&EPA
United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Cincinnati OH 45268
EPA-600/1 -80-030
July 1980
Research and Development
Effects of
Chloroform in the
Drinking Water of
Rats and Mice
Ninety-Day Subacute
Toxicity Study
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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 biornedical instrumentation and health research techniques utilizing ani-
mals — but always with intended application to human health measures.
I his document is available to the public through the National Technical Inforrna-
Springfield, Virginia 22161.
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EPA-600/1-80-030
July 1980
EFFECTS OF CHLOROFORM IN THE DRINKING WATER OF RATS AND MICE
Ninety-Day Subacute Toxicity Study
by
Ted A. Jorgenson and Carol J. Rushbrook
Mammalian Toxicology Program
SRI International
Menlo Park, California 94025
Contract No. 68-03-2616
Project Officers
Richard J. Bull
Merrel Robinson
Toxicological Assessment Branch
Health Effects Research Laboratory
Cincinnati, Ohio 45268
and
Carrie E. Whitmire
Carcinogenesis Testing Program
National Cancer Institute
Bethesda, Maryland 20014
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-
Cincinnati, U.S. Environmental Protection Agency, and approved for publica-
tion. 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.
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FOREWORD
The U.S. Environmental Protection Agency was created because of
increasing public and government concern about the dangers of pollution
to the health and welfare of the American people. Noxious air, foul
water, and spoiled land are tragic testimony to the deterioration of
our national environment. The complexity of that environment and the
interplay between its components require a concentrated and integrated
attack on the problem.
Research and development is that necessary first step in problem
solution and it involves defining the problem, measuring its impact, and
searching for solutions. The primary mission of the Health Effects Re-
search Laboratory in Cincinnati (HERL) is to provide a sound health
effects data base in support of the regulatory activities of the EPA.
To this end, HERL conducts a research program to identify, characterize,
and quantitate harmful effects of pollutants that may result from ex-
posure to chemical, physical, or biological agents found in the environ-
ment. In addition to the valuable health information generated by these
activities, new research techniques and methods are being developed that
contribute to a better understanding of human biochemical and physiological
functions, and how these functions are altered by low- level insults.
In this report the results are presented from Phase I (the sub-
chronic study) of the study on effects of chloroform in the drinking water
of rats and mice. This phase was initiated to provide data for setting
dose levels for the chronic-phase of testing chloroform in the drinking
water of rats and mice. More reliable data is being sought on the
chronic toxicity and carcinogen! city of chloroform to be used in risk
assessment and to validate EPA standards for trihalomethanes in drinking
water.
R. J. GARNER
Director
Health Effects Research Laboratory
111
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ABSTRACT
This research program was initiated with the overall objective of pro-
viding toxicologic and range-finding data sufficient for setting dose levels
for the chronic-phase testing of chloroform in drinking water of male rats
and female mice.
A 90-day subacute study was designed to investigate the effects of five
levels of chloroform (200, 400, 600, 900, 1800 ppm) in the drinking water of
rats and six dose levels of chloroform (200, 400, 600, 900, 1800, and 2700
ppm) in the drinking water of mice. Body weight, water consumption, chloro-
form concentration in blood serum, kidney fat-to-kidney weight ratios, and
gross and microscopic pathology findings were examined in rats. Body weight,
water consumption, organ fat/organ weight ratios, and gross and microscopic
pathologic findings were examined in mice.
Results showed that the body weights of rats receiving high chloroform
levels were affected by treatment, but the variances were less than 20%. A
dose-response was observed in water consumption, but intake at high chloroform
levels did not drop below the generally accepted maintenance level of 25 ml
per rat per day. No effect was observed on the percentage of kidney fat and
on the chloroform concentration in serum. Gross and microscopic pathology
findings generally were slight or mild in severity, not dose-related, and
either appeared adaptive (occurred in rats sacrificed after 30 or 60 days but
not in those sacrificed after 90 days) or were sporadic and (by nature and/or
incidence) not considered related to treatment.
Mice receiving 900, 1800, and 2700 ppm sustained body weight losses
during the first week, but thereafter all body weights were comparable to
those of controls. Considerable variability was evidenced in the water con-
sumption, and as a result, a dose-dependency was not evident. The percentage
of liver fat showed a statistically significant increase at the 2700 ppm level
throughout the study. Gross pathological examinations revealed occasional
hemorrhaging in the lungs of mice from all dose levels. Histologically,
centrolobular fatty changes in mouse livers appeared related to treatment.
Extramedullary hematopoesis in the liver and lymphoid atrophy of the spleen
were also observed. Other lesions were sporadic and (by nature and/or
incidence) not considered to relate to treatment.
IV
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CONTENTS
Foreword ill
Abstract iv
Tables vi
Acknowledgement vii
1. Introduction 1
2. Conclusions and Recommendations .' 3
3. Experimental Procedures . . 4
Compound Source and Identification 4
Preparation of Chloroform Water Solutions 4
Analytical Methods ... 5
Animals and Housing. . 9
Animal Groups and Dose Levels . 9
Observations 10
4. Results and Discussion 14
v
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TABLES
Number Page
1 Results of Analytical Chemistry Analyses - Rats 18
2 Results of Analytical Chemistry Analyses - Mice 19
3 Average Chloroform Levels in Rat Serum (ppb) 20
4 Liver Fat as a Percentage of Liver Weight - Mice 21
5 Kidney Fat as a Percentage of Kidney Weight - Rats ..... 22
6 Average Body Weights of Female Mice Receiving
Chloroform in Their Drinking Water (Grams) 23
7 Average Body Weights of Male Rats Receiving
Chloroform in Their Drinking Water 24
8 Average Water Consumption of Female Mice
Receiving Chloroform in Their Drinking Water 25
9 Average Water Consumption of Male Rats
Receiving Chloroform in Their Drinking Water 26
10 Average SCOT and LDH Values for Female Mice
Receiving Chloroform in Their Drinking Water 27
11 Summary of Clinical Chemistry Data for Rats
Receiving Chloroform in Their Drinking Water 28
Baseline and 30-Day Sacrifice
60-Day Sacrifice
90-Day Sacrifice
12 Summary Histopathological Findings in Mice 31
13 Summary Histopathological Findings in Rats 32
VI
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ACKNOWLEDGEMENT
This study was conducted in SRI International's Life Sciences Division
under the direction of Dr. David C. L. Jones, Director, Toxicology Laboratory
The experimental work in toxicology was directed by Ted A. Jorgenson, M.B.A.,
Manager, Mammalian Toxicology Program, who was Principal Investigator for
the project. The chemical and analytical work was directed by Dr. Ronald
J. Spanggord, Manager, Bio-Analytical Chemistry Program. Daniel L. Combs,
Organic Chemist, participated in the analytical work. Carol J. Rushbrook,
Toxicologist, assisted with the day-to-day toxicology program. Dr. Daniel
P. Sasmore, Director of Pathology Services and a board-certified veterinary
pathologist, supervised the necropsy procedures and preparation of histo-
pathological slides and interpreted the histologic results for this report.
Technical assistance and support was provided by Peter Gribling, Sandra
Phillips, Juan Dulude, Kathleen Dulude, Janice Brown, Sandra Green, and
Barbara Kirkhart.
The cooperation of Peninsula Medical Laboratory, Menlo Park, California,
in performing the hematology and serum chemistry determinations is gratefully
acknowledged.
Vll
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SECTION 1
INTRODUCTION
An aspect of environmental pollution of concern to the Environmental
Protection Agency is the purity, acceptability, and wholesomeness of drinking
water. In the determination of whether a problem exists, the first and most
important step is to assess chemically the drinking water supplies in some
major communities. This is being done, and, as the studies progress, the
extent and magnitude of water pollution are revealed. Priorities must be
established for further investigation, based on the concentrations of
particular pollutants, their nature, and their potential for causing biologic
harm.
One water pollutant, chloroform, is known to exist at concentrations of
up to 0.3 ppm in almost all drinking waters that are disinfected with chlorine.
A possible health hazard from this compound has been identified through a
study completed for the National Cancer Institute by Hazelton Laboratories
of America, Inc., of Vienna, Virginia. Chloroform was administered by corn
oil to male and female Osborne-Mendel rats by gavage at levels of 90 and
180 mg/kg. These doses produced 8% and 24% incidences, respectively, of
renal epithelial tumors in the male rats; no renal tumors were present in
the females or in the controls. In a similar study with B6C3F1 mice, chloro-
form produced dose-dependent hepatocellular carcinomas. Little doubt remains
that, under the conditions of these studies, chloroform can induce tumors in
rats and mice. In other studies conducted at Huntingdon Research Center
with rats, several strains of mice and dogs, somewhat lower doses produced
renal tumors in only the male ICI-Swiss mice.
Even under the most extreme condition (0.1 mg/liter of CHC1« and 1 liter
of drinking water drunk daily by a 10-kg child), the doses given rats in the
Hazelton study were 9,000 and 18,000 times the normal human use level. For
a 60-kg adult, the doses of 90 and 180 mg/kg are 22,500 and 45,000 times the
human use level. Testing of animals with such high doses (maximum tolerated
dose, MTD) is intended to facilitate identification of the target organs and
to increase the possibility of lesions developing. However, the testing of
animals at or near the use-level dose (with additional levels slightly
increased to provide a safety factor) may provide more meaningful data and,
ideally, a dose-response pattern.
Another point to emphasize is that in the Hazelton study, the laboratory
animals received chloroform not in their drinking water, but by gavage in
corn oil. This method of administration may have caused a strong daily
chloroform pulse, and the corn oil may have enhanced absorption.
