FUNDAMENTAL AND APPLIED TOXICOLOGY 3 2 , 2 2 4 - 2 3 2 (1996)
ARTICLE NO. 0125
Subchronic Toxicity of Ingested 1,3-Dichloropropene in Rats and Mice
K. T. HAUT, K. E. STEBBINS, K. A. JOHNSON, S. N. SHABRANG, AND W. T. STOTT1
Mammalian and Environmental Toxicology Research Laboratory, Health and Environmental Sciences,
The Dow Chemical Company, 1803 Building, Midland, Michigan 48674
Received October 6, 1995; accepted February 21, 1996
Several inhalation toxicity studies of relatively high purity
Subchronic Toxicity of Ingested 1,3-Dichloropropene in Rats 1,3-D formulations have been conducted in Fischer 344 rats
and Mice. HAUT, K. T., STEBBINS, K. E., JOHNSON, K. A., SHAand B6C3F1 mice. The respiratory and olfactory epithelia
BRANG, S. N., AND STOTT, W. T. (1996). Fundam. Appl. Toxicol.
of the nasal mucosa of both rats and mice were observed to
32, 224-232.
undergo degeneration and/or hyperplasia when exposed to
Male and female Fischer 344 rats and B6C3F1 mice (10/sex/ ^ 3 0 and >90 ppm vapor, respectively, 6 hr/day, 5 days/
dose group) were given 0, 5, 15, 50, or 100 mg/kg/day (rats) or 0, week, for 13 weeks (Stott et al, 1988). In addition, hyperpla15, 50, 100, or 175 (mice) mg/kg/day racemic 1,3-dichloropropene sia of the transitional epithelium of the urinary bladder was
(1,3-D), respectively, via their diets for 13 weeks. Satellite groups
of rats (recovery = 10 rats/sex/group) ingesting 0 or 100 mg/kg/ observed in female mice exposed to 3*90 ppm vapor. Under
day 1,3-D were provided control feed for an additional 4 weeks a similar exposure regimen, the chronic inhalation of 5^20
to examine recovery. The test material was stabilized in the feed by ppm 1,3-D vapors by Fischer 344 rats and B6C3F1 mice
microencapsulation in a starch/sucrose matrix (80/20). The body also resulted in degeneration and/or hyperplasia of nasal
weights of male and female rats ingesting 3=5 and s=15 mg/kg/ mucosa (Lomax et al, 1989). In addition, hyperplasia of
day, respectively, and of all treatment groups of mice were de- the urinary bladder and forestomach epithelia and changes
creased relative to controls. The terminal body weights of high consistent with decreased hepatocellular glycogen and lipid
dose group rats and mice were decreased approximately 13-16%. content of renal tubular cells were observed in one or both
A number of changes in serum biochemical parameters and de- sexes of mice inhaling 20 and/or 60 ppm vapor. The only
creases in organ weights accompanied the depressed body weights
of these animals. Histologically, the only treatment-related change neoplastic response observed was an increased incidence of
observed was a slight degree of basal cell hyperplasia and hyper- benign lung tumors in high exposure group male mice.
keratosis in the nonglandular portion of the stomachs of a majority
The limited repeated exposure oral toxicity data which
of male and female rats ingesting 3=15 mg/kg/day. After the 4- have been generated on 1,3-D have suffered from the use
week recovery period, most treatment-related changes were noted
of outdated product formulations and bolus dosing (gato be reversible in nature. No treatment-related histopathological
changes were observed in the tissues of treated mice. Based upon vage) techniques, the latter dictated by the relatively high
relatively slight depressions in body weights at the lowest dosages volatility and potential instability of 1,3-D. Til et al.
tested, the no-observed-adverse-effect levels for male rats and both (1973) administered up to 30 mg/kg/day of a relatively
sexes of mice were determined to be 5 mg/kg/day and 15 mg/kg/ impure formulation of 1,3-D (78% purity) to Wistar rats
day, respectively. A no-observed-effect level of 5 mg/kg/day was via gavage, 6 days/week, for 13 weeks. Liver weights of
established for female rats, c i
male and female Wistar rats administered > 10 and 30 mg/
kg/day dosages, respectively, were elevated. No histopathological changes accompanied the organ weight changes.
