completed Sep-
tember 15, 1978.
iv
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CONTENTS
Foreword ill
Abstract iv
Figures vi
Tables vii
Acknowledgment viii
1. Introduction 1
2. Conclusions 2
3. Materials and Methods 3
Tissue preparation and analysis techniques 3
Calculation of fibers per ug dry weight of tissue
and detection limit 5
4. Results and Discussion 6
References 14
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FIGURES
Number Page
1 Photomicrograph (600x) of a preparation of neonate baboon
spleen taken near the edge of a TEM grid 7
Photomicrograph (600x) of a preparation of neonate baboon
spleen taken in the center area of a TEM grid
Photomicrograph (800x) of a preparation of neonate baboon
spleen taken in the intermediate zone of a TEM grid ....
vi
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TABLES
Number Page
1 Frequency Distribution of Fiber Lengths of U.I.C.C. Canadian
Chrysotile and Chrysotile Fibers Found in the Kidney
Cortex ........................... 4
2 Blank Data .......................... 10
3 Results of Preliminary Scanning of Selected Tissues of
Neonate Baboons ...................... H
Preliminary Fiber Concentrations in Selected Tissues of
Neonate Baboons ...................... 12
Final Analysis of Kidney Cortex Tissue of Neonate Baboon
for Chrysotile Asbestos .................. 13
Length, Diameter, and Aspect Ratio Data for Chrysotile Fibers
Found in the Kidney Cortex of the Neonate Baboon ...... 13
vii
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ACKNOWLEDGEMENTS
The authors wish to acknowledge Patrick J. Clark and James R. Millette,
of the Health Effects Research Laboratory, for their assistance in providing
size characterization of the asbestos fibers.
viii
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SECTION 1
INTRODUCTION
Asbestos is being ingested by the general population with the primary
route of exposure being drinking water.1"6 An important environmental health
question concerns whether or not chronic exposure to ingested asbestos can
cause cancer at one or more sites. Recent studies indicate that ingested
asbestos fibers can penetrate through the gastrointestinal tract and migrate
to various organs.7 Fiber penetration is a matter of concern because trans-
located fibers may induce cancer at susceptible sites anywhere in the body.
Possible sites include the lung, gastrointestinal tract, plura, peritoneum,
and other sites.8 17
The main objective of the present study was to determine if fibers could
be recovered from various tissues of a neonate baboon which had been admin-
istered chrysotile asbestos in milk suspension. This protocol represents a
departure from all previous animal ingestion experiments which used a rat or
mouse mode. With the exception of the study by Cunningham, et al,7 previous
animal ingestion studies have provided inconclusive answers to the question of
fiber penetration of the gut wall. See Hallenbeck and Hesse18 for a review of
animal ingestion experiments which were published before 1976. In light of
Cook and Olson's (personal communication) work with humans, it became im-
portant to test the hypothesis of fiber penetration in a controlled animal
experiment which employed a species closely related to man. Hence the baboon
was selected primarily for phylogenetic reasons. In addition, the neonate
baboon can be taken from the mother at birth and readily bottle-fed a known
concentration of asbestos fibers suspended in milk formula.
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SECTION 2
CONCLUSIONS
The findings of this study strongly indicate that orally administered
chrysotile asbestos fibers penetrate through the gut wall of the neonate
baboon and that migration to the kidney cortex occurs. Also it appears that
fibers with a wide range of lengths penetrate through the gastrointestinal
tract of the neonate baboon and that no selection mechanism is operative.
Further work is in progress which involves the in-depth analysis of kidney
medulla, liver, spleen, and other tissues from the same test and control
animals sacrificed in the present study.
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SECTION 3
MATERIALS AND METHODS
GENERAL PROCEDURES
Two neonate baboons (Papio anubis), born exactly 8 days apart, were used
as control and test animals. Each animal was maintained in an Isolette infant
incubator under conditions of controlled temperature, humidity and air supply.
