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1,3-Dichloropropene: Two-generation inhalation reproduction study in Fischer 344 rats
FUNDAMENTAL
AND
APPLIED
TOXICOLOGY
12,129- 143 ( 1989)
1,3-Dichloropropene: Two-Generation Inhalation Reproduction Study in Fischer 344 Rats W. J. BRESLIN,’ H. D. KIRK, C. M. STREETER, Mammalian
J. F. QUAST,
AND J. R. SZABO~
and Environmental Toxicology Research Laboratory Health and Environmental Sciences, Dow Chemical Company, 1803 Building, Midland, Michigan 48674
Received February 8.1988; accepted June 6, I988 1,3Dichloropropene:
Two-Generation Inhalation Reproduction Study in Fischer 344 Rats. H. D., STREETER, C. M., QUAST, J. F., AND SZABO, J. R. ( 1989). Fundam. Appl. Toxicol. 12, 129-l 43. This study evaluated the effects of inhaled technical-grade 1,3-d& chloropropene (DCPT) on reproduction and neonatal growth and survival. Groups of 30 male and 30 female Fischer 344 rats, approximately 6 weeks of age, were exposed via inhalation to 0, lo,30 or 90 ppm DCPT for 6 hr/day, 5 days/week, for two generations. The parental f0 and f, generations were each bred twice. Reproductive and neonatal parameters evaluated included indices of fertility and pup survival, gestation length, litter size, pup body weight, and pup sex ratio. Gross and histologic examinations were conducted on all 5 and f, adults. In addition, randomly selected fib and fZsweanlings were given gross examinations. Parental effects were limited to rats exposed to 90 ppm DCPT and included decreased body weights and histopathologic effects on the nasal mucosa of adult male and female rata. The histopathologic effects consisted of slight, focal hyperplasia of the respiratory epithelium and/or focal degenerative changes in the olfactory epithelium. No adverse effects on reproductive parameters or neonatal growth or survival were observed in the f,. , fib, fin, or fZblitters even at an exposure concentration which produced effects in adult animals. Based on these results, it is concluded that inhalation exposure of rats up to 90 ppm DCPT for two successivegenerations did not adversely atfect the reproductive and neonatal parameters evaluated. Q 1989 kiety of Toxicology. BRESLIN,
W. J., KIRK,
Technical-grade 1,3dichloropropene (DCPT) is used as a preplant nematicide for a variety of agricultural crops. The present threshold limit value for DCPT, as adopted by the American Conference of Governmental Industrial Hygienists, is 1 ppm. Vapors of DCPT appear to be moderately toxic to rodents. The 4-hr LC50 value for rats is approximately 1000 ppm (Dow Chemical Co., unpublished data). In a subchronic inhalation toxicity study (Stott et al., 1988b) Fischer 344 rats were exposed to 0, 10,30,90 ’ To whom correspondence should be addressed. 2 Present address: Health and Environmental Sciences-Texas, Dow Chemi&l Company, Freeport, TX 75541.
or 150 ppm DCPT 6 hr/day, 5 days/week, for 13 weeks. Rats exposed to 90 or 150 ppm DCPT were observed to have a treatment-related depression in body weight gain. Treatment-related pathological changes noted in these rats were slight degeneration of the nasal olfactory epithelium ( 150-ppm-exposure group) and a slight hyperplasia of the nasal respiratory epithelium (90- and 150-ppm-exposure groups). In addition, a few male rats exposed to 30 ppm DCPT also had very slight hyperplasia of their nasal respiratory epithelium. Inhalation teratology studies with rats and rabbits indicated exposures as high as 120 ppm DCPT were not embryotoxic or teratogenie (Hanley et al., 1987). Maternal toxicity 129
0272-0590189 $3.00 Copyiigbt Q I989 by the %ci~ry of Toxicology. Au Mt.9 of reproduction in any form mewed.
130
BRESLIN
as manifested by decreases in body weight and/or body weight gain was observed in groups of rats exposed to 20,60, or 120 ppm and rabbits exposed to 60 or 120 ppm DCPT. The study described herein was conducted to evaluate the effects of inhaled technicalgrade DCPT on reproductive capability and neonatal growth and survival in rats. The inhalation route was chosen because it is the most likely route of potential human exposure in the manufacture and use of DCPT. MATERIALS
AND
METHODS
Test material. Technical-grade DCPT was obtained from The Dow Chemical Company, Freeport, Texas. The test material was analyzed by gas chromatography prior to initiation ofthe study, at approximately 6-month intervals during the course of the study, and following the last exposure. The purity of the test material was 92% DCPT, the remainder of the test material comprised chlorinated and unchlorinated alkanes and alkenes as well as approximately 2.0% nonvolatile stabilizer (epoxidized soybean oil). Test species and husbandry. Male and female Fischer 344 rats’ approximately 4 weeks of age were used. All rats were examined for health status by the laboratory veterinarian and acclimated to the laboratory environment for a minimum of 2 weeks prior to use. The rats were weighed and assigned to the exposure groups using a stratified, by weight, randomization procedure. This procedure was intended to increase the probability of uniform mean pretreatment body weights across groups. All rats were identified by uniquely numbered metal ear tags. Rats were housed singly in wire-mesh, stainless-steel cages during the exposure periods. During late gestation and lactation, females were housed in plastic shoe box cages provided with ground corn cob nesting material. Water was available ad libitum throughout the study. Feed4 was available ad libitum except during exposure periods. The animal rooms were designed to maintain temperature at approximately 22°C relative humidity at approximately 50%, and a 12-hr-light: 12-hr-dark photocycle. Vapor generation and exposure conditions. Inhalation exposures were conducted in 14.5m3 chambers with stainless-steel pyramid-shaped ceilings and epoxy-resincoated floors and walls. All chambers were operated un-
3 Charles River Breeding Laboratories, Kingston, NY. 4 Purina Certified Rodent Chow 5002, Ralston Purina Co., St. Louis, MO.