1,3-Dichloropropene (1,3-D) has been utilized as a soil Chronic oral gavage of a higher purity (approx. 90%) forfumigant over the last 50 years for the control of nematodes mulation of 1,3-D, stabilized with epichlorohydrin, in rats
in a number of agricultural applications. As reviewed by and mice resulted in a number of tumorigenic and nontuTorkelson (1994), 1,3-D has an LD50 in Fischer 344 rats of morigenic effects (NTP, 1985; Yang et al, 1986). Benign
220-300 mg/kg, is a moderate dermal irritant when applied and/or malignant tumors of the forestomach and liver were
(occluded) to the shaved backs of rabbits, and causes marked observed in rats administered 25 and/or 50 mg/kg/day 1,3redness and chemosis when instilled in the eyes of rabbits. D. Tumors of the forestomach, urinary bladder, and, possiMost repeated-dose toxicity data on 1,3-D in experimental bly, the liver were also observed in female mice adminisanimals have been generated using the inhalation route of tered 50 and/or 100 mg/kg/day 1,3-D.
administration due to its high vapor pressure.
The present studies were undertaken to provide subchronic oral toxicity data on 1,3-D in rats and mice utilizing
1
To whom correspondence should be addressed.
a more recent 1,3-D formulation and a nonbolus oral dosing
0272-0590/96 $18.00
Copyright O 1996 by the Society of Toxicology.
All rights of reproduction in any form reserved.
224
1,3-DICHROLOROPROPENE SUBCHRONIC ORAL TOXICITY
methodology. These studies were conducted following
global Good Laboratory Practice Guidelines.
MATERIALS AND METHODS
Test substance.
1,3-D, obtained from DowElanco, Indianapolis, Indiana, as TELONEII soil fumigant, was microencapsulated in a starch/sucrose
matrix (80/20%) by Midwest Research Institute (MRI), (Kansas City, MO).
Particle size ranged from 100 to 400 fim. The liquid 1,3-D submitted to
MRI for microencapsulation was determined to have a chemical purity of
95.8% (50.7% cis; 45.1% trans) via gas chromatography/infrared spectroscopy. The microencapsulated 1,3-D, received from MRI, was determined
both gravimetrically and analytically to have a percentage loading of approximately 40% w/w and a composition nearly identical to that of the
original material. Microspheres containing no 1,3-D (placebo) were also
provided by MRI.
Animals. Male and female Fischer 344 rats and B6C3F1 mice, 6 - 8
weeks of age at study start, were purchased from Charles River Research
Laboratories (Portage, MI for mice and Kingston, NY for rats) and were
utilized for this study. Upon arrival at the laboratory,2 the animals were
examined by the laboratory veterinarian and were acclimated to the laboratory environment for at least 7 days prior to the initiation of dosing. The
animals were assigned to control and dose groups using a computerized
randomization program based on body weights. Animals were uniquely
identified via an implanted microchip (Biomedic Data Systems, Inc., Maywood, NJ). All animals were individually housed in stainless steel cages in
rooms designed to maintain adequate environmental conditions (22°C, 50%
RH, 12-h photocycle). Cages contained a feed crock and a pressure-activated nipple-type watering system. The animals were provided Purina Certified Rodent Chow 5002 (Purina Mills, Inc., St. Louis, MO), which served
as the vehicle for the microencapsulated 1,3-D, and tap water ad libitum.
Dosage levels. Groups of rats and mice (10/dose/sex) were administered
the test substance via their diets for a 13-week period. The targeted dose
levels for the study were 0 (control), 5, 15, 50, or 100 mg/kg/day 1,3-D
for rats and 0 (control), 15, 50, 100, or 175 mg/kg/day 1,3-D for mice.
Placebo microspheres were added to rodent chow and served as the control.
In addition, satellite groups of 10 rats/sex were provided 0 or 100 mg/kg/
day for 13 weeks, followed by a 4-week recovery period during which time
all animals were provided control rodent chow. These groups of animals
were used to examine the reversibility of any treatment-related effects observed following 13 weeks of dosing.
Diet preparation.
Diets were prepared by serially diluting the microencapsulated 1,3-D with rodent feed. Initial concentrations of 1,3-D in the
diet were calculated from prestudy body weights and feed consumption
data. Subsequently, the concentrations of the test material in the feed were
adjusted weekly based upon the most recent body weight and feed consumption data. The placebo microspheres were mixed with rodent feed to serve
as an appropriate control. The amount of starch/sucrose microspheres added
to the feed of control animals was approximately equivalent to the amount
of starch/sucrose added to the high dose diets. Stability, homogeneity, and
concentration analyses were conducted during the study.