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Other factors are prompting investigators to define and, it is hoped,
solve the chloroform problem. Chlorination is viewed as the most effective
means of ensuring a wholesome drinking water supply, although other methods
are being investigated. Furthermore, a wide variety of other pollutants are
being found in drinking water. Interactions of chloroform with one or more
of these compounds may have biologic significance.
In the study reported here, we investigated the toxicity of chloroform
administered in the drinking water of male Osborne-Mendel rats and female
B6C3F1 mice for 90 days. The data derived from this study were used to
select the dose levels for a program to investigate the carcinogenic poten-
tial of chloroform in the drinking water of male Osborne-Mendel rats and
female B6C3F1 mice.
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SECTION 2
CONCLUSIONS AND RECOMMENDATIONS
Chloroform can be put into solution at the levels being used in this
program. Body weights of rats receiving higher dose levels were less than
20% below control weights. This difference should not affect the outcome
of a chronic program adversely. The animals did continue to gain weight and
appeared thrifty throughout. While a dose-response was evident in water
consumption, intakes remained satisfactory for animal maintenance. The
reduced water consumption did, however, slightly affect serum chemistry
values. Organ fat (kidney) was unaffected by treatment. Also, chloroform
does reach the blood stream and can be detected analytically at ppb levels.
Gross and microscopic pathology findings generally were slight or mild,
were not dose-related, and appeared adaptive or were sporadic and not
considered related to treatment.
After an initial acclimation period, body weights and water consumption
of mice were comparable to controls. There was a significant increase in
liver fat for mice given chloroform at 2700 ppm. Gross pathological
examination revealed no findings that could be attributed to treatment.
Histopathologic examination showed extramedullary hematopoesis in the liver,
lymphoid atrophy in the spleen and the centrolobular fatty changes in the
liver. Other lesions were sporadic and (by nature and/or incidence) not
considered treatment related.
All dose levels examined are suitable for inclusion in the chronic
phase. We recommend that the 200, 400, 900, and 1800 ppm levels be used
in the chronic phase of this program.
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SECTION 3
EXPERIMENTAL PROCEDURES
COMPOUND SOURCE AND IDENTIFICATION
SRI purchased a sufficient quantity of pesticide-quality chloroform
(Catalog # CX1052) from Matheson Coleman Bell (MCB). Lot No. 8G06 was used
throughout this study. Analysis for purity showed conformity with MCB
specifications. Approximately 30 ppb of diethyl carbonate (DEC) was detected
during the purity analysis. Because the presence of DEC was of concern
because of potential interference, the chloroform was distilled to remove
DEC. This distillation was performed twice a week and was restricted to
the quantities (100 ml) required for each dose-preparation period. Pro-
ceeding in this manner eliminated the need for routine monitoring of ethanol
and phosgene concentrations.
PREPARATION OF CHLOROFORM WATER SOLUTIONS
Twice weekly, on Mondays and Thursdays, fresh aqueous solutions of
chloroform were prepared and the old solutions were discarded. Each 10-liter
bottle (with stirring bar) was numbered, and a record was kept of the empty
and full weights, the calculated volume, and the amount of chloroform to be
added to achieve the desired concentration. Each bottle was filled with
distilled water to within several centimeters of the top and freshly
distilled chloroform was added using volumetric pipettes and/or microliter
syringes. The water level was brought to within 2.54 cm of the top, the
bottle was stoppered, and the stopper was secured with duct tape. The
bottle then was placed on a magnetic stirring unit (Vanlab Gyratherm UA/
#58922-054) for 16 to 24 hours of continuous mixing. No heat was used during
the stirring process.
When solutions were thoroughly mixed, they were transferred to animal
water bottles via a syringe-activated teflon siphon. For each chloroform
level prepared, two samples were removed for analysis of chloroform content,
the bottles were resealed and the solutions were saved for refilling any
animal water bottles that spilled.
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ANALYTICAL METHODS
Water Samples
Reagents:
Chloroform—from MCB, pesticide quality, distilled twice per
week, lot analyzed by mass spectrometry.
Acetonitrile—for internal standard, from Burdick and Jackson,
distilled in glass.
Water—filtered by reverse osmosis by a Milli-Pore Corporation
Milli-Q purification unit, then filtered through a charcoal
column (activated at 650°C for 12 hours), then continuously
purged with filtered nitrogen gas (Molecular Sieve-13X) in a
storage carboy.
Apparatus and Chromatographic Conditions
Gas Chromatograph (GD)—Varian model 2100 and Hewlett-Packard
5730.
Column—Chromosorb 101 Isothermal 140°C, 60/80 mesh, 6 ft
(1.8 m) x 4 mm I.D.
Flame lonization Detector (FID)—Attenuation Varian 2100,
1 x 10"11, Attenuation HP 5730A, 1 Range 10.
Integration—via Spectra-Physics Minigrator, Hewlett-Packard
3380A.
Standards
An internal standard solution of 50% acetonitrile in water was
prepared.
Twenty milliliters of purified water was sealed in a Teflon-capped vial
with a crimp-on metal ring. A small magnetic stirring bar was in the vial.
Chloroform was injected into the vial and dissolved by magnetic stirring
(0.5 hour), then 5 yl of the internal standard was added. Standard concen-
trations of chloroform were 200, 400, 600, 900, 1800, 2700 ppm, and that of
acetonitrile was about 100 ppm.
Samples
Samples were contained in sealed 20 ml vials. Five microliters of
internal standard solution was added to give a final acetonitrile concentra-
tion of 100 ppm. Both samples and standards were shaken thoroughly to
disperse the internal standard throughout the solution.
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Results
form. The Rf value, defined as
The FID responsiveness of acetonitrile was about 5 times that of chloro-
as
= Area CHC13 f ppm CH3CN
f ~ Area CH3CN * ppm CHC13 ,
was about 0.175. For quantitation of samples, an area ratio was defined as:
Area CHC13 ppm CH3CN
area ratio = Area CH3CN -- foo .
The division by 100 kept the numbers between 1 and 20.
Area ratio was plotted as a function of CHC13 (ppm) for the standards.
The inverse slope of this plot (which was always linear) gave a factor —
ppm CHCls/area ratio — that, when multiplied by the area ratio for the samples,
would give ppm CHC13. Blanks always gave an area ratio of zero. The
inverse of the slope factor, area ratio/ppm CHC13, times 100 gives an R^
value for all points on the best straight line through the standard points.
The Rf value of standards prepared every week did not change by more than 5%.
Serum Samples
Reagents
Chloroform — same as that for water samples.
Bromochlorome thane — from Aldrich Chemical Co .
Silicon Defoamer — Dow Corning, purchased from Arthur Thomas Co. Heated
a.t 80%C under reduced pressure for 20 min to remove volatiles that
would interfere with the GC analysis. The defoamer then was purged
overnight at room temperature with filtered nitrogen.
Out-of-Date Human Donor Serum — from American Red Cross. Purged to
remove volatile halogenated compounds and used for spiked standards.
Apparatus and Chromatographic Procedure
Purge Vessel — a) Standard EPA vessel with glass frit (5 ml) and
Carle gas valve; b) Hewlett-Packard Model 7675A Purge and Trap
Sampler.
Trap — Tenax GC 60/80 mesh, 15 cm x 2-5 mm I.D.
Purge Procedure — a) 95-100°C in oil or sand bath, 10 min with N2 or He
at 60 cc/min; b) room temperature, 10 minutes at 60 cc/min N2.
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Trap Desorption—2 min at 200°C.
GC Column—0.2% Carbowax 1500/Carbopack C 60/80, held at 20°C
during desorption, then increased to 160°C at 8-10°C/min.
Electron Capture Detector (BCD)—a) ScH3, Analog Technology
Corporation Model 140A, linear pulse, 250°C; b) Ni63, Hewlett-
Packard, linear pulse, 250°C.
GC—a) Varian 2100, N2 flow rate 37 cc/min; b) Hewlett-Packard
5730A, N2 flow rate 37 cc/min.
Integrator—a) Spectra-Physics Minigrator; b) Hewlett-Packard
3380A.
Attenuation—a) Base current 230, attn 214; b) attn 1.
Standards
An internal standard solution of C1CH2BR in water (0.28 ppm) was pre-
pared in a sealed vial.
A chloroform standard solution in water (0.10 ppm) was prepared in a
separate vial.
Standards for purging were prepared by injecting 20 yl of the internal
standard solution and 5, 10, 15, 20, and 25 yl of chloroform standard solu-
tion into 2 ml of purified water. Then 0.10 to 0.50 ml of human donor serum
was added to keep the viscosity and surface tension the same as those in
the sample solutions. Two to three drops of defoamer were added, the solu-
tion was purged onto the trap, and then desorption onto the analytical
column was accomplished by heating. The column was temperature-programmed
to elute the volatiles.
Samples
Serum samples were stored frozen. Just before analysis, they were
thawed at room temperature. Then 0.10 to 0.50 ml of thawed sample was added
to 2 ml of purified water containing 20 yl of the internal standard solution,
2 to 3 drops of defoamer were added, and the resulting solution was purged.
Results
Data were treated similarly to those for water samples. The area
ratio was defined as:
Area CHC13 m ,-,,, „ • -, ,_ •
Area ratio = — ciCH Br * nanograms ClCH2Br in solution.
This area ratio was calculated for the standard solutions and plotted as a
function of CHC13 (ng) in solution. Again the inverse of the slope gives
ng CHCla/area ratio. When a blank containing 2 ml of purified water, 2 to
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3 drops of defoamer, and 20 ul of internal standard solution was purged and
chromatographed, the area ratio was not zero, which means that some chloro-
form was in the samples. Therefore, the area ratio of the blank was sub-
tracted from the area ratios of the samples. This "corrected" area ratio
then was multiplied by the slope factor to give nanograms of CHC13 present
in the serum. Nanograms of chloroform present divided by ml ofkserum taken
for purging gave ppb chloroform.