ET AL. der dynamic airflow conditions at a slight negative pressure relative to the surrounding area. Control rats were placed in a chamber of the same design. The air supplied to the chambers was controlled by a system designed to maintain temperature and relative humidity at approximately 2 1°C and 50%. respectively; temperature and humidity were recorded daily. Test atmospheres containing DCPT were generated by metering liquid test material at controlled rates into glass J-tubes (Miller et al., 1980). The liquid was vaporized in the J-tubes with preheated compressed air and vapors were subsequently swept into the main chamber airstream where there was further dilution to the desired concentration. The compressed air was preheated with a flameless heat torch at the lowest temperature (<7o”C) necessary to facilitate complete vaporization of the liquid test material. Total chamber airflow was maintained at approximately 2500 liters/min. The concentration of the test material in each chamber was determined at least once per hour with a Miran 1A infrared spectromete? at a wavelength of 12.9 pm (peak absorption of tram isomer). The nominal concentration (ratio of DCPT used to total airflow through the chamber) was calculated for each chamber on a daily basis. The concentration of DCPT in the exposure chamber was determined by interpolation from a standard curve derived from vapor standards of known concentrations. Standard curves were run prior to the first exposure and approximately monthly thereafter. The infrared spectrometer was calibrated before each daily exposure using a known standard atmosphere of DCPT. Chamber vapor distribution was ascertained prior to the study start. Eight points at the extremes of the rat breathing zones were compared to the primary sample line (reference line) in each chamber in which DCPT was to be introduced. The mean concentration of DCPT, calculated from the eight distribution points per chamber, was approximately 90*5% of the primary sample line. Experimental design. Groups of 30 male and 30 female rats (fO)were exposed via inhalation to 0, 10, 30, or 90 ppm DCPT for 6 hr/day, 5 days/week, prior to breeding and 6 hrjday, 7 days/week, during breeding, gestation, and lactation. The exposure of f0 rats began at ap proximately 6 weeks of age. After 10 weeks of exposure (5 days/week), f0rats were mated, one male to one female of the respective treatment group, to produce the f,. litters. Breeding for the fib litter began 1 week after weaning ofthe last f,, litter. Following weaning ofthe last fib litter, 1 male and 1 female pup, when available from each litter, within a treatment group were randomly selected using a table of random numbers and assigned to the respective exposure groups to become parents for the next generation. In treatment groups with fewer than 30 litters, additional pups were randomly selected from litters of the same treatment group until 30 male and 30 female pups
’ Foxboro Analytical, Norwalk, CO.
1,3-DICHLOROPROPENE 300
131
REPRODUCTION
A
280 1 260 1 240 220 200 180 160 OPPM ,D
120 100
-
PPM
3opPM WPPM
1
80 I 601 - 18 -11 -4
r. 3
n = I 7, - 1 -1 10 17 24 31 38
9 1 . , =I 45 52 59
-1 66
-,
-1
70
77
73
80
Days on test 320 300 280 260 240 220 200 180 160 I-o-
140
WPpn
120 0
7
14
21
28
35
42
49
56
63
84
Days on test FIG. 1. Premating body weights for&,(A) and f, (B) adult male rats exposed to DCPT vapors. The number of male rats per exposure group was 29 or 30. *Statistically different from control by Dunnett’s test, a = 0.05, two-sided.
132
BRESLIN ET AL.
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Days on test 280 -B 260 _ 240 _
220 _ 200 _ 180 _
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1,
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Gest.
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Lat.
,
1’1’1’1
7
‘I
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Gest.
‘I
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Lat.
Days on test FIG. 2. Body weights for f0 (A, B) and f, (C, D) adult female rats exposed to DCPT vapors. The number of female rats per exposure group was equal to 30 during the premating period and ranged from 19 to 27 during gestation and lactation (B, D). *Statistically different from control by Dunnett’s test, (Y= 0.05, twosided.
I ,3-DICHLOROPROPENE
133
REPRODUCTION
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15
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g
13
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11 10 9 8 0
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28
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42
56
49
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Days on test
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28 7
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128
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Days on Test FIG. 2-Continued. were obtained. After approximately 12 weeks of exposure (5 days/week, excluding holidays), the f, adults were bred to produce the fra litters. Breeding for the frs litters began 1 week after weaning of the last fr, litter. Exposure
of f0 and fr adult rats to DCPT was continued until the adults were sacrificed. Rats were housed continuously in exposure chambers except during late gestation and lactation when females
BRESLIN ET AL.
134
TABLE 1 FERTILITY
INDICES AMONG
RATS EXFQSED
TO DCPT
Generation DCPT (mm)
N
f la
f Ib
f 2a
f 2b
93.3 96.7 90.0 96.7
90.0 93.1 83.3 100.0
85.7 79.3 70.4 93.1
85.2 92.6 80.0 86.2
Female mating index” 0 10
30 90
30 29 or 30 30 29 or 30
100.0 96.7 90.0 96.7
96.7 100.0 90.0 96.7
Female conception index” 0 10 30 90
27-30 27-30 25-27 29
0
22-25 22-25 19-23 25-27
73.3 75.9 81.5 89.7
86.2 80.0 85.2 86.2
Female gestation index ’ 10
30 90
96.0
100.0 100.0 100.0 100.0
100.0 100.0 100.0
100.0 100.0 100.0 100.0
95.7 100.0 100.0 100.0
Male mating indexd 0 10
30 90
30 29 or 30 30 30
93.3 76.7 80.0 83.3
80.0 90.0 76.7 86.7
76.7 80.0 66.7 90.0
76.7 72.4 63.3 83.3
91.3 83.3 75.0 92.6
87.0 95.2 84.2 92.0
Male conception index’ 10
0
23-28 21-27
30 90
25-27
19-24
78.6 78.3 83.3 92.0
95.8 85.2 95.7 92.3
Cohabitation time (days) required for mating/ 0 10 30 90
27-30 27-30 25-27 29
4.3 f 3.2 5.3 AZ4.4 5.2 + 5.8 5.3 rt 4.6
5.4 f 5.1 3.8 k 2.5 4.2f3.1 4.5 + 4.9
3.9 4.9 6.8 7.0
f 4.6 + 3.7 + 6.8 + 7.3
4.6 f 3.6 f 4.3 iI 4.7 +
4.5 2.7 4.1 5.3
Note. No values differed significantly from control value by Fischer’s exact probability test, (Y= 0.05, with Bonferroni correction. UNumber of females with a sperm-positive vaginal smear/total number females (percentage). b Number of females delivering a litter/number of sperm-positive females (percentage). ’ Number of females delivering a live litter/number of females delivering a litter (percentage). d Number of males which mated resulting in a sperm-positive vaginal smear/total number of males (percentage). ’ Number of males which sired a litter/number of males mated resulting in a sperm-positive vaginal smear (percentape). ‘Average number of days of male/female cohabitation required to produce a sperm-positive vaginal smear. 8 Mean 2 SD.