In-life data. Animals were carefully examined each day for signs of
toxicity. In addition, a weekly clinical examination was conducted on all
animals which included a detailed, hands-on examination. Body weights
and feed consumption data were collected and calculated, respectively, for
all rats and mice twice during the prestudy period and then weekly during
the dosing and recovery (rats only) periods.
Clinical pathology.
Urinalysis (rats only), clinical chemistry, and hematological evaluations were conducted on all rats and mice. In rats, param-
2
Fully accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC).
225
eters identified as significantly different than controls during the 13-week
study were also evaluated in the recovery group rats following the 4-week
recovery period. Urine samples for rats were collected from nonfasted animals during the week prior to the scheduled necropsy. Urine pH, bilirubin,
glucose, protein, ketone, occult blood and urobilinogen content, specific
gravity, color, and appearance were determined using a Clinitek 200 urine
chemistry analyzer (Ames Division, Miles Laboratory, Elkhart, IN) and a
Goldberg Refractometer (American Optical Company, Instrument Division,
Buffalo, NY).
Blood samples were collected during the scheduled necropsies. The animals were anesthetized with methoxyflurane and blood samples were obtained via puncture of the orbital sinus. Hematology samples were mixed
with EDTA and blood smears were prepared and stained with Wright's
stain. Hematocrit, hemoglobin concentration, erythrocyte count, total leukocyte count, and platelet count were determined using an ELT-8 (Ortho
Instruments, Westwood, MA). Leukocyte differential counts were conducted manually and erythrocyte, leukocyte, and platelet morphology were
evaluated.
Blood samples for clinical chemistry determinations were held on ice
and the serum was harvested. Enzyme activities of alkaline phosphatase,
alanine aminotransferase, aspartate aminotransferase, creatine phosphokinase (rat only), and concentrations of urea nitrogen, creatinine, total protein,
albumin, globulin (calculated), glucose, total bilirubin, electrolytes (Na, K,
P, Cl, Ca), cholesterol, and triglycerides were determined using a Monarch
2000 chemistry analyzer (Instrument Laboratories, Lexington, MA).
Anatomic pathology. Rats and mice were anesthetized with methoxyflurane, the tracheas were exposed and clamped, and the animals were
euthanized by decapitation. Terminal body weights (rats, fasted; mice, nonfasted) were recorded for all animals at the scheduled necropsy. Brain, liver,
kidneys, heart, adrenals (rats only), and testes or ovaries (rats only) were
weighed and organ-to-body weight ratios calculated.
A complete gross examination of tissues was conducted on all animals.
The nasal cavity was flushed and the lungs distended to an approximately
normal inspiratory volume with neutral, phosphate-buffered 10% formalin.
In addition, the urinary bladder was moderately distended with neutral,
phosphate-buffered 10% formalin. Representative samples of over 65 tissues from each animal were collected and preserved in formalin solution.
All preserved tissues from control and high dose group animals were
processed by standard procedures and embedded in paraffin. Tissues were
sectioned at approximately 6 ^m, stained with hematoxylin and eosin, and
examined by light microscopy by a veterinary pathologist The lungs, liver,
kidneys, stomach, mesenteric tissues (female rats only), any tissues having
grossly observable lesions, and any target tissues were processed and examined from intermediate and low dose group animals. Potential target tissues
identified following 13 weeks, kidneys, liver, stomach, and mesenteric tissues, were examined histologically from recovery group rats.
Statistics. Descriptive statistics (means and standard deviations) were
reported for feed consumption, leukocyte differential counts, and nucleated
erythrocyte counts. Body weights, organ weights, clinical chemistry data,
appropriate hematologic data, and urinary specific gravity were evaluated
by Bartlett's test (a = 0.01; Winer, 1971) for equality of variances. Based
on the outcome of Bartlett's test, exploratory data analysis was performed
by a parametric (a = 0.10; Steel and Tome, 1960) or nonparametric (a =
0.10; Hollander and Wolfe, 1973) analysis of variance (ANOVA), followed
respectively by Dunnett's test (a = 0.05, two sided; Winer, 1971) or the
Wilcoxon rank-sum test (a = 0.05, two sided; Hollander and Wolfe, 1973)
with a Bonferroni correction (Miller, 1966) for multiple comparisons.