Standards were run every day or every other day and were run for heated
and unheated purges (no differences in recovery or Rf were noted).
Chloroform in Air Samples
The same principles used above were applied to the analysis of chloro-
form in air samples in the animal rooms. Air samples were drawn through a
Tenax trap by a calibrated metering pump and desorbed into a gas chromato-
graph as described above.
Organ Fat
Chloroform increases lipids in liver and kidney tissues, so monitoring
this parameter in test animals is important. The procedure used to perform
this assay was as follows. The organ tissue was tamped dry on the exterior
and weighed on an analytical balance. The tissue then was homogenized with
4 ml of high-purity water (Milli-Q) using a Tektron, Inc., Polytron homo-
genizer. The homogenate was added to a separatory funnel containing 49 ml
of chloroform:methanol, 2:1, the mixture shaken for 30 seconds, and 8 ml
of 0.018 N H2SOi, added. The resulting mixture was shaken for 15 seconds, and
the total contents were added to a 150 ml centrifuge bottle, which was spun
at 2000 rpm for 20 minutes. The resulting suspension contained two layers
separated by a thin white protein disk. The upper aqueous layer was drawn
off by suction, and a 20 ml fraction of the bottom layer (chloroform) was
evaporated to dryness in a tared 3 g test tube in a water bath at 57°C.
Nitrogen gas was used to remove final traces of the solvent. The dry test
tube was placed in a dessicator overnight, and the net weight, representing
lipids, was determined.
Chloroform in Feed
The method used involved vacuum distillation of a feed and water slurry.
The distillate was collected in a dry ice/acetone cold trap (-77°C) and a
liquid nitrogen cold trap (-192°C). Chloroform solidifies at -63°C. The
contents of the traps then were rinsed out with cold water, and the water
was analyzed for chloroform by the purge-trap technique.
An aliquot of feed was ground up using a Waring blender. Ten grams were
added to a 250 ml boiling flask containing 25 ml of high-purity organic-free
water. The contents were heated (while being stirred) to 85°C for 1 hour.
The vacuum was applied by means of a water aspirator.
At the end of the distillation, each trap was rinsed out with 25 ml of
high-purity water.
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The water rinse was analyzed by a purge trap using a Hewlett-Packard
7675A Purge and Trap Sampler with a Tenax Trap. The trap was desorbed at
150°C. For the analysis a Hewlett-Packard 7430A GC containing Carbowax
1500 (0.22)/Carbopack C column was used. The temperature was programmed
to increase from 30°C to 160°C at 8°C/min, the detector was a Ni63 electron
capture detector (BCD) and the purge gas and carrier gas was nitrogen.
Pesticides, PCBs, and Phthalates in Feed
Feed (Purina Laboratory Chow) was analyzed and cleaned up in two
fractions. Each sample was analyzed by gas chromatography using ECD for
the chlorinated pesticides and PCBs, and a nitrogen phosphorus specific
detector for the organophosphorus pesticides. A packed column was used to
separate components. The results of this analysis indicated that malathion
was present. To confirm its presence, OS-mass spectrometry analysis was
done. Only phthlates (dibutyl and dioctyl) were found. Other compounds
searched for included p,p'-DDT, mirex, methoxychlor, endrin, dieldrin,
aldrin, lindane, malathion, and aroclor 1254.
ANIMALS AND HOUSING
SRI purchased 250 male Osborne-Mendel rats from Camm Research Institute,
Wayne, New Jersey, and received 300 female B6C3F1 mice from the Charles River
Breeding Laboratory, at the request of NCI. The rats were born on 9/4/78
and received at SRI at 4 weeks of age on 10/3/78; the mice were born on
9/5/78 and received at SRI at 4 weeks of age on 10/4/78. After two weeks
of quarantine, they were released for the tests at six weeks of age.
Rats were housed two per cage in 19" x 10-1/2" x 8" polycarbonate cages
containing hardwood chip bedding and the mice were housed five per cage in
19" x 10-1/2" x 6" polycarbonate cages. Cages were changed twice a week
for the rats and once a week for the mice by the biological technicians at
the time the water bottles were changed. At the same time, each cage was
rotated two positions to the right on the rack shelf and each rack was moved
one position clockwise in the room. In this way, no cage was located in the
same position on the rack during the 13-week study, and all racks were in
each room position at least twice.
Control animals were housed in one room; chloroform-treated animals
were housed in a similar but separate animal room. The temperature and
humidity of each room was monitored by a hygrothermograph (Model #311,
Weather Measure Corporation, Sacramento, California).
ANIMAL GROUPS AND DOSE LEVELS
The rats and mice were assigned to experimental groups by the following
procedure. The animals were numbered consecutively. Odd-numbered rats had
a picric acid mark on the back; even-numbered rats were unmarked. The
mice were distinguished by different colored felt pen markers on the tail.
Cage cards identified each pair of rats and group of mice. A table of random
numbers was used to select the animal to be allocated to experimental groups.
As each was selected, it was transferred to the appropriate labeled test
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cage. The first rat in each cage received one notch clipped in the right
ear; the second rat did not. Thus, the two rats assigned to each cage were
distinguishable. The five mice in each cage were distinguished by the first
mouse having one notch in the right ear; the second mouse having two notches
in the right ear; the third mouse having one notch in the left ear; the
fourth mouse having two notches in the left ear; and the fifth mouse having
no notches in either ear.
The first two rats and five mice selected using the table of random
numbers were assigned to the first cage of group 1, and the allocation
proceeded until all cages in group 1 had been filled. Group 2 then was
allocated in a like manner, and so forth until all groups were complete.
The test groups, levels of chloroform, and the number of animals
assigned to each were:
Group No.
Dose Level (ppm)
No. of Animals
1 0 (Control)
2 0 (Matched Control) t
3 200
4 400
5 600
6 900
7 1800
8 2700
*80
60
60
60
60
60
60
30
(40
(30
(30
(30
(30
(30
(30
mice
rats;
rats ;
rats;
rats;
rats;
rats;
rats ;
40
30
30
30
30
30
30
mice)
mice)
mice)
mice)
mice)
mice)
mice)
* Includes 10 of each strain for day 0 sacrifice.
t See text under "Observations" for explanation.
OBSERVATIONS
Each animal was observed daily for abnormalities in appearance, activity,
and general behavior. When an animal was moribund or lost weight steadily,
it was observed closely throughout the day, and when it appeared unlikely
to survive until the next day, it was sacrificed and necropsied.
Twice each week, bottles of freshly prepared chloroform/water solutions
were placed on each cage. Bottle weights were recorded at the beginning
and end of each semi-weekly measurement period. The difference in bottle
weight was recorded as grams of test solution consumed, and the average
grams consumed per day was then calculated. An exception to this procedure
was made for the matched control groups. For each cage receiving 1800 ppm
10
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(rats), or 2700 ppm (mice), a corresponding 0 ppm (matched control) cage was
put on test 1 day later. Although the water bottles containing 1800 ppm or
2700 ppm chloroform were changed only twice a week, they were weighed daily
to calculate each day's water consumption. The amount consumed by the
animals in each cage then was allotted to the corresponding matched control
cage for the following day. When deaths caused uneven numbers of mice in
matched cages, the allotment was determined by calculating the amount of
2700 ppm water consumed per mouse, and multiplying this by five for the mice
in 0 ppm matched cage. When an allotment value was indeterminable (e.g.
due to spillage), the previous day's allotment was used.
At the same time as the semi-weekly bottle change, animals were trans-
ferred to clean cages containing new bedding.
Once each week, during the bottle-change procedure, samples were taken
from five water bottles at each chloroform level, including 0 ppm. These
samples were analyzed for chloroform concentration.
Once each week, on the alternate bottle-change day. animals were
weighed and individual body weights were recorded. When an animal's weight
seemed irregular, it was reweighed.
Ten rats and ten mice were sacrificed before the start of the study.
The rats were used to determine baseline values of kidney fat-kidney weight
ratios, serum chloroform, serum biochemistry, and gross and microscopic
tissue pathology findings. The mice were used to determine baseline values
of liver fat-liver weight ratios, serum LDH and SCOT activity, serum
chloroform, and gross and microscopic pathologic findings.
Before all sacrifices, urine samples were obtained from 10 per group
(10 total for the baseline group). The urine samples were evaluated for
specific gravity, pH, protein, glucose, ketone, bilirubin, occult blood,
and color. The specific gravity was measured on a refractometer, and the
remaining measurements, except for color, were made using Ames Bililabstix.®
All samples also were examined microscopically for casts, cells, crystals,
and other inclusions.
Ten animals from each of the groups were sacrificed within several days
after weeks 4, 8, and 13 of the test (days 30, 60, and 90). At each
sacrifice period, every animal was first bled for analysis of chloroform in
the blood, and the rats had serum analyses of the following:
SCOT C02
SGPT Cl~
LDH Albumin
BUN Calcium
Triglycerides Balance [Na(Cl+C02)]
Total cholesterol Total bilirubin
Creatinine Uric acid
Total protein Globulin
11
-------
Inorganic phosphorus Total iron
Na+ A/G ratio
K4" Glucose
Alkaline phosphatase
The mice were bled first for serum analyses of LDH and SCOT. Also, a
peripheral blood smear was prepared, air-dried, and fixed in absolute
methanol.