1,3-DICHLOROPROPENE TABLE 2 GESTATION AND POSTNATAL SURVNAL AMONG LITTERS OF RATS EXPOSED TO DCPT Generation DCPT (m-W
Number of litters
f la
fib
fi,
fZb
100.0 98.8 100.0 99.6
92.8 99.1 100.0 98.8
Gestation survival index” 0 10 30 90
22-25 22-25 19-23 25-27
99.1 99.5 100.0 99.7
98.1 99.6 99.3 100.0
Postnatal survival index, 4-day b 0 10 30 90
22-25 22-25 19-23 25-27
99.1 99.5 100.0 100.0
100.0 98.0* 99.6 99.3
99.5 99.6 98.2 99.3
98.6 99.1 95.9 99.2
98.8 100.0 99.3 99.0
98.7 99.4 98.4 100.0
Postnatal survival index, 28day’ 0 10 30 90
22-25 22-25 19-23 25-27
100.0 100.0 100.0 96.1
99.5 98.2 99.5 99.5
’ Number of pups alive at birth/number of pups born (percentage). b Number of pups surviving for 4 days/total number of livebom pups (percentage). ’ Number of pups surviving for 28 days/total number of live pups after culling on Day 4 (percentage). *Significantly different from control value by Fischer’s exact probability test, OL= 0.05, with a Bonferroni correction.
were removed following the exposure period. Parturition and housing of pups necessitated the utilization of nesting cages with solid bottoms and sides. The solid-bottom/ sided cages were incompatible with exposure conditions due to difficulties in maintaining adequate airflow and a homogeneous DCPT vapor distribution. Thus, following the daily exposure periods during late gestation and lactation, the females were transferred to cages outside of the exposure chambers. Mated females remained in the solid bottom/sided cages and were not exposed to DCPT from Gestation Day 2 1 through Lactation Day 4 to allow for parturition and early lactation. The removal of maternal animals from the exposure chambers for this short period would not be expected to significantly diminish potential treatment-related e&&s on parturition or early pup viability, as the dams were exposed to DCPT for 13 to 16
REPRODUCTION
135
weeks prior to delivery. In addition, to maximize exposure during critical reproductive events, exposure of dams to vapors of DCPT was extended to 7 days per week during breeding, Days 0 through 20 of gestation and Days 5 through 28 of lactation. Due to their small size and the need for bedding material, the pups were not placed in the exposure chambers, but remained in the nesting cages separated from the maternal rats, for 6 hr/ day, on Lactation Days 5 through 28. Historical control data generated in our laboratory indicate that removal of dams from lactating pups for 6 hr/day during Lactation Days 5 through 28 has no effect on pup survival but does result in slightly decreased pup growth. Exposure of pups to DCPT vapors began at weaning. Breeding procedure. Breeding of f0 and f, adults commenced after 10 weeks and 12 weeks, respectively, of exposure. Each breeding program consisted of three 7day cohabitation periods with one female and one male of the respective treatment group. For the f, mating, cohabitation of male and female littermates was avoided. Females that failed to mate during the first 7-day mating period were placed with an alternate randomly selected male from the respective treatment group for the second 7-day period. This procedure was repeated for unmated females during the third 7-day mating period. Males that did not mate during the first week of mating may not have received a second opportunity to mate. During the breeding period daily vaginal smears were evaluated for the presence of sperm, an indication of mating. The day on which vaginal sperm were detected was considered Day 0 of gestation. Sperm-positive females were separated and returned to their original cage. Following exposure on Day 18 of gestation the females were housed in solid-bottom cages containing ground corn cob bedding during nonexposure periods. Parental data. Each rat on study was observed daily for evidence of treatment-related alterations in behavior or demeanor. All adult rats found dead or moribund were submitted for pathologic examination. All parental animals were weighed weekly during the prebreeding treatment period. Following the prebreeding period, males were weighed weekly throughout the duration of the study. Sperm-positive females were weighed on Days 1,7, 14, and 2 1 of gestation. Lactating females with pups were weighed on Days 1,4,7, 14,2 1, and 28 of lactation. Nonpregnant females and females without litters were not weighed during the gestation and lactation periods. Litter data. All litters were examined on the day of parturition. The following parameters were recorded on each of the litters: date of parturition, numbers of live and dead pups on Days 0, 1,4,7, 14,2 1, and 28 postpartum, sex of the pups on Days 1, 4, and 28 postpartum, weight of the litters on Days 1, 4 (before and after culling), 7, 14, and 21 of lactation, and individual body weights for each pup on Day 28 of lactation. Any visible physical abnormalities or demeanor changes in the neo-
136
BRESLIN ET AL TABLE 3
Generation DCPT (mm)
Number of litters
f la
fib
Pup body weight (g)-Day 0 10
30 90
22-24 22-25 19-23 25-21
5.5 + 5.4 + 5.5 f 5.3 f
0.3” 0.5 0.4 0.4
0
22-24 22-25 19-23 25-27
8.1 kO.5 7.9 + 0.7 1.9 f 0.6 7.8 + 0.7
0
20-24 21-25 18-23 23-26
48.3 41.5 41.2 48.3
k 3.3 +- 4.1 + 4.3 f 2.8
0
10 30 90
21-24 22-25 17-23 23-27
50.7 49.1 49.7 50.4
+ 3.9 f 6.8 f 4.5 + 3.8
5.2 5.2 5.2 5.1
+ 0.4 + 0.6 f 0.4 kO.5
7.5 + 1.3 + 7.4 + 7.4 f
1.3 0.9 0.9 1.0
7.5 f 0.9 8.1 + 1.2 7.5 f 1.1 7.4-+ 1.1
43.3 43.7 46.3 43.4
+ 6.3 I!I4.8 zk4.1 + 4.9
47.8 zk 5.7 51.7?5.2* 46.3 + 4.7 47.6 t 4.6
44.9 46.4 47.7 45.5
+ + f +
50.6 54.8 49.2 48.8
28, females
49.6 47.1 48.3 48.6
Pup body weight (g)-Day
5.3 + 0.6 5.1 t- 0.4 5.0 I!z0.5 5.0 f 0.5
4’
7.1 f 1.2 7.3 + 1.1 1.3 f 0.6 1.5 + 0.9
Pup body weight (g)-Day 10 30 90
1
5.2 f 0.5 5.1 kO.5 5.1 kO.4 5.2 k 0.6
Pup body weight (g)-Day 10 30 90
f 2b
f2a
+ f k +
6.2 5.7 4.3 3.9
28, males
51.7 f 6.5 49.8 f 5.2 51.0 + 4.7 5l.Ok5.5
6.8 4.9 4.4 6.6
+ + k +
5.7 5.4* 5.0 5.8
’ Means f SD of litter means. * After culling. * Statistically different from control value by the Dunnett’s test (alpha = 0.05). nates were recorded during the lactation period. To reduce the variation in the growth of the pups, litters with more than eight pups were reduced by random selection on Day 4 postpartum to eight pups (four male and four female if possible). In litters with fewer than four pups of a particular sex, additional pups of the opposite sex were retained to achieve a litter size of eight. Litters with fewer than eight pups were not culled. Any weanlings not held for the next generation or for pathologic examination were sacrificed without further examinations. Gross necropsy and histopathology on adult rats. All adults from the f0 and f, generations were subjected to a complete gross necropsy examination. The adults were fasted overnight, weighed, anesthetized with methoxyflurane, and sacrificed by decapitation. Eyes were examined in situ using a glass slide technique with fluorescent illumination. An extensive list of tissues were collected