RESULTS
Dosing. The microcapsule technology used in this study
was developed by MRI to provide a means by which volatile,
reactive, or unpalatable chemicals can be administered to
226
HAUT ET AL
1 2
3
4
5
10
11
12
OMO/KO/DAY
OMO/KQ/DAY
3UO/XO/DAY
5MQ/WVOAY
ISUO/KO/QAY
19 UQ/KOtlAY
SOUOAOCMY
SOUO/KOOAY
100 WVKO/DAY
100 UO/KO/DAY
13 14 15 16 17
1 2
3
4
5
10 11
12 13 14 15 16 17
FIG. 1. Mean body weights of (a) male rats and (b) female rats ingesting 1,3-D for 13 weeks and control feed for a 4-week recovery period (means
of 10-20 rats/sex/dose).
test animals via their diet. The relative reactivity and volatility (Vp = 28 mm Hg at 25°C) of 1,3-D precluded the addition
of liquid 1,3-D to the feed. After encapsulation by MRI, the
percentage loading of 1,3-D in the starch/sucrose microsphere matrix (i.e., weight of microcapsules contributed by entrapped 1,3-D) was determined to be 39.1 ± 0.2% w/w of
1,3-D. The microencapsulated 1,3-D proved to be stable for
several years with room temperature storage and stable in
the diet for at least 21 days postmixing (5=90% of target)
and could provide a homogeneous mixture with animal feed.
Analyses of the test diets during the study established that
all animals received approximately their targeted dosages of
1,3-D. The mean concentrations of 1,3-D in test diets ranged
from 88 to 109% of target for rats and 96 to 114% of target
for mice. In addition, a separate study has demonstrated that
the bioavailability of ingested microencapsulated 1,3-D is
similar to that of neat 1,3-D (W. Stott, unpublished data).
These data establish the use of microencapsulation technology as a valid method of oral administration of 1,3-D to test
animals.
weeks of dosing (Figs. 3a and 3b). By the end of the 4-week
recovery period, high dose level rats ingested amounts of
feed approximately equivalent to those of control rats. Feed
consumption by treated mice was relatively unchanged and
only occasionally identified as depressed at the higher dose
levels relative to controls during the study (data not shown).
Antemortem data. There were no treatment-related clinical signs of toxicity noted in rats or mice at any dose level
over the course of the study. Body weight data are presented
in Figs. 1 and 2. A dose-related, statistically identified depression in body weights (6-16%) was observed in male
rats ingesting dosages of 5=5 mg/kg/day and female rats
(5-11%) ingesting 5*15 mg/kg/day, respectively, relative to
control values. Following the 4-week recovery period, the
body weights of high dose males and females were only
11.4 and 8.7% lower than controls, indicating some recovery.
Body weights of male and female mice ingesting dosages
of 5= 15 mg/kg/day were depressed in a dose-related manner
at the termination of the study by 5-15 and 5-13%, respectively.
Feed consumption was consistently decreased for high
dose group male and female rats and occasionally depressed
at lower dosages relative to control values during the 13
Also shown in Table 1, statistically identified differences
in clinical chemistry parameters were limited to slight decreases in serum alkaline phosphatase (5=50 mg/kg/day), triglyceride levels (s= 15 mg/kg/day), and increased cholesterol
levels (high dose) in male rats and minimal decreases in the
total protein, albumin, and globulin levels in 50 and/or 100
mg/kg/day dose group females. Following a 4-week recovery
period, most values had returned to near control levels
though several were still statistically identified as different
from controls (Table I). The only statistically identified
changes observed in mice were decreases in the serum glucose levels in high dose group males and triglyceride levels
in females ingesting 5=15 mg/kg/day 1,3-D (Table 1). With
the possible exception of alkaline phosphatase levels, clinical
chemistry changes in either species were consistent with the
lower body weights and reduced nutritional status of affected
animals.
Clinical pathology. There were no treatment-related
changes identified in any urinalysis parameters measured
in treated rats following 13 weeks. The only statistically
identified changes observed in hematological parameters in
either species tested were increases in the platelet count of
male rats ingesting 3=50 mg/kg/day and a decrease in the
white blood cell count of male mice ingesting 175 mg/kg/
day (Table 1); however, these changes were not dose-related
and/or lacked a histopathologic correlate. No treatment-related changes in WBC differential counts were noted in any
treatment groups of animals (data not shown). PLAT counts
had returned to control values following the 4-week recovery
period (Table 1).