A complete necropsy was performed according to the Guidelines for
Carcinogen Bioassay in Small Rodents (NCI-CG-TR-1, Technical Report Series
#1, February 1976). The following tissues were examined and preserved in
10% neutral buffered formalin:
Gross lesions Lungs and bronchi
Tissue masses Heart
Suspect tumors Thyroid glands
Regional lymph nodes Parathyroid glands
Skin Esophagus
Mandibular lymph node Stomach
Mammary gland Small intestine
Salivary gland Spleen
Larynx Kidneys
Trachea Adrenals
Cecum Bladder
Colon Seminal vesicles
Rectum Prostate
Mesenteric lymph node Testes
Liver Ovaries
Thigh muscle Uterus
Sciatic nerve Nasal cavity
Sternebrae, vertebrae, or Brain
femur (plus marrow) Pituitary gland
Costochondral junction, ribs Eyes
Thymus gland Spinal cord
Pancreas Gall bladder
One kidney from each rat in the 0, matched 0, and 1800 ppm groups was
reserved for determination of kidney fat/kidney weight ratios and a section
of liver from each mouse in the 0, matched 0, and 2700 ppm groups was
reserved for determination of liver fat/liver weight ratios as described
previously.
Generally the following tissues were processed, stained, and examined
microscopically:
Suspected tumors and gross Adrenal glands
lesions Spleen
Liver Stomach
Kidney Small intestine
Regional lymph nodes Colon
12
-------
Urinary bladder Lung
Esophagus Thymus
Pancreas Testes
Trachea
Seminal vesicles
13
-------
SECTION 4
RESULTS AND DISCUSSION
Analysis of the chloroform for Identity and purity showed that we had
received chloroform and that its purity met the manufacturer's specifications.
During this analysis, we found 30 ppb of diethyl carbonate (DEC). Subse-
quently, each aliquot of chloroform used in the preparation of the drinking
water was subjected to another simple distillation just before it was placed
in solution.
Tables 1 and 2 present the results of analyses of samples taken from
the first chloroform stock preparations made each week and of samples from
bottles collected each Friday from selected animal cages in each treatment
group. Occasionally, the analysis of bottle samples yielded values lower
than we would have preferred (see 1/3 and 11/10). Generally, however, all
values were well within acceptable limits, taking into account the number
of times each preparation was handled and allowing for a reasonable analytical
error.
Table 3 summarizes the results of analyses of blood serum for chloroform
concentrations. Unfortunately, the 90-day samples were inadvertently con-
taminated with extraneous chloroform before analysis. A review of the
procedures and of other findings from the 90-day data indicates that the
values are approximately tenfold greater than expected. A serum analysis
is scheduled to be conducted when the rats are sacrificed at 90 days on the
chronic study. This sampling should verify the interpretation we have made
of the present data. We demonstrated, however, that rats receiving several
levels of chloroform (200 to 1800 ppm) in their drinking water show chloro-
form in their blood after day 30 of the test. Analyses of air samples
collected in animal rooms showed levels of chloroform ranging from 0.4 to
56.3 ppb. Additional analyses were invalid because the blank decomposed.
Table 4 provides organ fat as a percentage of organ weight data from
mouse livers collected at the 30-, 60-, and 90-day sacrifices. The liver fat
was increased significantly in mice at the 2700-ppm level at all sampling
periods. The significant findings in the matched control group at 90 days
is not totally understood and cannot be fully explained.
Table 5 provides the amount of lipid as a percentage of organ weight
from rat kidneys collected after the 30-, 60-, and 90-day sacrifices. No
changes in lipids were observed in rat kidneys from the 1800-ppm dose level.
The only significant increase in percent lipids occurred in the matched
control group from the 90-day sacrifice.
14
-------
Analysis of the block Purina Laboratory Chow for chloroform showed trace
amounts of chloroform. Levels observed were in the ppb range and were not
considered capable of influencing the outcome of this program. Further
analysis of feed for pesticides and PCBs showed only dibutyl and dioctyl
phthalates. These are not considered capable of interfering with the results
of this study.
During the first week of treatment the animals receiving chloroform
appeared generally depressed. The higher the dose level, the more apparent
was the depression. During this initial three weeks, seven mice died (one
at 600 ppm, two at 900 ppm, and four at 2700 ppm). For these animals, we
observed body weight losses down to 11 grams, then death. From our daily
observations, we attributed these losses to a refusal to drink the chloroform-
treated water.
Table 6 presents the average body weights for female B6C3F1 mice during
the 90-day study. Significant body-weight losses were observed during the
first week in the mice receiving 900, 1800, and 2700 ppm and in the matched
control group. After this initial acclimation period, all body weights were
generally comparable to controls even though there were scattered significant
differences occurring during weeks 2, 3, and 6.
Table 7 presents the average body weights for the rats during the 90-day
study. Only the 1800-ppm group and the matched control group showed body
weight gains consistently lower than those of the controls throughout the
study.
Food consumption, observed weekly throughout the program, was considered
to be normal.
Table 8 presents the average water consumption for female mice. Con-
siderable variability and acclimation to the levels of chloroform were
evident during the first eight sampling periods. After the eighth period,
consumption became more consistent. Because of the variability in consump-
tion, dose-dependency was not evident. Using average body weight and
average water consumption data to calculate the average mg/kg/day chloroform
intake showed that the mice had actual intake levels of from 148 to 175% of
the intended levels. We had intended to provide 20, 40, 60, 90, 180, and
270 mg/kg/day of chloroform in the drinking water of mice. Our observations
showed that the B6C3F1 female mouse is extremely active and has a relative
low body weight. This, combined with a relatively consistent water consump-
tion, accounts for the actual chloroform intake being higher than that
predicted at the beginning of the program.
Table 9 presents the average water consumption for male rats. For
these rats, the average water consumption for the chloroform-treated and
matched control groups was consistently less than that of the control
group. The decrement appears to be dose-dependent. The greatest reduction
occurred in the 1800-ppm group and, as expected, in the corresponding
matched control group. Using average body weight and average water consump-
tion data to calculate the average chloroform intake in mg/kg/day showed an
extremely close correlation to intended chloroform levels. It was intended
15
-------
to provide chloroform doses at 20, 40, 60, 90, and 180 mg/kg/day in the
drinking water of rats, and the actual cumulative intakes were 20, 38, 57,
81, and 160 mg/kg/day.
Table 10 summarizes the SCOT and LDH for female mice. These data vary
so much that their usefulness in this program is questionable; they should
be reviewed thoroughly before they are included in the chronic phase.
Summary data for the serum chemistry analyses for rats are presented in
Table 11. Generally, glucose, creatinine, BUN, Na+, Cl~, calcium, balance,
cholesterol, total bilirubin, SCOT, SGPT, LDH, alkaline phosphatase, total
iron, total protein, albumin, globulin, and A/G ratio values in the treated
groups were comparable with control values. Further, blood urea nitrogen
(BUN), creatinine, uric acid, Cl~, K+, calcium, inorganic phosphorus,
cholesterol, total protein, SGPT, albumin, and A/G ratio all showed signifi-
cant increases on at least one dose level (generally the 1800-ppm level but
there were some at the 400, 600, and 900-ppm levels) during the study.
Values, lower than those in controls, were observed for C02, triglycerides,
and lactic dehydrogenase (LDH). These reductions were generally confined to
the 1800-ppm level. However, triglycerides were reduced at the 600-ppm level
after 30 days and in the 900-ppm group after 60 days. The various changes
that occurred in the matched control group were consistent with those
expected from animals deprived of normal fluid intake (restricted water
consumption).
Evaluation of urinalysis data of the rats showed a slight reduction in
pH for chloroform-treated groups when compared with that in the control
and/or matched control groups. Changes in protein observed in the urine
were consistent with the changes in overall water consumption among the
different experimental groups.
Microscopic examination of the sediment showed a rare to occasional
red and/or white cell in all groups; an occasional hyaline and/or granular
cast; and the sperm, triple phosphate crystals, and amorphous phosphates and
urates expected in rat urine.
Gross pathologic examinations on each mouse revealed a very slight
hemorrhaging of the lungs of several animals at all treatment levels.
Histologic examination of selected mouse tissues revealed the following.
Centrolobular fatty change in mouse livers occurred most often at the
two highest dose levels (1800 ppm and 2700 ppm) but appeared to be mild and
reversible. This lesion was evident 'at 400 ppm at 30 days but not at 60
or 90 days. The lesion is related to treatment but the occurrences at 400
ppm at 30 days may have been adaptive, reversible, and transitory.
Extramedullary hematopoesis in the liver was observed at virtually all
dose levels at 30 days, and also is not related to treatment.
Lymphoid atrophy of the spleen was seen in 2 animals each at 60 and 90
days in the 2700 ppm mice. This was also noted in one animal each at 600
16
-------
and 900 ppm at 90 days. Relationship to treatment appears likely, but this
lesion is mild and unimpressive.
Other lesions were sporadic and (by nature and/or incidence) not
considered related to treatment. Table 12 provides a summary of the histo-
pathological findings in mice.
Gross pathological examination of each rat revealed the following
findings, which were scattered among all treatment levels (including
controls): fibrin-like material in urinary bladder; slight to moderate
hemorrhaging of thymus, lungs, stomach and lymph nodes; hydronephrosis; and
lung congestion. Histopathologic examination revealed an embryonal nephroma
in the kidney of one control rat and lymphocytic lymphosarcoma in the thymus
of another control rat. Tubular hyperplasias usually occurred in the kidneys
of rats at the lower dose levels. These were mild, small, and circumscribed.
Hepatosis, nephropathy, and tracheitis were increased over controls at all
sacrifice intervals. Most lesions were slight or ngLld in severity, were not
dose-related, appeared adaptive (occurred in 30- or 60-day sacrifices but
not in 90-day sacrifices), or were sporadic and (by nature and/or incidence)
not considered related to treatment. Table 13 provides a summary of the
histopathological findings in rats.