and preserved in neutral, phosphate-buffered 10% formalin. The lungs were infused with neutral, phosphatebuffered 10% formalin to their approximate normal inspiratory volume. The nasal cavity was flushed with formalin via the pharyngeal duct to ensure rapid fixation of the tissue. Histologic examination of reproductive tissues (cervix, coagulating glands, epididymides, ovaries, oviducts, prostate, seminal vesicles, testes, uterus, and vagina) and apparent target tissues (lacrimal/Harderian glands, larynx, liver, lungs, nasal, stomach, trachea, and bladder) was performed on the 0- and 90ppm-exposure groups. Based upon the findings in the 90-ppm-exposure group, nasal tissues and stomachs from rats in the lo- and 30-ppm-exposure groups were also examined. Gross necropsy on weanling rats. At the time of weaning, 10 pups/sex/exposure level from the fib and fib litters
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TABLE 4 LITTER SIZE AT BIRTH AMONG RATS EXPOSED TO DCPT Generation DCPT (mm)
Number of litters
f ,a
f lb
fh
f ztl
10+4 10+4 1252 11+3
9+4 11+2 11+3 10+3
10*4 9+3 10*4 lo-+4
5446 52~48 49:51 51:49
41:53 50:50 5644 41:53
55:45 47:53 51:49 51:49
Number of live pups 0 10 30 90
22-25 22-25 19-23 25-27
10&2” 10+3 11+2 11+2 Pup sex ratio (%)
0 10 30 90
22-25 22-25 19-23 25-21
45:55b 49:51 54:46 49:51
Note. No value significantly decreased in comparison to the control value by Dunnett’s test. a Mean + SD. b Male:female.
were randomly selected for a complete gross necropsy examination. The sacrifice and collection of tissues were conducted as described for adults. Statistical evaluation. Body weights and body weight gains were evaluated by Bartlett’s test for equality of variances, a = 0.01 (Winer, 197 1). Based upon the outcome of Bartlett’s test, parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA), a = 0.10, was performed. Cohabitation time required to produce a sperm-positive vaginal smear, gestation length, and litter size were analyzed by a nonparametric ANOVA. If the ANOVA was significant Dunnett’s test, (Y = 0.05, two-sided (Winer, 1971), or Wilcoxon’s rank-sum test, a = 0.05, two-sided (Hollander and Wolfe, 1973) with Bonferroni’s correction (Miller, 1966), was performed. The fertility indices (mating, conception, and gestation) were analyzed by the Fischer exact probability test, (Y= 0.05, one-sided (Siegel, 1956). Evaluation of the neonatal sex ratio was performed by the binomial distribution test, (Y= 0.05, twosided (Steel and Torrie, 1960). Neonatal survival indices were analyzed using the litter as the experimental unit by the censored Wilcoxon test, (Y = 0.05, one-sided, as modified by Haseman and Hoe1 (1974). Because numerous measurements were statistically compared in the same group of animals, the overall falsepositive rate (type I errors) was much greater than the cited (Ylevels would suggest. Thus, the final interpretation of numerical data considered statistical analyses along with other factors such as dose-response relation-
ships and whether the results were significant in the light of other biologic and pathologic findings.
RESULTS
Chamber conditions. The average analytical concentrations of DCPT present in the chambers during exposures of the f0 and f, generations were 10, 30, or 89 ppm and 10, 30, or 90 ppm, respectively. Mean chamber temperature and relative humidity ranged from 2 1 to 23°C and 44 to 60%, respectively. Clinical observations. No treatment-related effects on general appearance or demeanor were observed in any rats during the exposure periods. A total of four adult rats, one f0 and one f, 10 ppm male, one fi 10 ppm female, and one f, 90 ppm female, died during the test period. The cause of death of these animals was not related to exposure to DCPT. Body weights.The mean body weights offs and f, adult male rats exposed to 90 ppm DCPT were statistically decreased through-
138
BRESLIN ET AL.
FIG. 3. Photomicrographs of the nasal turbinate from control adult f, male (A) and fra adult f, male exposed to 90 ppm DCPT (B). The respiratory mucosa (arrows) of the control rat is one two cd ooidal to column nar cells thick. The hyperplastic mucosa (arrows) of the treated rat is three: to five :ll layersi thick and has a pseudostratified appearance. H & E, 440X.
1,3-DICHLOROPROPENE
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139
FIG. 3-Continued.
out most of the treatment periods (Fig, 1). sure periods, with occasional statistically The mean body weights off,, and fi adult fe- identified decreases occurring during the premale rats in the 90-ppm-exposure group were mating, gestation, and lactation periods (Fig. also decreased throughout most of the expo- 2). No significant effects on body weights
140
BRESLIN
were observed in adult males or females exposed to 10 or 30 ppm DCPT at any time during the study. Reproductive data. No adverse treatmentrelated effects on the mating, conception, or gestation indices (Table l), cohabitation time required for mating (Table l), pup survival indices (Table 2), pup body weights (Table 3), or litter size (Table 4) were observed at any exposure level in the fi,, fib, fZa, or fZblitters. During the first 24 hr postpartum, five pups in the IO-ppm-exposure group from the fib litters died (one pup from each of five litters), resulting in a statistically identified decrease in the Day 1 (data not presented) and Day 4 (Table 2) pup survival indices. This effect was considered spontaneous in view of the lack of an effect at this dose level in the fr,, fZa, or fZb litters or in the 30- or 90-ppm-exposure groups. Similarly, no adverse treatment-related effects on gestation length, pup sex ratio (Table 4), or external alterations of pups were observed in this study (alteration data not presented). Gross and histopathology pathology. No adverse treatment-related histopathologic effects were observed on male or female reproductive tissues. However, adult male and female rats from the 90-ppm-exposure group showed treatment-related effects on the nasal mucosa. Lesions in the respiratory epithelium occurred in the nasal turbinate section taken at the level of the first ruga palatina (Fig. 3). This alteration, involving a small percentage of the respiratory mucosa, consisted of disorganization of the respiratory epithelium at the tip of the dorsal concha; the respiratory hyperplasia was limited to a slight, uniform thickening which occasionally produced a pseudostratified appearance. Histopathologic changes in the olfactory epi-
ET AL.