The test animal weighed 920 grams and was bottle fed with a suspension of UICC
Canadian chrysotile in milk formula. See Table 1 for the frequency distri-
bution of UICC fiber lengths. The suspension was prepared by swirling 10 mg
of asbestos in 500 ml of distilled water which had been previously filtered
through a 0.1 ym Millipore membrane. It was mixed 1:1 with concentrated milk
formula (Similac, Ross Laboratories) prior to feeding to give 10 mg of chryso-
tile per liter. Assuming 106 fibers per nanogram, the feeding suspension
contained 1013 fibers per liter (f/£). A total of 2.8 liters was provided ad
libitum over a period of 9 days beginning 24 hours after birth. Therefore,
the total dose for the test animal was 3.0 x 1013 F/kg. The control animal
weighed 961 g and was bottle-fed with a one to one mixture of concentrated
milk formula and distilled, filtered water; 2.6 liters were consumed over 9
days. Both animals were sacrificed at the end of the 10-day trial. The
following tissues were collected from each animal after perfusion and bleeding:
gastrointestinal tract, spleen, liver, kidney, urine, lymph nodes, blood, and
heart.
Tissues preparation and analysis technique
1. Comparable weights of control and test tissues were dried in a vacuum
oven at 90°C until weights became constant.
2. Dried tissue was ashed in a low temperature plasma asher at 80 watts
for 3 hours.
3. The resultant ash was suspended in 10 ml of 1% acetic acid and ultra-
sonicated at 50 kHz for 1 minute to obtain dispersion without breakage.
Treatment with acid improved the removal of organics in the subsequent
ashing step. A check using reference UICC Canadian chrysotile indicated
that acid treatment did not disturb the morphology or diffraction pattern
under transmission electron microscopy (TEM).
4. The suspension was filtered through a 0.1 ym Nucleopore membrane and the
latter was ashed in the plasma unit at 80 watts for 3 hours. The residue
was suspended in 5 ml of filtered (0.2 ym Fluoropore) acetone and soni-
cated for one minute.
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TABLE 1 FREQUENCY DISTRIBUTION OF FIBER LENGTHS OF U.I.C.C. CANADIAN
CHRYSOTILE AND CHRYSOTILE FIBERS FOUND IN THE KIDNEY CORTEX
Length range
(in ym)
0.2-0.5
0.5-1
1-2
2-5
5-10
10-25
25-50
50-100
100-200
u.i.c.c.20
%
30.6
33.4
19.8
13.2
1.76
0.93
0.24
0.07
0.00
Kidney cortex
%
37.6
26.6
23.0
5.5
3.7
2.7
0.9
0
0
5. 6 yl were immediately withdrawn and deposited on a carbon and Formvar
coated TEM grid. Rapid drying was effected by the use of an infrared
lamp (250 watts at 5 inches from the grid).
6. Chrysotile fibers were identified by morphology, and randomly selected
area electron diffraction (SAED) patterns were obtained with a Philips
300 TEM. Grids were scanned at a magnification of 19,000 and in all cases
an equal number of test and control grid holes were scanned.
Calculation of concentration and detection limit
The concentration of fibers per mg day weight of tissue was calculated
using the following equation:
Fibers/mg dry weight of tissue = ¥ x A1 x V1 = 7 x 1()5 ^
A2 x V2 x M M
where F = average number of fibers per grid square
Ax = 0.07 cm2 = area of a 200 mesh TEM grid
A2 = 8 x 10 5 cm2 = area of one grid square
Vi = 5 ml = volume of sample suspension
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V2 = 0.006 ml = volume of suspension deposited on grid
M = mass of tissue prepared (mg dry weight)
The detection limit of this analytical technique can be calculated by sub-
stituting F = 0.1 and a particular dry weight, say M = 100 mg, in the above
equation. This substitution yields a detection limit of 700 fibers per mg
(dry weight) of tissue.