In summary, there were both similarities and differences in the effects
of chloroform on the two species. During the first week of administration,
treated rats and mice exhibited signs of depression and decreased body weight,
In mice, the weight decrements were transient, with recovery evident begin-
ning in the second week. In rats, significant weight decrements persisted
longer, lasting throughout the study at the highest dose level. Water con-
sumption was decreased initially in treated animals of both species. In
mice, decreased water consumption persisted for only about four weeks, while
in rats it continued throughout the study. Food consumption appeared to be
unaffected by chloroform administration in both species. The only serum
constituents evaluated in both species, SCOT and LDH, were also unaffected.
Fat content as a percentage of target organ weight was increased in the liver
in treated mice but was normal in the kidney of treated rats. The only
treatment-related pathologic finding was the occurrence of fatty changes in
the liver in the mice.
17
-------
TABLE 1. RESULTS OF ANALYTICAL CHEMISTRY ANALYSES - RATS
Chloroform 1978
Level (ppm) 10/17
10/24
PERCENTAGE OF
200
400
600
900
1800
83.3
83.8
92.0
101.8
84.6
75.3
78.1
80.8
82.0
85.4
PERCENTAGE OF
200
400
600
900
1800
10/20t
96.8
82.6
87.4
87.4
88.5
10/27
86.5
83.1
81.5
82.2
83.8
10/31
11/7
EXPECTED VALUE
83.0
88.6
84.8
95.8
97.0
81.8
69.4
72.8
84.6
84.5
EXPECTED VALUE
11/3
61.5
63.1
67.8
67.3
66.8
11/10
81.1
64.9
68.9
78.0
80.5
11/14
FOR
88.3
89.4
94.8
92.4
91.7
FOR
11/17
77.3
77.1
80.7
80.5
77.1
Date Sample Taken
11/21 11/28 12/5 12/12
CHLOROFORM IN
109.3
90.5
92.7
89.9
95.1
87.0
81.6
81.6
82.9
82.4
CHLOROFORM IN
11/24
68.9
70.6
72.3
71.8
75.9
12/1
88.7
85.3
81.5
74.7
66.5
12/19
PREP SAMPLES (Average
82.5
101.9
86.8
81.9
84.9
BOTTLE
12/Bt
87.9
92.3
89.3
80.4
86.8
103.3
90.6
88.8
92.2
80.6
SAMPLES"
12/15
98.8
78.2
84.8
80.9
74.5
108.3
109.3
97.5
95.8
93.9
12/22
89.6
90.6
98.0
96.3
88.9
12/26
of two
87.3
104.5
103.9
105.6
98.7
12/29
99.6
104.2
102.6
101.6
94.2
1979
1/2
1/9
1/16
samples)
123.0
115.9
112.0
109.8
111.5
1/5
93.3
88.4
86.5
88.6
85.4
104.8
98.3
91.3
91.9
90.5
1/12
99.6
90.1
85.8
89.6
83.6
98.5
94.8
96.6
96.7
95.3
*The first 4 weeks represent an average of 10 samples, the next 4 weeks represent an average of 9 samples,
and the remaining 5 weeks represent an average of 7 samples for all levels.
tSome samples were lost when refrigerator malfunctioned; some bottles froze and broke.
-------
TABLE 2. RESULTS OF ANALYTICAL CHEMISTRY ANALYSES - MICE
Chloroform 1978
Level (ppm) 10/17
10/24
PERCENTAGE OF
200
400
600
900
1800
2700
83.3
83.8
92.0
101.8
84.6
97.2
75.3
78.1
80.8
82.0
85.4
91.7
PERCENTAGE OF
200
400
600
900
1800
2700
ll/20t
96.8
82.6
87.4
87.4
88.5
92.7
10/27
86.5
83.1
81.5
82.2
83.8
81.7
10/31
11/7
EXPECTED VALUE
83.0
88.6
84.8
95.8
97.0
82.2
81.8
69.4
72.8
84.6
84.5
79.6
EXPECTED VALUE
11/3
61.5
63.1
67.8
67.3
66.8
72.5
11/10
81.1
64.9
68.9
78.0
80.5
76.7
11/14
FOR
88.3
89.4
94.8
92.4
91.7
93.2
FOR
11/17
77.3
77.1
80.7
80.5
77.1
75.9
Date Sample Taken
11/21 11/28 12/5 12/12
CHLOROFORM IN
109.3
90.5
92.7
89.9
95.1
97.5
87.0
81.6
81.6
82.9
82.4
87.2
CHLOROFORM IN
11/24
68.9
70.6
72.3
71.8
75.9
79.1
12/1
88.7
85.3
81.5
74.7
66.5
76.0
12/19
PREP SAMPLES (Average
82.5
101.9
86.8
81.9
84.9
93.1
103.3
90.6
88.8
92.2
80.6
92.8
108.3
109.3
97.5
95.8
93.9
92.6
12/26
of two
87.3
104.5
103.9
105.6
98.7
92.2
1979
1/2
1/9
1/16
samples)
123.0
115.9
112.0
109.8
111.5
107.7
104.8
98.3
91.3
91.9
90.5
89.5
98.5
94.8
96.6
96.7
95.3
85.9
BOTTLE SAMPLES*
12. 8t
87.9
92.3
89.3
80.4
86.8
83.4
12/15
98.8
78.2
84.8
80.9
74.5
70.6
12/22
89.6
90.6
98.0
96.3
88.9
72.3
12/29
99.6
104.2
102.6
101.6
94.2
94.4
1/5
93.3
88.4
86.5
88.6
85.4
82.0
1/12
99.6
90.1
85.8
89.6
83.6
86.1
*The first 4 weeks represent an average of 10 samples, the next 4 weeks represent an average of 9 samples,
and the remaining 5 weeks represent an average of 7 samples for all levels except 2700 ppm. The 2700-ppm
level represents 5 samples during the first 4 weeks, 4 samples during the next 4 weeks, and 2 samples
during the last 5 weeks.
tSome samples were lost when refrigerator malfunctions; some bottles froze and broke.
-------
TABLE 3. AVERAGE CHLOROFORM LEVELS IN RAT SERUM (ppb)
Group
0 Day
Baseline Control
0 ppm
0 ppm
Matched Control
200 ppm
400 ppm
600 ppm
900 ppm
1800 ppm
30-Day (SD)t
60-Day (SD)
90-Day (SD)t
692.2
0.
0.
0.
1.
1.
7.
4.
60
89
86
26
12
18
17
(0.
(0.
(0.
(1.
(0.
(9.
(1.
19)
34)*
31)*
30)
30)**
50) *
66)**
1.
0.
1.
0.
1.
1.
5.
45
96
07
92
15
34
89
(0.
(0.
(0.
(0.
(0.
(1.
(10
93)
35)
86)
39)
78)
02)
.52)
165
182
162
187
257
237
294
.7
.2
.7
.5
.8
.6
.6
(110.28)
(77.59)
(91.03)
(162.40)
(151.14)
(154.61)
(283.24)
tSamples found to have been contaminated with extraneous chloroform.
*Significant at P < 0.05.
**Signifleant at P < 0.01.
20
-------
TABLE 4. LIVER FAT AS A PERCENTAGE OF LIVER WEIGHT - MICE
Treatment Group
Control
Matched Control
2700 ppm
30- Day
Sacrifice
3.52
3.19
8.22**
60-Day
Sacrifice
2.18
2.36
5.56**
90-Day
Sacrifice
4.08
6.80**
6.52**
**Significant at P < 0.01.
21
-------
TABLE 5. KIDNEY FAT AS A PERCENTAGE OF KIDNEY WEIGHT - RATS
30-Day 60-Day 90-Day
Treatment Group Sacrifice Sacrifice Sacrifice
Control 3.24 3.62 2.82
Matched Control 3.10 3.31 4.07**
1800 ppm 3.33 3.67 3.28
**Significant at P < 0.01.
22
-------
TABLE 6. AVERAGE BODY WEIGHTS OF FEMALE MICE RECEIVING
CHLOROFORM IN THEIR DRINKING WATER (Grams)
Week of
Test
Initial
1
2
3
4
5
6
7
8
9
10
11
12
13
0
20
(30)t
20
21
21
21
22
(20)
23
23
23
24
(10)
24
25
24
24
200
18
(30)
20
20
21
22
22
(20)
23
23
23
23
(10)
23
24
24
24
400
19
(30)
20
19
20
22
22
(20)
22
22
23
23
(10)
23
24
25
25
Chloroform Concentration
600 900 1.800
18
(30)
19
18*
(29)
20*
21
22
(19)
22
22
22
22
(9)
23
24
24
25
18*
(30)
17*
19
(29)
19*
(28)
21
22
(18)
22
22
22
22
(9)
23
23
24
24
20
(30)
17*
19*
20
21
22
(20)
22
23
22
23
(10)
23
24
24
25
(ppm)
2700 0 (Matched)
19
(30)
14*
(29)
19*
(26)
20
22
23
(16)
22
23
23
23
(8)
24
24
24
25
20
(30)
16*
19*
19
22
22
(20)
21*
22
22
22
(10)
23
23
25
25
^Number of mice included in average.
and interim sacrifices.
^Significant at P < 0.05.
Reduction in numbers due to mortalities
23
-------
TABLE 7. AVERAGE BODY WEIGHTS OF MALE RATS RECEIVING
CHLOROFORM IN THEIR DRINKING WATER (Grams)
Week of
Test
Initial
1
2
3
4
5
6
7
8
9
10
11
12
13
0
191
(30)t
233
274
306
330
346
(20)
365
370
381
397
414
(10)
425
433
428
200
186
(30)
228
266
302
325
344
(20)
358
371
380
394
420
(10)
432
444
435
Chloroform Concentration (ppm)
400 600 900 1800
186
(30)
228
265
299
320
345
(20)
358
375
380
394
397
(10.)