thelium (Fig. 4) characterized by focal degenerative processes in the neuronal layer and loss of sustentacular cell cytoplasm were also observed in adult male and female rats in the 90-ppm-exposure group. An increased incidence of active and/or healing ulcers was also observed in adult f0 and fi female rats in the 90-ppm-exposure group. These ulcers were considered a result of stress secondary to exposure to DCPT and not a direct effect of the test material. There were no gross or histopathologic changes observed in the adult rats from the lo- or 30-ppm-exposure groups. In addition, no gross pathologic changes were observed in pups from the fib or fZb litters at any exposure level.
DISCUSSION Inhalation of DCPT vapors produced parental toxicity in both f0 and fi adult rats. Male and female f0 and f, adults from the 90ppm-exposure group exhibited significant decreases in body weight during the treatment period. The body weight effect was most pronounced in males. The decreased body weights in males and females at 90 ppm were consistent with previous reports of decreased body weight and/or body weight gain in males and females exposed to vapors of DCPT at concentrations of 60 ppm and higher (Hanley et al., 1987; Stott et al., 1988a,b). Treatment-related effects of the nasal mucosa were also observed in f0 and f, adult rats in the 90-ppm group. These effects included a slight, focal hyperplasia of the respiratory epithelium of the tip of the dorsal concha and/or focal degenerative changes in the olfactory epithelium. The increased inci-
FIG. 4. Photomicrograph of olfactory mucosa, dorsomedial portion of nasal cavity at the level of the second palatal ridge, from a control adult f, female (A) and an adult f, female exposed to 90 ppm DCPT (B). The mucosa of the control rat comprises columns of six to eight neurons and a uniform layer of sustentacular cell cytoplasm adjacent to the surface (arrow). Focal degeneration of the mucosa (between arrows) in the treated rat is characterized by disorganization and thinning of the neuronal layer and loss of sustentacular cell cytoplasm. S, nasal septum. H & E, 270X.
1,3-DICHLOROPROPENE
REPRODUCTION
141
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BRESLIN
ET AL.
1 ,fDICHLOROPROPENE
dence of stomach ulcers observed in female rats from the 90-ppm treatment group was attributed to the combined stresses of pregnancy, lactation, and chemical exposure, and was not considered a direct result of DCPT exposure. This interpretation is supported by the results reported by Stott et al. (1988a,b) in which no increase in gastric lesions was observed in male or female Fischer 344 rats following inhalation exposure to 150 ppm DCPT for 90 days or to 60 ppm DCPT for 2 years. In addition, gastric ulcerations are reported to commonly occur in the rat (Burek, 1978; Goodman et al., 1979), the pathogenesis of which may be mucosal damage from the endogenous products of stress (Brodie and Valitski, 1963; Senay and Levine, 1967). No adverse treatment-related effects on body weight or gross or histopathology were observed in adults from the lo- or 30-ppm exposure group. Although exposure to 90 ppm DCPT resulted in parental toxicity, no adverse treatment-related effects were observed on reproductive parameters or neonatal growth or survival at any exposure concentration tested. The statistically significant decrease in Day 1 and 4 pup survival observed in the fib litters at 10 ppm did not occur in previous or subsequent litters and was not observed in the 30- or 90-ppm exposure groups and thus was not considered a result of exposure to DCPT. In conclusion, inhalation exposure of male and female Fischer 344 rats to 10, 30, or 90 ppm DCPT for two generations did not adversely affect reproduction or neonatal growth or survival. Hence, the reproductive no-observed-effect level was 90 ppm. Although no reproductive effects were observed, exposure to 90 ppm DCPT resulted in parental toxicity as indicated by significant
143
REPRODUCTION
decreases in body weights and histopathologic effects on the nasal mucosa of adult male and female rats. REFERENCES BRODIE, D. A., AND VATLITSKI, L. S. (1963). Production ofgastric hemorrhage in rats by multiple stresses.Proc. Sot. Exp. Biol. Med. 133,998-1001. BUREK, J. D. (1978). Age-associated pathology. In Puthology of Aging Rats, Chap. 4, pp. 29-167. CRC Press, West Palm Beach, FL. GOODMAN, D. G., WARD, J. M., SQUIRE, R. A., CHU, K. C., AND LINHART, M. S. (1979). Neoplastic and nonneoplastic lesions in aging F344 rats. Toxicol. Appl. Pharmacol. 48,237-248. HANLEY, T. R., JOHN-GREEN, J. A., YOUNG, J. T., CALHOUN, L. L., AND Uo, K. S. ( 1987). Evaluation of the effects of inhalation exposure to 1,3-dichloropropene on fetal development in rats and rabbits. Fundam. Appl. Toxicol. 8,562-570. HASEMAN, J. K., AND HOEL, D. G. (1974). Tables of Gehan’s generalized Wilcoxon test with fixed point censoring. J. Statist. Comput. Simul. 3, 117-135. HOLLANDER, M., AND WOLFE, D. A. (1973). Nonparametric Statistical Methods. Wiley, New York. MILLER, R. G., JR. (1966). Simultaneous Statistical Inference. McGraw-Hill, New York. MILLER, R. R., LETTS, R. L., POTTS, W. J., AND McHENNA, M. J. (1980). Improved methodology for generating controlled test atmospheres. Amer. Ind. Hyg. Assoc. J. 41,844-846. STRAY,E. C., AND LEE, R. J. (1967). Synergism between cold and r&mint for rapid production of stressulcers in rats.Proc.Soc. Exp. Biol.Med. 124,1221-1223. SIU;EL, S. (1956). Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, New York. STEEL, R. G. D., AND TORRIE, J. H. (1960). Principles and Procedures of Statistics. McGraw-Hill, New York. STOTT, W. T., JOHNSON, K. A., LOMAX, L. G., AND CALHOUN, L. L. (1988a). Chronic toxicity and oncogenicity of inhaled technical grade 1,3dichloropropene (DCP) in rats and mice. Toxicologist 8( l), 177. STOTT, W. T., YOUNG, J. T., CALHOUN, L. L., AND BATTJES, J. E. (1988b). Subchronic toxicity of inhaled technical grade 1,3dichloropropene in rats and mice. Fundam. Appl. Toxicol., in press. WINER, B. J. (197 1). Statistical Principles in Experimental Design, 2nd ed. McGraw-Hill, New York.