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SECTION 4
RESULTS AND DISCUSSION
Kidney, spleen, and liver tissues from control and test neonates were
prepared for TEM analysis. The preparation technique described above resulted
in a uniform distribution of particles on the grid surface; this can be seen
in the photomicrographs of the edge, intermediate, and center areas of one
grid (see Figures 1-3). Also, analysis of blank data revealed no chrysotile
fibers in 10 grid squares. See Table 2 for a description of blank data.
Preliminary screening of kidney, spleen and liver tissues (see Tables 3
and 4) indicated that the concentration of fibers in the kidney cortex was
significantly greater than its control (P = 0.005). The statistical evaluation
of data such as that shown in Table 3 has been discussed by Hallenbeck, et al.2
In order to make a final analysis of the kidney cortex preparation, an ad-
ditional 62 grid squares were scanned and the results are shown in Table 5.
Final analysis of the kidney cortex preparations revealed a statistically sig-
nificant excess of fibers in the test preparation as compared to control pre-
paration (P < 0.005). See Tables 1 and 6 for detailed information concerning
the length, diameter, and aspect ratio of fibers found in the kidney cortex.
It can be seen in Table 1 that there is very little difference in the frequency
distributions of fiber lengths of UICC Canadian chrysotile and fibers found in
the kidney cortex. Hence, there appears to be no fiber selection process oc-
curring in the gastrointestinal tract of the neonate baboon. Additional data
is being collected on kidney medulla, spleen, liver, and other tissues in order
that final analyses can be made concerning the level of chrysotile fibers in
these tissues.
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Figure 1. Photomicrograph (600x) of a preparation of neonate baboon spleen
taken near the edge of a TEM grid. Note the uniform distribution
of particulates. The same preparation was used in Figures 1-3.
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Figure 2. Photomicrograph (600x) of a preparation of neonate baboon spleen
taken in the center area of a TEM grid. Note the uniform distri-
bution of particulates. The same preparation was used in Figures
1-3. ,
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Figure 3. Photomicrograph (800x) of a preparation of neonate baboon spleen
taken in the intermediate zone of a TEM grid. Note the uniform dis-
tribution of particulates. The same preparation was used in Figures
1-3.
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TABLE 2. BLANK DATA
Grid square
number
1
2
3
4
5
6
7
8
9
10
Total fibers
Blank 1*
0
0
0
0
0
0
0
0
0
0
0
Blank 2*
0
0
0
0
0
0
0
0
0
0
0
Blank 1 was prepared by placing 6 yl of acetone (previously filtered through
a 0.2 ym Fluoropore membrane) on a TEM grid with a carbon coated Formvar
substrate. Blank 2 was prepared by placing 6 yl of an acetone suspension
(5 ml) of an ashed Nucleopore membrane (0.1 ym) on a TEM grid with a carbon
coated Formvar substrate.
10
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TABLE 3. RESULTS OF PRELIMINARY SCANNING OF
SELECTED TISSUES OF NEONATE BABOONS
Number of chrysotile fibers
Grid square
number
1
2
3
4
5
6
7
8
9
10
Total fibers
Kidney cortex
ca
0
0
0
0
0
0
0
0
0
0
0
T3
0
0
0
0
0
1
6
2
0
1
10C
Kidney
C
0
0
0
0
0
0
0
0
0
0
0
medulla
T
2
0
0
0
2
0
0
0
0
0
4d
Liver
C
0
0
0
0
0
0
0
0
0
0
0
T
0
0
0
0
0
0
0
0
0
0
0
Spleen
C
0
0
0
0
0
0
0
0
0
0
0
T
0
0
0
0
0
0
b
1
0
0
0
1
T = test neonate; C = control neonate
""The single fiber observed in the spleen of the test neonate was composed of a
bundle of 40-50 fibrils.
:P = 0.005
*P = 0.10.