227
1,3-DICHROLOROPROPENE SUBCHRONIC ORAL TOX1CITY
OUO/KQ/DAY
OUQXQflMY
13MQ/KQAIAY
15MQ/KQ/DAY
UUQ/KQJDAY
00 MQ/KQ/DAY
100 MQ/KQ/DAY
100 MG/KO/OAY
175MQ/KQ/tMY
175 UO/KQ/DAY
10
11
12
13
FIG. 2. Mean body weights of (a) male mice and (b) female mice ingesting 1,3-D for 13 weeks (means of 10 mice/sex/dose).
Postmortem data. Consistent with the depressed body
weights of treated animals during the dosing period, terminal
body weights of fasted rats at the mid and high dose levels
and nonfasted mice at all dose levels were decreased 6-16
and 5-15% relative to controls, respectively (Tables 2 and
3). A number of statistically identified organ weight changes
were also observed in treated rats and mice (Tables 2 and
3). In general, absolute weights were decreased while organ
to body weight ratios were increased consistent with the
depressed body weights of these animals.
No gross pathologic lesions that were attributable to 1,3D ingestion were observed at necropsy for rats or mice at
any dose level. Decreased body fat was observed in most
female rats ingesting 5=50 mg/kg/day 1,3-D following 13
weeks of dosing; however, this observation was also consistent with the depressed body weights of these animals.
The most significant histopathological change observed
in either species ingesting 1,3-D was in the mucosa of the
v
vn
16 i
r v^
14
6
0 UQfl<Q/OAY
•
8UQWODAY
nonglandular portion of the stomach of rats. A minimal
degree of basal cell hyperplasia was observed in both male
and female rats at dosages of 3= 15 mg/kg/day 1,3-D (Table
4). As shown in Fig. 4B, this effect was characterized by
prominence of the basal or deepest layers of the mucosa
due to increased cytoplasmic basophilia along with an
apparent increased number of cell layers in the basilar
portion of the mucosa. The normally round nuclei found
in this location were oval with the long axis perpendicular
to the basement membrane and were crowded together
into a layer generally two to three cells thick rather than
the single layer normally present. Additionally, there was
a slight prominence of mononuclear cells at the basement
membrane. These cells appeared to consist of endothelial,
fibroblast, and inflammatory cells. Overall, basal cell hyperplasia was of minimal severity even at the high dosages. It is noteworthy that the nonglandular gastric mucosa
of a control rat also had a very slight degree of basal cell
u v
12 •
10
— f i — OUO/KQ/IMY
•
12
5UQ/KO/OAY
8
10 -
*
50UQ/KO/DAY
D
100 MOKOfOAY
8
3
4
5
7
8
9
10
11
12
13
14
15
16
17
19UO/KOQAY
*
50UO/KOOAY
D
100UQ/KQAMY
6
4
1 2
0
•
1 2
1
1
1
3
4
1
5
1
1
1
1
1
1
1
I
1
1
1
1
7
8
9
10
11
12
13
14
15
16
17
WEEK
FIG. 3. Mean feed consumption values of (a) male rats and (b) female rats ingesting 13-D for 13 weeks and control feed for a 4-week recovery
period (means of 10-20 rats/sex/dose).
228
HAUT ET AL.
TABLE 1
Selected Hematology and Clinical Chemistry Data for Male and Female Rats and Mice Ingesting 1,3-D for 13 Weeks
(Mean and Standard Deivations of 10 Animals/Sex/Dose)
Rats
Males
Dose
(mg/kg/day)
0
5
15
50
100
0 (recov.)
100 (recov.)
Alkaline
phosphatase
(g/dl)
Platelets
(Xl0E3/Cu mm)
460
470
460
500
503
451
458
±
±
±
±
±
±
±
29
38
20
28*
33*
43
36
142 ±
136 ±
133 ±
120 ±
I19±
147 ±
156 ±
Mice
11
10
11
8*
8*
10
5*
Females
Cholesterol
(mg/dl)
Triglyceride
(mg/dl)
Albumin
(g/dl)
±
±
±
±
±
±
±
9 2 ± 21
87 ± 22
72 ± 8*
58 ± 11*
59 ± 14*
91 ± 20
77 ± 14
3.4 ± 0.