AND
APPLIED
TOXICOLOGY
12,129- 143 ( 1989)
1,3-Dichloropropene: Two-Generation Inhalation Reproduction Study in Fischer 344 Rats W. J. BRESLIN,’ H. D. KIRK, C. M. STREETER, Mammalian
J. F. QUAST,
AND J. R. SZABO~
and Environmental Toxicology Research Laboratory Health and Environmental Sciences, Dow Chemical Company, 1803 Building, Midland, Michigan 48674
Received February 8.1988; accepted June 6, I988 1,3Dichloropropene:
Two-Generation Inhalation Reproduction Study in Fischer 344 Rats. H. D., STREETER, C. M., QUAST, J. F., AND SZABO, J. R. ( 1989). Fundam. Appl. Toxicol. 12, 129-l 43. This study evaluated the effects of inhaled technical-grade 1,3-d& chloropropene (DCPT) on reproduction and neonatal growth and survival. Groups of 30 male and 30 female Fischer 344 rats, approximately 6 weeks of age, were exposed via inhalation to 0, lo,30 or 90 ppm DCPT for 6 hr/day, 5 days/week, for two generations. The parental f0 and f, generations were each bred twice. Reproductive and neonatal parameters evaluated included indices of fertility and pup survival, gestation length, litter size, pup body weight, and pup sex ratio. Gross and histologic examinations were conducted on all 5 and f, adults. In addition, randomly selected fib and fZsweanlings were given gross examinations. Parental effects were limited to rats exposed to 90 ppm DCPT and included decreased body weights and histopathologic effects on the nasal mucosa of adult male and female rata. The histopathologic effects consisted of slight, focal hyperplasia of the respiratory epithelium and/or focal degenerative changes in the olfactory epithelium. No adverse effects on reproductive parameters or neonatal growth or survival were observed in the f,. , fib, fin, or fZblitters even at an exposure concentration which produced effects in adult animals. Based on these results, it is concluded that inhalation exposure of rats up to 90 ppm DCPT for two successivegenerations did not adversely atfect the reproductive and neonatal parameters evaluated. Q 1989 kiety of Toxicology. BRESLIN,
W. J., KIRK,
Technical-grade 1,3dichloropropene (DCPT) is used as a preplant nematicide for a variety of agricultural crops. The present threshold limit value for DCPT, as adopted by the American Conference of Governmental Industrial Hygienists, is 1 ppm. Vapors of DCPT appear to be moderately toxic to rodents. The 4-hr LC50 value for rats is approximately 1000 ppm (Dow Chemical Co., unpublished data). In a subchronic inhalation toxicity study (Stott et al., 1988b) Fischer 344 rats were exposed to 0, 10,30,90 ’ To whom correspondence should be addressed. 2 Present address: Health and Environmental Sciences-Texas, Dow Chemi&l Company, Freeport, TX 75541.
or 150 ppm DCPT 6 hr/day, 5 days/week, for 13 weeks. Rats exposed to 90 or 150 ppm DCPT were observed to have a treatment-related depression in body weight gain. Treatment-related pathological changes noted in these rats were slight degeneration of the nasal olfactory epithelium ( 150-ppm-exposure group) and a slight hyperplasia of the nasal respiratory epithelium (90- and 150-ppm-exposure groups). In addition, a few male rats exposed to 30 ppm DCPT also had very slight hyperplasia of their nasal respiratory epithelium. Inhalation teratology studies with rats and rabbits indicated exposures as high as 120 ppm DCPT were not embryotoxic or teratogenie (Hanley et al., 1987). Maternal toxicity 129
0272-0590189 $3.00 Copyiigbt Q I989 by the %ci~ry of Toxicology. Au Mt.9 of reproduction in any form mewed.
130
BRESLIN
as manifested by decreases in body weight and/or body weight gain was observed in groups of rats exposed to 20,60, or 120 ppm and rabbits exposed to 60 or 120 ppm DCPT. The study described herein was conducted to evaluate the effects of inhaled technicalgrade DCPT on reproductive capability and neonatal growth and survival in rats. The inhalation route was chosen because it is the most likely route of potential human exposure in the manufacture and use of DCPT. MATERIALS
AND
METHODS
Test material. Technical-grade DCPT was obtained from The Dow Chemical Company, Freeport, Texas. The test material was analyzed by gas chromatography prior to initiation ofthe study, at approximately 6-month intervals during the course of the study, and following the last exposure. The purity of the test material was 92% DCPT, the remainder of the test material comprised chlorinated and unchlorinated alkanes and alkenes as well as approximately 2.0% nonvolatile stabilizer (epoxidized soybean oil). Test species and husbandry. Male and female Fischer 344 rats’ approximately 4 weeks of age were used. All rats were examined for health status by the laboratory veterinarian and acclimated to the laboratory environment for a minimum of 2 weeks prior to use. The rats were weighed and assigned to the exposure groups using a stratified, by weight, randomization procedure. This procedure was intended to increase the probability of uniform mean pretreatment body weights across groups. All rats were identified by uniquely numbered metal ear tags. Rats were housed singly in wire-mesh, stainless-steel cages during the exposure periods. During late gestation and lactation, females were housed in plastic shoe box cages provided with ground corn cob nesting material. Water was available ad libitum throughout the study. Feed4 was available ad libitum except during exposure periods. The animal rooms were designed to maintain temperature at approximately 22°C relative humidity at approximately 50%, and a 12-hr-light: 12-hr-dark photocycle. Vapor generation and exposure conditions. Inhalation exposures were conducted in 14.5m3 chambers with stainless-steel pyramid-shaped ceilings and epoxy-resincoated floors and walls. All chambers were operated un-
3 Charles River Breeding Laboratories, Kingston, NY. 4 Purina Certified Rodent Chow 5002, Ralston Purina Co., St. Louis, MO.