11
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TABLE 4. PRELIMINARY FIBER CONCENTRATIONS IN
SELECTED TISSUES OF NEONATE BABOONS
Kidney Spleen Liver
Medulla Cortex
CS Ta C T C T C T
Dry weight (mg) 38 57 133 158 168 168 297 328
Chrysotile fibers/ing ,
dry weight BD 2,456 BD 4,430° BD 417 BD BD
C = control neonate; T = test neonate, bottle-fed a total dose of 3.0 x 1013
chrysotile fibers/kg body weight in a suspension of milk formula.
BD = below detection
CP = 0.005
r = 0.10. Because of the low mass of medullar tissue 12 yl were deposited on
the TEM grid rather than 6 yl.
12
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TABLE 5. FINAL ANALYSIS OF KIDNEY CORTEX TISSUE OF
NEONATE BABOON FOR CHRYSOTILE ASBESTOS
No. of grid squares scanned
No. of chrysotile fibers found
Dry weight (mg)
Chrysotile fibers/mg dry weight
Control Tissue
70
0
133
BDb
Test Tissue
72
119a
158
7,3223
P <0.005
BD = below detection
TABLE 6. LENGTH, DIAMETER, AND ASPECT RATION DATA FOR CHRYSOTILE
FIBERS FOUND IN THE KIDNEY CORTEX OF THE NEONATE BABOON
Average
Standard deviation
Maximum
Minimum
Median
Geometric mean
Length
ym
1.7
4.38
35
0.24
0.5
0.73
Width
ym
0.04
0.04
0.34
0.03
0.03
0.04
Aspect ratio
39.5
89.5
800
5
16.7
20.4
13
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REFERENCES
1. Asbestos in the Great Lakes Basin, with Emphasis on Lake Superior. A re-
port to the International Joint Commission from the Great Lakes Research
Advisory Board, 1975, p. 35.
2. A Study of the Problem of Asbestos in Water. The American Water Works As-
sociation Research Foundation, Denver, Colorado, J. Am. Water Works Assoc.
(2):l-22, 1974.
3. Cunningham, H.M., Pontefract, R.: Asbestos fibers in beverages and drink-
ing water. Nature. 232:332-333, 1971.
4. Durham, R.W., Pang, T.: Asbestos fibers in Lake Superior, Water Quality
Parameters, ASTM STP 573, American Society for Testing and Materials.
1975, pp. 5-13.
5. Cook, P.M., Glass, G.E., Tucker, J.H.: Asbestiform amphibole minerals
detection and measurement of high concentrations in municipal water sup-
plies. Science. 185:853-855, 1974.
6. Kay, G.H.: Asbestos in drinking water. J. Amer. Water Works Assoc.
66:513-514, 1974.
7. Cunningham, H.M., Moodie, E., Lawrence, G., and Pontefract, R.D.: Chronic
effects of ingested asbestos in rats. Arch. Environ. Contain. Toxicol.
6:507, 1977.
8. Selikoff, I.J., Hammond, E.G., Seidman, H.: Cancer risk of insulation
workers in the United States. Bogovski, P., Timbrell, V., Gilson, J.C.,
Wagner, J.C., Davis, W. (ed): The proceedings of the conference on the
Biological Effects of Asbestos, Lyon, France, International Agency for
Research on Cancer, WHO, 1972, pp. 209-216.
9. Selikoff, I.J.: Epidemiology of gastrointestinal cancer. Environ. Health
Perspect. 9:299-305, 1974.
10. Enterline, P., DeCoufle, P., Henderson, V.: Mortality in relation to oc-
cupational exposure in asbestos industry. J. Occ. Med. 14(12): 897-903,
1972.
11. Newhouse, M.L.: Cancer among workers in the asbestos textile industry.
Bogovski, P., Timbrell, V., Gilson, J.C., Wagner, J.C., Davis, W., (ed):
The proceedings of the conference on the Biological Effects of Asbestos,
Lyon, France, International Agency for Research on Cancer, WHO, 1972,
pp. 203-208.
14
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12. Newhouse, M., Berry, G., Wagner, J.C., Turok, M.E.: A Study of the mor-
tality of female asbestos workers. Br. J. Ind. Med. 29:134-141, 1972.