399
414
407
186
(30)
223
261
296
319
339
(20)
354
369
373
387
376
(10)
391
399
399
187
(30)
221*
261
294
317
347
(20)
362
379
385
396
405
(10)
422
429
430
186
(30)
204*
239*
273*
288*
305*
(20)
316*
326*
333*
342*
345*
(10)
357*
364*
361*
0 (Matched)
193
(30)
207*
248*
274*
303*
317*
(20)
327*
340*
349*
354*
362*
(10)
371*
380*
377*
tNumber of rats included in average.
interim sacrifices.
*Significant at P < 0.05.
Periodic reduction to scheduled
24
-------
TABLE 8. AVERAGE WATER CONSUMPTION OF FEMALE MICE RECEIVING
CHLOROFORM IN THEIR DRINKING WATER
(Grams per Mouse per Day)
Sampling
Periodf
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
0
4.
4.
4.
4.
3.
5.
5.
4.
4.
3.
4.
4.
4.
4.
4.
4.
4.
4.
4.
4.
4.
4.
4.
3.
3.
3.
0
6
3
6
8
3
1
9
7
9
2
3
1
4
5
4
2
1
2
1
2
3
0
9
7
9
200
2.1
4.1
3.7
3.9
3.7
4.1
3.5
3.9
4.3
3.2
3.9
4.1
3.2
4.1
4.2
4.1
3.0
4.1
4.6
4.2
3.1
4.7
3.7
3.9
4.2
3.4
Chloroform Concentration (ppm)
400 600 900 1800 2700
2.0
3.6
3.0
3.2
3.9
3.6
3.9
4.2
4.2
3.5
3.1
3.9
3.2
3.9
3.8
3.7
4.7
3.5
3.8
4.3
3.2
4.0
3.6
3.7
3.8
3.4
0.8
2.9
2.7
3.1
3.5
3.5
3.2
2.9
3.6
3.4
3.0
3.6
3.2
3.4
3.6
3.6
3.6
3.8
3.2
3.5
3.4
4.0
4.0
3.6
3.6
3.7
0.6
2.1
2.7
3.0
3.0
3.1
2.4
3.9
3.3
3.2
3.1
3.1
3.5
3.4
3.4
3.6
2.7
3.6
3.4
3.8
3.1
3.8
3.6
3.2
3.5
3.3
0.6
1.8
2.9
2.8
2.9
3.0
2.9
3.2
3.0
3.4
3.0
3.3
3.5
3.7
3.4
3.8
3.4
3.6
3.6
3.7
3.3
3.8
3.6
3.9
3.3
3.7
0.
1.
2.
3.
3.
3.
3.
3.
2.
3.
3.
3.
3.
3.
3.
3.
3.
3.
3.
3.
3.
3.
4.
3.
3.
3.
6
2
3
4
8
4
0
6
8
5
4
2
4
6
2
7
4
6
6
8
3
7
1
4
5
6
0 (Matched)
0.
1.
2.
3.
3.
3.
3.
3.
2.
3.
3.
3.
3.
3.
3.
3.
3.
3.
3.
3.
3.
3.
4.
3.
3.
3.
6
2
3
3
8
4
0
6
8
5
4
1
4
6
2
9
5
6
6
8
2
7
1
5
5
6
tOdd-numbered sampling periods are recordings of 3 days of water consumption
and even-numbered sampling periods are recordings of 4 days of water consump-
tion. These correspond with the number of days water bottles remained on
the cages without changing. The chloroform solutions were prepared twice
weekly.
25
-------
TABLE 9. AVERAGE WATER CONSUMPTION OF MALE RATS RECEIVING
CHLOROFORM IN THEIR DRINKING WATER
(Grams per Rat per Day)
Sampling
Periodt
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
0
37.
40.
40.
42.
45.
39.
38.
40.
39.
38.
38.
40.
40.
42.
42.
42.
39.
40.
44.
41.
44.
41.
42.
42.
40.
40.
3
6
0
3
3
7
6
1
7
9
5
4
1
2
1
3
9
7
1
2
2
6
6
3
5
6
200
29.5
34.5
33.1
34.1
34.0
33.0
32.4
35.8
35.5
32.0
30.5
32.4
30.9
32.0
33.8
33.4
32.4
33.3
35.7
35.0
36.1
35.1
34.7
33.9
35.2
32.8
Chloroform Concentration (ppm)
400 600 900 1800
26.
33.
31.
31.
31.
31.
31.
35.
32.
31.
30.
30.
28.
30.
31.
30.
29.
30.
-31.
32.
31.
29.
32.
33.
31.
28.
5
3
1
7
9
9
6
0
6
0
2
3
8
7
6
7
3
0
7
0
5
6
7
1
7
9
24.7
33.3
29.1
30.9
29.5
30.1
30.3
31.7
32.2
31.5
30.1
30.9
29.5
30.9
31.0
31.2
31.7
30.5
31.4
28.8
31.5
32.9
32.2
29.8
31.8
28.8
24.3
32.3
30.0
30.2
28.6
29.3
30.1
32.5
32.1
31.7
30.2
30.3
28.8
29.5
29.2
30.5
29.0
29.3
29.1
29.0
30.6
31.1
31.0
29.0
31.9
27.3
16.
25.
25.
27.
26.
27.
25.
27.
27.
26.
25.
26.
25.
27.
26.
28.
25.
25.
26.
27.
27.
27.
27.
27.
27.
24.
9
3
5
1
4
1
5
4
0
2
7
3
5
2
3
1
4
4
5
5
2
2
6
4
4
4
0 (Matched)
16
25
25
26
25
27
25
27
26
26
26
26
25
27
26
26
25
25
26
27
27
27
27
27
27
24
.4
.4
.4
.6
.9
.2
.4
.4
.9
.3
.0
.3
.4
.1
.4
.8
.2
.4
.5
.5
.1
.4
.5
.6
.6
.0
tOdd-numbered sampling periods are recordings of 3 days of water consumption
and even-numbered sampling periods are recordings of 4 days of water consump-
tion. These correspond with the number of days water bottles remained on the
cages without changing. The chloroform solutions were prepared twice weekly.
26
-------
TABLE 10. AVERAGE SCOT AND LDH VALUES FOR FEMALE MICE
RECEIVING CHLOROFORM IN THEIR DRINKING WATER
Treatment
Group (ppm)
SCOT
(U/L)
LDH
(U/L)
Treatment SCOT
Group (ppm) (U/L)
LDH
(U/L)
Baseline (Day zero)
0
918
47
30-Day interim sacrifice
0
200
400
600
900
1800
2700
0 (Matched)
328
225
233
196
246
298
156*D
522
909
738
767
601
913
898
653
1525*
60-Day interim sacrifice
0 623
200 400
400 414
600 257
900 383
1800 136*D
2700 619
0 (Matched) 349
1010
996
965
1114
1018
548**D
1318
1413*
90-Day terminal sacrifice
0
200
400
600
900
1800
2700
0 (Matched)
353
609
201
392
234
176*D
190
620*
1222
1520
767*D
1197
1160
731**D
765**D
1506
-"Significant at P < 0,05.
**Signifleant at P < 0.01.
D, decreased below controls.
27
-------
Table 11
SUMMARY OF CLINICAL CHEMISTRY DATA FOR RATS RECEIVING CHLOROFORM IN THEIR DRINKING WATER
(Ten Rats per Treatment Group)
30-Day Sacrifice
Day Zero
Parameter Examined
Glucose (mg 7.)
BUN (mg "/.)
Creatinine (mg '/.)