3.3 ± 0.
3.3 ± 0.
3.3 ± 0.
3.2 ± 0. *
3.5 ± 0.1
3.4 ± 0 1
65
63
65
71
78
52
49
7
6
4
7
5*
4
2*
Globulin
(g/dl)
3.4
3.4
3.3
3.2
3.1
3.3
3.2
±
±
±
±
±
±
±
0.2
0.3
0.1
0.2*
0.2*
0.1
0.2*
Total proL
(g/dl)
6.8 ± 0.3
6.7 ± 0.3
6.5 ± 0.2
6.5 ± 0.2*
6.3 ± 0.3*
7 1 ± 0.3
6.7 ± 0.3*
Males
Dose mg/
kg/day
Leukocytes
(Xl0E3/Cu mm)
0
5
15
50
100
2.0
2.0
1.7
1.5
1.1
±
±
±
±
±
Females
Triglyceride
(mg/dl)
Glucose
(mg/dl)
190
186
184
178
162
0.5
0.6
0.6
0.6
0.5*
±
±
±
±
±
29
27
22
24
16*
92
87
72
58
59
±
±
±
±
±
21
22
8*
11*
14*
' Statistically identified as different than controls by Dunnett's test (a = 0.05).
TABLE 2
Mean Terminal Body Weights and Selected Organ Weight Data for Rats Ingesting 1,3-D for 13 Weeks
(Means of 10 Animals/Sex/Dose)
Body
Dose
(mg/kg/day)
weight
(g)
Brain weight
(g)
(g/100)
Heart weight
(g)
Kidney weight
(g/100)
(g)
(g/100)
Liver weight
Testes weight
(g)
(g/100)
(g)
g/100)
8.001
7.725
7.640
6.990**
7.072**
2.749
2.795
2.798
2.846**
2.889**
3.033
3.030
3.072
2.785
3 072
.043
.098*
.126*
136
258*
Males
0
5
15
50
100
291.0
276.3
273.0**
245.6**
244.9**
.906
.890
890
.847**
.848**
0.656
0.685
0.693
0.753**
0.758**
0.872
0.843
0.865
0.774**
0.764**
0.300
0.305
0.317
0.315
0.313
.928
.860
.845
.721**
707**
0.662
0.674
0.676
0.700
0.699
Females
Adrenal weights"
0
5
15
50
100
159.2
158.9
155.1
148.6**
143.2**
.727
.698
.719
.716
.691
1.088
1.070
1.109
1.155**
1.183**
0.619
0.607
0.598
0.583
0.550**
0.388
0.382
0.386
0.399
0.384
.192
.153
.170
.159
.127
0.749
0.725
0.755
0.780
0.787
4 502
4.411
4.350
4.304
4.146**
" Male adrenal weights not presented as they were not statistically identified as different from control values.
* Statistically different from control mean by Dunnett's test, a = 0.05.
** Statistically different from control mean by Wilcoxon's test, a = 0.05.
2.827
2.776
2.804
2.898
2.894
0.054
0.053
0.052
0.048
0.046**
0.034
0.033
0.033
0.033
0.032
229
1,3-DlCHROLOROPROPENE SUBCHR0N1C ORAL TOXICITY
TABLE 3
Mean Temrinal Body Weights and Selected Organ Weight Data for Mice Ingesting 1,3-D for 13 Weeks
(Means of 10 Animals/Sex/Dose)
Body
Dose
(mg/kg/day)
weight
(g)
Brain weight
(g)
Heart weight
(g/100)
Kidney weight
(g/100)
(g)
is)
(g/100)
Liver weight
(g)
Testes weight
(g/100)
(g)
(g/100)
0.230
0.238
0.231
0.232
0.220
0.859
0.934
0.941*
0.975*
0.963*
Males
0
15
50
100
175
26.8
25.4*
24.6*
23.9*
22.8*
0.506
0.4%
0.485
0.490
0.485
1.894
1.952
1.978
2.056*
2.129*
0.542
0.577
0.558
0.581
0.547
0.146
0.147
0.137
0.139
0.125*
0.500
0.495
0.478
0.452*
0.407*
1.867
1.943
1.945
1.894
1.784
1.366
1.300
1.225*
1.148*
1.079*
5.059
5.105
4.987
4.811
4.730
0.365
0.348
0.341*
0.344*
0.313*
1.446
1.460
1.482
1.547
1.429
1.370
1.228*
1.202*
1.151*
1 110*
5.431
5.136
5214
5.163
5.071*
Females
0
15
50
100
175
25.2
23.9*
23.1*
22.3*
21.9*
0.500
0.496
0.486
0.497
0.497
1.985
2.083
2.113*
2.235*
2.272*
0.139
0.125**
0.128**
0.123**
0.117**
0.549
0.526
0.559
0.553
0.536
* Statistically different from control mean by Dunnett's test, a = 0.05.