13. Enterline, P.E.: Mortality among asbestos products workers in the United
States. Ann. N.Y. Acad. Sci. 132:156-165, 1965.
14. McDonald, J.C.: Cancer in chrysotile mines and mills. Bogovski, P.,
Timbrell, V., Gilson, J.C., Wagner, J.V., Davis, W. (ed): The proceedings
of the conference on the Biological Effects of Asbestos, Lyon, France,
International Agency for Research on Cancer, WHO, 1972, pp. 189-194.
15. Enterline, P.E., and Kendrick, M.A.: Asbestos dust exposures at various
levels and mortality. Arch. Environ. Health. 15:181, 1967.
16. Hammond, E.G., Selikoff, I.J., and Churg, J.: Neoplasia among insulation
workers in the United States with Special reference to intra-abdominal
newplasia. Ann. N.Y. Acad. Sci. 132:519, 1965.
17. Selikoff, I.J., Churg, J., and Hammond, E.G.: Asbestos exposure and neo-
plasia. J. Amer. Med. Assoc. 188:22, 1964.
18. Hallenbeck, W.H., Hesse, C.S.: A review of the Health Effects of ingested
asbestos. Reviews on Environmental Health. 2:157-166, 1977.
19. Hallenbeck, W.H., Chen, E.H., and Wolff, A.H.: Precision of analysis for
waterborne chrysotile asbestos by transmission electron microscopy.
Bull. Environ. Contam. Toxicol. 17:551-558, 1977.
20. Rendall, R.E.G.: The data sheets of chemical and physical properties of
the U.I.C.C. standard reference samples. In Pneumonconiosis Proceedings
of the International Conference. (Johannesburg), edited by Shapiro, H.A.,
Oxford University Press, pp. 23-27, 1970.
15
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
REPORT NO.
EPA-600/1-78-069
2.
TITLE AND SUBTITLE
Fate of Ingested Chrysotile Asbestos Fiber
in the Newborn Baboon
REPORT DATE
December 1978
6. PERFORMING ORGANIZATION CODE
AUTHOR(S)
William H. Hallenbeck and Kusum Patel-Mandlik
8. PER
PERFORMING ORGANIZATION NAME AND ADDRESS
School of Public Health
University of Illinois
Chicago, IL 60680
10. PROGRAM ELEMET
6l4B(d)
11. CONTRACT/GRANT NO.
Order No. CA-7-3159-J
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory - Cinn, OH
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268 .
13. TYPE OF REPORT AND PERIOD COVERED
Final - 9/8/77-9/15/78
14. SPONSORING AGENCY CODE
EPA/600/10
15. SUPPLEMENTARY NOTES
To be submitted to:
J. of Environmental Pathol. Toxicol
16. ABSTRACT
The objective of this research was to determine if orally administered chrysotile
asbestos fibers could penetrate through the gastrointestinal tract of the neonate
baboon and be recovered in selected tissues. A neonate baboon was given a cumulative
dose of 3 x 1013 chrysotile fibers per kilogram. At the end of a 9-day feeding
period test and control animals were sacrificed. The following tissues were collected
kidney, liver, spleen, heart, lymph nodes, urine, blood, and gastrointestinal tract.
Many methodological problems were encountered in preparing tissues for evaluation by
electron microscopy. After developing a satisfactory preparation technique, tissue
samples of kidney cortex, kidney medulla, spleen, and liver were analyzed. Definitive
data are presented which demonstrate the recovery of chrysotile asbestos from kidney
cortex tissue. Data concerning the kidney medulla, spleen and liver tissues must be
considered preliminary as more definitive work is in progress concerning these and
other tissues.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Asbestos, Serpentine,
Ingestion (biology), Laboratory animals,
Potable water
Health Effects
06 F
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
20. SECURITY CLASS (This page)
UNCLASSIFIED
21. NO. OF PAGES
-24
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS COITION is OBSOLETE
o US. SMJOMDITrarrBGOfflCL B79 -657-060/1545
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