Uric acid (mg %)
Na"1" (meq/liter)
K"1" (meq/liter)
C02 (meq/liter)
Cl~ (meq/liter)
Calcium (mg %)
Inorganic
phosphorus (mg%)
CO Balance
Na-[Cl+C02]
Cholesterol (mg %)
Triglycerides (.mg %)
Total Bilirubin (mg %)
SCOT (my/ml)
SGPT (ran/ml)
LDH (my/ml)
Alkaline
phosphatase (mu/ml)
Total iron (meq %)
Total protein (g %)
Albumin (A) (g %)
Globulin (G) (g %)
A/G
Baseline — SDt
162
20
0.6
2.0
141
5.3
27
98
10.6
9.4
17
77
37
0.2
66
125
1388
616
423
4.7
2.5
2.18
1.14
(12.06)
(1.70)
(0.05)
(0.26)
(1.20)
(0.34)
(1.78)
(1.35)
(0.20)
(0.45)
(1.70)
(3.56)
(14.07)
(0.04)
(22.57)
(72.60)
(331.82)
(77.85)
(57.60)
(0.10)
(0.05)
(0.06)
(0.03)
0 — SD
193
20
0.6
2.2
142
5.4
29
95
10.3
8.4
19
76
87
0.1
174
63
1593
264
260
5.3
2.6
2.69
0.96
(35.12)
(1.69)
(0.09)
(1.09)
(1.29)
(0.76)
(1.83)
(2.58)
(0.31)
(0.75)
(2.41)
(29.27)
(58.22)
(0.04)
(89.72)
(21.13)
(503.83)
(50.38)
(49.64)
(0.25)
(0.19)
(0.20)
(0.10)
200— SD
231
22
0.7
2.9
142
6.1
28
95
10.5
8.4
19
69
80
0.1
202
83
1651
254
243
5.3
2.7
2.61
1.02
(72.89)
(2.55)
(0. 10)
(1.80)
(0-99)
(1.59)
(1.85)
(2.37)
(0.65)
(0.89)
(3.06)
(5.46)
(23.43)
(0.03)
(73.84)
(33.05)
(360.78)
(42.44)
(30.20)
(0.20)
(0.10)
(0.11)
(0.02)
Chloroform Concentration (ppm)
400— SD 60
252
23
0.7
5.5
142
7.2
26
96
11.1
9.1
20
82
96
0.2
144
63
1270
285
301
5.6
2.7
2.87
0.95
(56.58)* 242
(2.88)** 22
(0.11)* 0.7
(1.90)**3.5
(1.99) 141
(1.49)**6.3
(1.69)** 28
(1.85) 97
(0.55)**10.7
(0.49)* 8.3
(2.88) 17
(44.89) 70
(98.15) 60
(0.05) 0.2
(36.96) 161
(10.63) 60
(224.75) 1577
(62.10) 266
(52.33) 293
(0.25)**5.5
(0.22) 2.7
(0-22) 2.75
(0.11) 0.99
(91.55)
(3.82)
(0.09)*
(2.25)
(1.34)
(1.89)
(1.83)
(2.51)
(0.89)
(1.25)
(3.67)
(4.42)
(10.00)
(0.05)
(51.49)
(17.40)
(302.73)
(40.82)
(66.83)
(0.18)*
(0.11)*
(0.11)
(0.05)
900-SD
172
25
0.6
2.1
141
5.4
28
98
10.3
8.2
16
108
313
0.2
151
83
1275
249
264
5.3
2.6
2.74
0.95
(30.75)
(9.94)
(0.12)
(1.25)
(1.78)
(0.86)
(2.42)
1800 — SD 0 (Matched) — SD
220
26
0.5
3.9
142
7.4
24
(2.05)** 99
(0.30)
(1.20)
(4.68)
(127.03)
(832.05)
(0.08)*
(66.76)
(63.58)
(359.54)
(75.24)
(90.98)
(0.32)
(0.35)
(0.25)
(0.15)
10.8
9.7
20
75
43
0.2
251
112
1616
277
283
5.7
2.9
2.75
1.06
(60.77) 240
(1.60)** 28
(0.11) 0.7
(2.32) 3.3
(2.00) 145
(2.16)* 6.2
(2.98)** 26
(2.07)**100
(0.69)*10.4
(1.64)* 7.9
(2.72) 17
(5.85) 100
(8.69)* 123
(0)** 0.1
(125.33) 166
(53.08) 62
(482.38) 1484
(52.18) 230
(48.64) 230
(0.28)**5.6
(0-14)**2.7
(0.16) 2.91
(0.05)* 0.84
(101.80)
(9.50)*
(0.13)
(2.04)
(1.51)**
(1.59)
(1.60)**
(3.10)**
(0.71)
(0.90)
(2.91)
(112.30)
(210.43)
(0.05)
(64.19)
(16.39)
(287.41)
(50.38)
(52.78)
(0.20)**
(0-29)
(0.28)
(0.15)
tSD ~ Standard Deviation, in parentheses.
*Significant at P i 0.05.
"Significant at P <. 0.01.
-------
Table 11 (continued)
60-Day Sacrifice
Chloroform Concentration (ppm)
Parameter Examined
Glucose (mg %)
BUN (mg %)
Creatinine (mg %)
Uric acid (mg %)
Na4" (meq/liter)
K+ (meq/liter)
C02 (meq/liter)
Cl~ (meq/liter)
Calcium (mg %)
Inorganic
phosphorus (mg %)
Balance
Na-[Cl+C02]
Cholesterol (mg %)
Trlglycerid.es (mg %)
Total bilirubin (mg %)
SCOT (mu/ml)
SGPT (ran/ml)
LDH (mu/ml)
Alkaline
phosphatase (mg/ml)
Total iron (meq %)
Total protein (g %)
Albumin (A) (g %)
Globulin (G) (g %)
A/G
0 — SDf
190
22
0.7
1.8
142
5.3
29
97
10.0
7.0
16
62
77
0.1
138
65
1370
204
209
5.4
2.6
2.78
0.94
(51.20)
(1.70)
(0.07)
(1.29)
(1.20)
(1.05)
(2.18)
(2.46)
(0.45)
(0.37)
(3.06)
(6.98)
(25.60)
(0.03)
(48.42)
(17.89)
(442.24)
(36.81)
(27.35)
(0.24)
(0.15)
(0.14)
(0.06)
20'
192
23
0.7
2.2
141
5.8
28
97
9.8
6.7
16
68
68
0.1
129
66
1370
232
199
5.4
2.6
2.02
0.91
(72.29)
(1.89)
(0.08)
(1.71)
(2.81)
(1.36)
(2.10)
(3.06)
(0.59)
(0.94)
(2.88)
(7.87)
(15.83)
(0)
(28.39)
(12.83)
(368.27)
(45.56)
(22.00)
(0.22)
(0.11)
(0.12)
(0.02)
400— SD
165
22
0.6
1.8
141
5.5
29
97
9.7
6.7
16
71
86
0.1
117
64
1190
226
197
5.5
2.6
2.87
0.92
(33.95)
(2.63)
(0.09)
(1.34)
(1.96)
(0.99)
(3.59)
(2.38)
(0.45)
(0.67)
(3.17)
(6.56)
(20.67)
(0.03)
(23.98)
(15.17)
(349.15)
(41.50)
(15.52)
(0.11)
(0.11)
(0.07)
(0.05)
600— SD
177
23
0.6
1.4
143
5.2
28
99
9.9
7.1
16
ft* 75
81
0.1
122
66
1079
191
211
5.5
2.6
2.84
0.93
(25.52)
(1.03)
(0.07)
(0.45)
(2.64)
(0.51)
(2.67)
(2.72)
(0.22)
(0.47)
(2.76)
(10.20)
(30.29)
(0.04)
(37.18)
(16.58)
(328.35)
(33.92)
(22.76)
(0.24)
(0.14)
(0.16)
(0.06)
90
203
23
0.7
2.5
141
6.0
27
90
10.0
7.6
17
ft* 78
62
0.1
124
65
1014
202
229
5.4
2.7
2.71
0.98
,(-i c n
'U — bL)
(47.24)
(1.27)
(0.10)
(1.53)
(4.02)
(1.38)
(3.68)
(2.80)
180
219
25
0.7
2.8
141
6.3
26
98
(0.39) 10.2
(0.54)**
(2.36)
(11.26) **
(24.73)
(0.05)
(45.55)
(13.20)
(460.63)
(55.39)
(27.71)
(0.31)
(0.21)
(0.12)
(0.05)
7.6
17
79
36
0.2
104
65
676
196
235
5.5
2.8
2.76
1.01
(94.92)
(2.58)
(0.12)
(2.52)
(2.67)
(2.39)
(2.81)
(2.84)
(0.70)
(1.15)
(2.91)
(9.55)
(15.45)
(0.05)
(30.03)
(19.87)
0 (Mat
162
* 26
0.7
1.9
145
4.8
* 28
97
9.4
6.2
21
** 67
** 53
** 0.1
142
65
(269.17)**1467
(30.99)
(38.90)
(0.21)
(0.11)
(0.13)
(0.05)
177
179
5.4
* 2.7
2.70
* 1.00
chsd ) — bJJ
(21.11)
(1.66)**
(0.09)
(0.75)
(3.26)*
(0.55)
(1.81)
(3.07)
(0.37)*
(0.43)**
(1.90)**
(8.01)
(13.28)*
(0)
(34.74)
(11.29)
(318.89)
(22.68)
(19.97)*
(0.27)
(0.09)
(0.21)
(0.06)
-tSD = Standard Deviation, in parentheses.
*Significant at P <. 0.05.
**Signif leant at P <_ 0.01.
-------
Table 11 (concluded)
Terminal Sacrifice
Chloroform Concentration (ppm)
Parameter Examined
Glucose (mg %)
BUN (mg 7.)
Creatinine (mg 7.)
Uric acid (mg %)
Na+ (meq/liter)
K+ (meq/liter)
C02 (meq/liter)
Cl~ (meq/liter)
Calcium (mg %)
Inorganic
phosphorus (mg %)
Balance
Na"[Cl+C02]
Cholesterol (mg 7.)