** Statistically different from control mean by Wilcoxon's test, a = 0.05.
TABLE 4
Histopathologic Incidence Data for Rats and Mice Ingesting 1,3-D for 13 Weeks
Rats"
Males (mg/kg/day)
15
Stomach, basal cells
Nonglandular portion
Hyperplasia (minimal)
Main study
Recovery group
Hyperkeratosis (minimal)
Main study
Recovery group
Females (mg/kg/day)
50
100
10
10
15
50
100
10
10
6
5
0
Mice"
Females (mg/kg/day)
Males (mg/kg/day)
15
50
100
175
Kidneys
(Valuolation of tubules)
Decreased, very slight
Liver
(Hepatocytes, size)
Decreased, very slight
" Number of animals having observation per 10 animals/group/sex examined. (—) Not evaluated.
15
50
100
175
230
1,3-DICHROLOROPROPENE SUBCHRONIC ORAL TOXICITY
hyperplasia. Basal cell hyperplasia of the mucosa of the
nonglandular stomach was also observed following the 4week recovery for rats; however, the severity was somewhat diminished compared to the 13-week findings, indicating the reversible nature of this lesion (Table 4).
Hyperkeratosis of the mucosa of the nonglandular stomach was also noted in several male and female rats ingesting
2*50 mg/kg/day and in a single male ingesting 15 mg/kg/
day 1,3-D (Table 4). Also shown in Fig. 4b, this effect was
characterized by a slight thickening of the most superficial
layer of the nonglandular mucosa, the keratinized layer, suggesting a response to the potentially irritating 1,3-D. No
effects were noted in the glandular portion of the gastric
mucosa. Following the 4-week recovery period, no incidences of hyperkeratosis of the nonglandular stomach were
noted.
Histologic changes were noted in the livers and kidneys of
male mice ingesting 1,3-D (Table 4); however, these changes
were considered secondary to the lowered body weights and
reduced nutritional status of a majority of these animals. The
liver change was observed in a number of male mice from all
of the treatment groups and was characterized as a minimal,
diffuse decrease in hepatocellular size consistent with decreased cytoplasmic glycogen. The kidney change was characterized as decreased vacuolation of tubular epithelial cells
in a few males ingesting 175 mg/kg/day 1,3-D consistent
with decreased cytoplasmic lipid content. There were no
microscopic liver or kidney changes in female mice, although statistically identified decreases in absolute liver and
kidney weights were noted in females ingesting 5=50 mg/
kg/day 1,3-D. Female mice normally appear to have less
glycogen and cytoplasmic vacuolation in hepatocytes and
renal tubular cells, respectively, upon microscopic examination, and therefore minimal changes are more difficult to
recognize.
DISCUSSION
1,3-D, a volatile and potentially unstable chemical, was
successfully administered to rats and mice for 13 weeks by
mixing a microencapsulated formulation of this chemical
into animal feed. Analyses of test diets during the course of
the study ensured that the microencapsulated 1,3-D was stable in feed and that test animals ingested targeted dosages
of the test material. Additional work has shown that once
ingested, microencapsulated 1,3-D is as readily absorbed
as neat 1,3-D (W. Stott, unpublished data). Utilizing this
technology, the oral dosages administered in the present
study were in excess of those previously administered to
231
rats and mice using bolus dosing (gavage), especially if the
frequency of dosing is taken into consideration. Rats were
administered up to only 30 mg/kg/day low purity 1,3-D for
5 days/week by Til et al. (1973), while rats and mice were
administered up to 50 and 100 mg/kg/day of higher purity
1,3-D, respectively, for only 3 days/week in the NTP (1985)
gavage bioassay (Yang et al., 1986), albeit for a much longer
period of time.