ET AL. der dynamic airflow conditions at a slight negative pressure relative to the surrounding area. Control rats were placed in a chamber of the same design. The air supplied to the chambers was controlled by a system designed to maintain temperature and relative humidity at approximately 2 1°C and 50%. respectively; temperature and humidity were recorded daily. Test atmospheres containing DCPT were generated by metering liquid test material at controlled rates into glass J-tubes (Miller et al., 1980). The liquid was vaporized in the J-tubes with preheated compressed air and vapors were subsequently swept into the main chamber airstream where there was further dilution to the desired concentration. The compressed air was preheated with a flameless heat torch at the lowest temperature (<7o”C) necessary to facilitate complete vaporization of the liquid test material. Total chamber airflow was maintained at approximately 2500 liters/min. The concentration of the test material in each chamber was determined at least once per hour with a Miran 1A infrared spectromete? at a wavelength of 12.9 pm (peak absorption of tram isomer). The nominal concentration (ratio of DCPT used to total airflow through the chamber) was calculated for each chamber on a daily basis. The concentration of DCPT in the exposure chamber was determined by interpolation from a standard curve derived from vapor standards of known concentrations. Standard curves were run prior to the first exposure and approximately monthly thereafter. The infrared spectrometer was calibrated before each daily exposure using a known standard atmosphere of DCPT. Chamber vapor distribution was ascertained prior to the study start. Eight points at the extremes of the rat breathing zones were compared to the primary sample line (reference line) in each chamber in which DCPT was to be introduced. The mean concentration of DCPT, calculated from the eight distribution points per chamber, was approximately 90*5% of the primary sample line. Experimental design. Groups of 30 male and 30 female rats (fO)were exposed via inhalation to 0, 10, 30, or 90 ppm DCPT for 6 hr/day, 5 days/week, prior to breeding and 6 hrjday, 7 days/week, during breeding, gestation, and lactation. The exposure of f0 rats began at ap proximately 6 weeks of age. After 10 weeks of exposure (5 days/week), f0rats were mated, one male to one female of the respective treatment group, to produce the f,. litters. Breeding for the fib litter began 1 week after weaning ofthe last f,, litter. Following weaning ofthe last fib litter, 1 male and 1 female pup, when available from each litter, within a treatment group were randomly selected using a table of random numbers and assigned to the respective exposure groups to become parents for the next generation. In treatment groups with fewer than 30 litters, additional pups were randomly selected from litters of the same treatment group until 30 male and 30 female pups
’ Foxboro Analytical, Norwalk, CO.
1,3-DICHLOROPROPENE 300
131
REPRODUCTION
A
280 1 260 1 240 220 200 180 160 OPPM ,D
120 100
-
PPM
3opPM WPPM
1
80 I 601 - 18 -11 -4
r. 3
n = I 7, - 1 -1 10 17 24 31 38
9 1 . , =I 45 52 59
-1 66
-,
-1
70
77
73
80
Days on test 320 300 280 260 240 220 200 180 160 I-o-
140
WPpn
120 0
7
14
21
28
35
42
49
56
63
84
Days on test FIG. 1. Premating body weights for&,(A) and f, (B) adult male rats exposed to DCPT vapors. The number of male rats per exposure group was 29 or 30. *Statistically different from control by Dunnett’s test, a = 0.05, two-sided.
132
BRESLIN ET AL.
Q E
140130-
+20-
E
.zp llO$ loo6 8 go80
-
6011,,,,,,,,,.,,,,,,,,,,,,,,, -11 -4 3
10
17
24
31
38
45
52
59
66
73
80
Days on test 280 -B 260 _ 240 _
220 _ 200 _ 180 _
160
1,
,
,
7
21
Gest.
,
, ,
,
1
, 28
Lat.
,
1’1’1’1
7
‘I
21
Gest.
‘I
1
‘1
‘1
28
Lat.
Days on test FIG. 2. Body weights for f0 (A, B) and f, (C, D) adult female rats exposed to DCPT vapors. The number of female rats per exposure group was equal to 30 during the premating period and ranged from 19 to 27 during gestation and lactation (B, D). *Statistically different from control by Dunnett’s test, (Y= 0.05, twosided.
I ,3-DICHLOROPROPENE
133
REPRODUCTION
18
3 ; l6 9
15
5
l4
g
13
6
12
0
m
11 10 9 8 0
7
14
21
28
35
42
56
49
63
70
77
84
Days on test
3 E E c!
E g P 6 $
250
230 210
lgo: 4 170
,:,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, 7 21 1
Gest.
28 7
21
Gest.
Lat.
128
Lat.
Days on Test FIG. 2-Continued. were obtained. After approximately 12 weeks of exposure (5 days/week, excluding holidays), the f, adults were bred to produce the fra litters. Breeding for the frs litters began 1 week after weaning of the last fr, litter. Exposure
of f0 and fr adult rats to DCPT was continued until the adults were sacrificed. Rats were housed continuously in exposure chambers except during late gestation and lactation when females
BRESLIN ET AL.
134
TABLE 1 FERTILITY
INDICES AMONG
RATS EXFQSED
TO DCPT
Generation DCPT (mm)
N
f la
f Ib
f 2a
f 2b
93.3 96.7 90.0 96.7
90.0 93.1 83.3 100.0
85.7 79.3 70.4 93.1
85.2 92.6 80.0 86.2
Female mating index” 0 10
30 90
30 29 or 30 30 29 or 30
100.0 96.7 90.0 96.7
96.7 100.0 90.0 96.7
Female conception index” 0 10 30 90
27-30 27-30 25-27 29
0
22-25 22-25 19-23 25-27
73.3 75.9 81.5 89.7
86.2 80.0 85.2 86.2
Female gestation index ’ 10
30 90
96.0
100.0 100.0 100.0 100.0
100.0 100.0 100.0
100.0 100.0 100.0 100.0
95.7 100.0 100.0 100.0
Male mating indexd 0 10
30 90
30 29 or 30 30 30
93.3 76.7 80.0 83.3
80.0 90.0 76.7 86.7
76.7 80.0 66.7 90.0
76.7 72.4 63.3 83.3
91.3 83.3 75.0 92.6
87.0 95.2 84.2 92.0
Male conception index’ 10
0
23-28 21-27
30 90
25-27
19-24
78.6 78.3 83.3 92.0
95.8 85.2 95.7 92.3
Cohabitation time (days) required for mating/ 0 10 30 90
27-30 27-30 25-27 29
4.3 f 3.2 5.3 AZ4.4 5.2 + 5.8 5.3 rt 4.6
5.4 f 5.1 3.8 k 2.5 4.2f3.1 4.5 + 4.9
3.9 4.9 6.8 7.0
f 4.6 + 3.7 + 6.8 + 7.3
4.6 f 3.6 f 4.3 iI 4.7 +
4.5 2.7 4.1 5.3
Note. No values differed significantly from control value by Fischer’s exact probability test, (Y= 0.05, with Bonferroni correction. UNumber of females with a sperm-positive vaginal smear/total number females (percentage). b Number of females delivering a litter/number of sperm-positive females (percentage). ’ Number of females delivering a live litter/number of females delivering a litter (percentage). d Number of males which mated resulting in a sperm-positive vaginal smear/total number of males (percentage). ’ Number of males which sired a litter/number of males mated resulting in a sperm-positive vaginal smear (percentape). ‘Average number of days of male/female cohabitation required to produce a sperm-positive vaginal smear. 8 Mean 2 SD.