Triglycerides (mg %)
Total bilirubin (mg %)
SCOT (n.M/rol)
SGPT (mp/ml)
LDH (mu/ml)
Alkaline
phosphatase (mM/ml)
Total iron (meq %)
Total protein (g SO
Albumin (A) (g %)
Globulin (G) (g%)
A/G
0— SDt
196
22
0.6
1.8
143
5.1
27
97
9.4
6.4
18
62
78
0.1
155
62
2016
195
195
5.4
2.7
2.77
0.96
(43.11)
(0.79)
(0.08)
(1.00)
(0.95)
(0.75)
(1.65)
(2.91)
(0.33)
(0.79)
(2.79)
(15.59)
(32.83)
(0.04)
(88.50)
(7.35)
(1581.98)
(38.09)
(14.32)
(0.20)
(0.11)
(0.18)
(0.08)
200— SD
176
22
0.6
1.7
143
5.2
28
97
9.6
6.1
17
73
93
0.1
107
59
1859
192
194
5.5
2.7
2.79
0.97
(40.10)
(2.04)
(0.08)
(0.36)
(1.37)
(0.91)
(1.06)
(1.51)
(0.30)
(0.51)
(2.10)
(10.59)
(42.80)
(0.05)
(17.48)
(7.32)
(699.94)
(42.99)
(19.93)
(0.16)
(0.12)
(0.19)
(0.09)
400— SD
190
22
0.6
2.2
142
5.5
27
98
9.7
6.3
16
75
91
0.1
110
72
1547
202
215
5.6
2.7
2.88
0.93
(64.15)
(1.60)
(0.07)
(2.06)
(1.43)
(1.40)
(1.65)
(2.32)
(0.51)
(0.70)
(2.62)
(18.76)
(53.24)
(0.05)
(34.53)
(32.89)
(455.41)
(33.31)
(28.34)
(0.20)
(0.14)
(0.12)
.(0.06)
600— SD
171
23
0.6
1.4
142
5.2
27
99
9.7
6.4
17
73
80
0.1
119
64
1091
186
204
5.3
2.6
2.73
0.94
(36.55)
(1.97)
(0.10)
(0.77)
(0.99)
(0.63)
(2.26)
(1.83)
(0.25)*
(0.73)
(2.59)
(10.80)
(24.34)
(0.03)
(38.91)
(14.06)
(299.91)
(28.74)
(17.25)
(0.24)
(0.12)
(0.16)
(0.05)
900— SD
194
22
0.6
1.6
141
6.1
27
98
9.9
6.8
16
76
93
0.2
107
61
818
177
226
5.4
2.6
2.80
0.95
(41.86)
(2.73)
(0.10)
(1.52)
(1.43)**
(2.02)
(2.07)
(3.85)
(0.51)*
(0.72)
(3.92)
(8.72)*
(18.14)
(0.04)
(18.37)
(15.69)
(404.95)
(20.35)
(28.56)**
(0.21)
(0.11)
(0.16)
(0.06)
1800— SD
214
25
0.7
3.1
141
7.1
26
98
10.4
7.7
17
86
38
0.2
153
84
1016
160
216
5.5
2.8
2.73
0.93
(83.79)
(1.51)**
(0.09)*
(1.79)
(2.23)
(2.64)*
(2.28)
(2.94)
(0.79)**
(18.13)
(3.68)
(12.69)**
(21.41)**
(0.05)
(51.18)
(36.67)
(390.87)
(44.52)
(17.17)**
(0.24)
(0.14)*
(0.14)
(0.06)*
0 (Matched) — SD
263
25
0.9
3.7
145
6.9
24
104
9.9
5.8
18
87
84
0.1
183
74
1383
165
175
5.7
2.7
3.02
0.90
(118.20)
(4.59)
(0.15)**
(2.31)*
(3.00)*
(2.12)*
(2.49)**
(2.65)**
(0.80)
(0.55)
(5.29)
(43.21)
(45.94)
(0.04)
(81.24)
(20.64)
(592.22)
(31.97)
(33.52)
(0.22)**
(0.23,
(0.29)*
(0.13)
tSD = Standard Deviation, in parentheses.
*Signiflcant at f ^ 0.05.
"Significant at P <_ 0.01.
-------
Table 12
SUMMARY HISTOPATHOLOGICAL FINDINGS IN MICE
30-Day Sacrifice
60-Day Sacrifice
90-Day Sacrifice
Base- Chloroform Concentration ^ppm) Chloroform Concentration (ppm) _Ch 1 grof orm__Cpncen tra t i on (pp m)
Microscopic line Matched Matched Matched
Finding 0 0 200 400 600 900 1800 2700 0 Q 200 400 600 900 1800 2700 Q 0 200 400 600 900 1800 2700 0
Lung
Hemorrhage
Congestion
Liver
Extramedullary
hematopoiesis
Centrilobular
fatty change
Focal necrosis
Focal
autolysis
Kidney
Tubular cyst
Focal
congestion
Focal
nephritis
4421 2
3 256
Lymphoid
atrophy
Adrenal
Extracapsular
adrenal tissue
Lymphoid
infiltration
-------
M
Table 13
SUM11ARY HISTOPATHOLOGICAL FINDINGS IN RATS
30-Day Sacrifice 60-Day Sacrifice
90-Day Sacrifice
Chloroform Concentration (ppm) Chloroform Concentration (ppm)
Microscopic Baseline Matched Matched
Findings 0 0 200 400 600 900 1800 0 0 200 400 600 900 1800 0
Lung
Alveolar collapse 1 2 131122
Alveolar dilata-
tion 2 1
Congestion 1 1
Hemorrhage 11121 1
Exudative pneu-
monia
Liver
Pericholangitis 1 11 1 11122
Necrosis 11 1
Hepatosis :
Centrilobular 1222 323212
Mid-zonal 3 222 164332
Peripherolobular 1111 1
Thyroid
Thyroiditis 1 1
Duodenum
Congestion 1
Focal ectopic
pancreas 2
Focal hemorrhage 1
Autolysis 1
Ileum
Autolysis 113 132211122
Mucosal hemorrhage
Jejunum
Autolysis 111 1 1
Chloroform Concentration (ppm)
Matched
0 200 400 600 900 1BOO 0
1 11
1 1
1 12
1
2 1211 1
43 5 45
56411
14 2
121 22
1
Stomach
Glandular dilatation
Parakeratosis
-------
Table 13 (continued)
30-Day Sacrifice
60-Day Sacrifice
90-Day Sacrifice
Microscopic
Findings
Thymus
Hemorrhage
Lympho sarcoma
Baseline
0 0
1
Chloroform Concentration (ppm)
Matched
'200 400 600 900 1800 0
2
Chloroform Concentration (ppm)
Matched
0 200 400 600 900 1800 0
Chloroform Concentration (ppm)
Matched
0 200 400 600 900 1800 0
1
GJ
UJ
Casts 3
Chronic inter-
stitial
nephritis 3
Mineralization 1
Pelvic hemorrhage 1
Tubular cyst 1
Adenomatous
Cellular casts
Cyst
Glomerulosclerosis
Healed infarction
Hematuria
Hydronephrosis
Nephropathy—
Slight focal
Moderate focal
Marked focal
Moderate diffuse
Marked diffuse
Kephrosis
Papillary cystadenoma
Proteinuria
Pyelitis
Tubular cystic
hyperplasia
Tubular dilatation
Tubular hyperplasia
Embryonal nephroma
Glomerular hyalinization
Unilateral hypoplasia
1
1 2
2 3
1
10
132
-------
30-Day Sacrif ice
Table 13 (continued)
60-Day Sacrifice
90-Day Sacrifice
Microscopic
Findings
Urinary Bladder
Autolysis
Chronic- cystitis
Serosal minerali-
zation
Lymph node
Hemorrhage
Lymphoid hyper-
plasia
Chloroform Concentration (ppm) Chloroform Concentration (ppin) Chloroform Concentration (ppm)
Baseline Matched Matched Matched
0 0 200 400 600 900 1800 0 0 200 400 600 900 1800 0 0 200 400 600 900 1800 0
1
1 1
1
11 1
1
Pancreas
Interstitial pan-
creatitis
Periductal inflammation
Duct hyperplasia
Periductal fibrosis
Subacute pancreatitis
Lymphoid hyperplasia
Peritonitis
Increase pigmentation
Congestion
Siderotic plaque
Testes
Hyperplasia
Interstitial cell hyperplasia
Trachea
Glandular ectasia
Hemorrhage
Tracheitis
Cystic gland
Glandular dilatation
2
2 3
1
-------
to
Ln
Table 13 (concluded)
30-Day Sacrifice 60-Day Sacrifice 90-Day Sacrifice
Microscopic Baseline
Findings 0
Adrenal
Cortical nodular
hypertrophy
Cortical vacuolation
Extracapsular cortical
nodules
Colon
Contained blood
Submucosal hemorrhage
Seminal Vesicles
Chloroform Concentration (ppm) Chloroform Concentration (ppm) Chloroform Concentration (ppm)
Matched Matched Matched
0 2QO 400 600 900 1800 0 0 200 400 600 900 1800 0 0 200 400 600 900 1800
1 1
2 2 1111
1
1
1
Chronic vesiculitis
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
REPORT NO.
EPA-600/1-80-030
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Effects of Chloroform in the Drinking Water of Rats
and Mice
Ninety-Day SuTDacute Toxicity Study
5. REPORT DATE
I .TTTT.Y IQaO ISSUING. DA.TK.
5. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Ted A. Jorgenson
arol J. Rushbrook
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
SRI, International
333 Ravenswood Avenue
Menlo Park, California 94025
10. PROGRAM ELEMENT NO.
C60C1C Task 013
11. CONTRACT/GRANT NO.
68-03-2616
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
Final report, Phase I
14. SPONSORING AGENCY CODE
EPA/600/10
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This research was initiated to provide toxicologic and range finding data
sufficient for setting dose levels for the chronic phase testing of chloroform in
drinking water of male Osborne-Mendel rats and female B6C3F1 mice. A 90-day subchronic
study was designed to investigate the effects of chloroform in drinking water at levels
of 200, 400, 600, 900, and 1800 ppm in both the rats and mice, and 2700 ppm level in the
mice. Body weights, water consumption, chloroform concentration in blood serum, kidney
fat-to-kidney weight ratios (for rats), liver fat-to-liver weight ratios (in mice), and
gross and microscopic pathology findings were examined.
Results showed that the body weights of both rats and mice receiving the high
chloroform levels were affected by treatment, but the variances were low and the mice
body weights returned to that of controls after one week. There was no effect on the
percentage of kidney fat (rats) but a significant increase in liver fat (mice) was seen
in the 2700 ppm group.
Data in this study indicated that all chloroform levels examined in the 90-day
study are acceptable for use in the chronic study.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDEDTERMS
c. COS AT I Field/Group
Chloroform
Toxicity
Oral
Rats
Mice
Subchronic
Range finding
In Drinking Water
18. DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (This Report)
Unclassified
21. NO. OF PAGES
44
20. SECURITY CLASS (This page)
Unclassified
22. PRICE
EPA Form 2220 —I (Rev. 4 — 77) PREVIOUS EDITION is OBSOLETE
36
U.S. GOVERNMENT PRINTING OFFICE: 1980--657-165/0041
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