The most significant treatment-related effect noted in rats
or mice ingesting 1,3-D was a dose-related depression in
body weights over much of the dosing period. In rats, doserelated decreases in feed consumption suggest a possible
palatability problem with the test diets; however, a similar
decrease was not noted in treated mice. The present study
was not designed to unequivocally determine palatability of
test diets (i.e., the preference of control over test diets);
thus decreased feed consumption may simply have reflected
decreased body weights. It is important to note that despite
decreases in the feed consumption of some animals, correction of the concentration of 1,3-D in the diets ensured that
target dosages were still maintained throughout the dosing
period.
The only significant histopathological change noted in
these animals was a minimal degree of hyperplasia and hyperkeratosis of the nonglandular stomach mucosa of roughly
half the rats ingesting 3=50 mg/kg/day 1,3-D. These findings
are consistent with the irritant properties of this chemical.
Hyperplasia of the nonglandular stomach mucosa was also
reported in rats administered 1,3-D via gavage in the NTP
study within 9 months of dosing (NTP, 1985; Yang et al.,
1986). Further evidence of a portal of entry effect was noted
in rats and mice administered inhaled 1,3-D vapors in which
hyperplasia of the nasal mucosa was found to be a target
tissue in these animals (Lomax et al., 1989; Stott et al.,
1988). Upon removal of the test material from the diet, some
recovery from gastric mucosal lesions was observed in both
sexes of rats.
The results of the mouse study contrast with those of
earlier studies of 1,3-D in that the transitional epithelium of
the urinary bladders of mice were not affected at dosages
roughly 3 - 7 times higher than those causing significant hyperplasia and tumors, in the gavage B6C3F1 mouse study
conducted by NTP, 50 or 100 mg/kg/day (NTP, 1985; Yang
et al., 1986). Obviously, the longer dosing duration of the
latter study, even with a 3 days/week dosing regimen, could
have affected lesion formation. However, a similar, significant degree of hyperplasia was noted in the urinary bladder
mucosa of B6C3F1 mice inhaling >90 ppm 1,3-D vapor at
a similar dosage and duration of dosing as in the present
FIG. 4. Photomicrographs of a section of the nonglandular ponion of the stomach of (A) a control male rat and (B) a male rat ingesting 100 mg/
kg/day 1,3-D for 13 weeks (H&E; 365x magnification). Note increased numbers of basal cells ([]) and slightly increased amounts of keratin (*) in the
treated rat.
232
HAUT ET AL.
study. Dosages administered in the latter study at 90- and
150-ppm exposure levels are calculated to have been approximately 160-210 and 260-350 mg/kg/day, respectively (5
days/week; assuming respiratory minute volumes 40-54 ml/
min and 80% absorption of vapor) (Chang et al., 1981; Guyton, 1947). A possible explanation for this phenomenon may
lie in differences in the levels of 1,3-D metabolites in the
urine of mice. Stott et al. (1992) speculated that specific,
species-specific breakdown products of glutathione conjugates of 1,3-D were responsible for bladder mucosa irritation
and subsequent hyperplasia in mice. The concentration(s) of
these products would be expected to reach higher levels
following bolus (gavage) administration of 1,3-D or a 6hr inhalation exposure than would occur during the more
prolonged, yet lower, intake of 1,3-D during normal feeding.
1,3-D itself is not excreted in the urine of mice even at high
bolus dosages (Dietz et al., 1984; J. Waechter, unpublished
data) and is thus not believed to play a direct role in bladder
mucosal effects (Stott et al, 1992).
In conclusion, nonbolus oral administration of 1,3-D to
rats and mice was successfully conducted using microencapsulated 1,3-D added to the animal's diet. Treatment-related
microscopic changes noted in treated animals were limited
to portal-of-entry effects in the nonglandular stomach mucosa of rats consistent with the potential irritating properties
of 1,3-D. The no-observed-adverse-effect level (NOAEL)
for male rats, based upon minimal in-life body weight depression (=s6%), and the no-observed-effect level for female
rats were determined to be 5 mg/kg/day. No specific target
tissues were identified in mice; however, based on minimal
body weight changes (*£5%), the NOAEL was determined
to be 15 mg/kg/day in both sexes.
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