1,3-DICHLOROPROPENE TABLE 2 GESTATION AND POSTNATAL SURVNAL AMONG LITTERS OF RATS EXPOSED TO DCPT Generation DCPT (m-W
Number of litters
f la
fib
fi,
fZb
100.0 98.8 100.0 99.6
92.8 99.1 100.0 98.8
Gestation survival index” 0 10 30 90
22-25 22-25 19-23 25-27
99.1 99.5 100.0 99.7
98.1 99.6 99.3 100.0
Postnatal survival index, 4-day b 0 10 30 90
22-25 22-25 19-23 25-27
99.1 99.5 100.0 100.0
100.0 98.0* 99.6 99.3
99.5 99.6 98.2 99.3
98.6 99.1 95.9 99.2
98.8 100.0 99.3 99.0
98.7 99.4 98.4 100.0
Postnatal survival index, 28day’ 0 10 30 90
22-25 22-25 19-23 25-27
100.0 100.0 100.0 96.1
99.5 98.2 99.5 99.5
’ Number of pups alive at birth/number of pups born (percentage). b Number of pups surviving for 4 days/total number of livebom pups (percentage). ’ Number of pups surviving for 28 days/total number of live pups after culling on Day 4 (percentage). *Significantly different from control value by Fischer’s exact probability test, OL= 0.05, with a Bonferroni correction.
were removed following the exposure period. Parturition and housing of pups necessitated the utilization of nesting cages with solid bottoms and sides. The solid-bottom/ sided cages were incompatible with exposure conditions due to difficulties in maintaining adequate airflow and a homogeneous DCPT vapor distribution. Thus, following the daily exposure periods during late gestation and lactation, the females were transferred to cages outside of the exposure chambers. Mated females remained in the solid bottom/sided cages and were not exposed to DCPT from Gestation Day 2 1 through Lactation Day 4 to allow for parturition and early lactation. The removal of maternal animals from the exposure chambers for this short period would not be expected to significantly diminish potential treatment-related e&&s on parturition or early pup viability, as the dams were exposed to DCPT for 13 to 16
REPRODUCTION
135
weeks prior to delivery. In addition, to maximize exposure during critical reproductive events, exposure of dams to vapors of DCPT was extended to 7 days per week during breeding, Days 0 through 20 of gestation and Days 5 through 28 of lactation. Due to their small size and the need for bedding material, the pups were not placed in the exposure chambers, but remained in the nesting cages separated from the maternal rats, for 6 hr/ day, on Lactation Days 5 through 28. Historical control data generated in our laboratory indicate that removal of dams from lactating pups for 6 hr/day during Lactation Days 5 through 28 has no effect on pup survival but does result in slightly decreased pup growth. Exposure of pups to DCPT vapors began at weaning. Breeding procedure. Breeding of f0 and f, adults commenced after 10 weeks and 12 weeks, respectively, of exposure. Each breeding program consisted of three 7day cohabitation periods with one female and one male of the respective treatment group. For the f, mating, cohabitation of male and female littermates was avoided. Females that failed to mate during the first 7-day mating period were placed with an alternate randomly selected male from the respective treatment group for the second 7-day period. This procedure was repeated for unmated females during the third 7-day mating period. Males that did not mate during the first week of mating may not have received a second opportunity to mate. During the breeding period daily vaginal smears were evaluated for the presence of sperm, an indication of mating. The day on which vaginal sperm were detected was considered Day 0 of gestation. Sperm-positive females were separated and returned to their original cage. Following exposure on Day 18 of gestation the females were housed in solid-bottom cages containing ground corn cob bedding during nonexposure periods. Parental data. Each rat on study was observed daily for evidence of treatment-related alterations in behavior or demeanor. All adult rats found dead or moribund were submitted for pathologic examination. All parental animals were weighed weekly during the prebreeding treatment period. Following the prebreeding period, males were weighed weekly throughout the duration of the study. Sperm-positive females were weighed on Days 1,7, 14, and 2 1 of gestation. Lactating females with pups were weighed on Days 1,4,7, 14,2 1, and 28 of lactation. Nonpregnant females and females without litters were not weighed during the gestation and lactation periods. Litter data. All litters were examined on the day of parturition. The following parameters were recorded on each of the litters: date of parturition, numbers of live and dead pups on Days 0, 1,4,7, 14,2 1, and 28 postpartum, sex of the pups on Days 1, 4, and 28 postpartum, weight of the litters on Days 1, 4 (before and after culling), 7, 14, and 21 of lactation, and individual body weights for each pup on Day 28 of lactation. Any visible physical abnormalities or demeanor changes in the neo-
136
BRESLIN ET AL TABLE 3
Generation DCPT (mm)
Number of litters
f la
fib
Pup body weight (g)-Day 0 10
30 90
22-24 22-25 19-23 25-21
5.5 + 5.4 + 5.5 f 5.3 f
0.3” 0.5 0.4 0.4
0
22-24 22-25 19-23 25-27
8.1 kO.5 7.9 + 0.7 1.9 f 0.6 7.8 + 0.7
0
20-24 21-25 18-23 23-26
48.3 41.5 41.2 48.3
k 3.3 +- 4.1 + 4.3 f 2.8
0
10 30 90
21-24 22-25 17-23 23-27
50.7 49.1 49.7 50.4
+ 3.9 f 6.8 f 4.5 + 3.8
5.2 5.2 5.2 5.1
+ 0.4 + 0.6 f 0.4 kO.5
7.5 + 1.3 + 7.4 + 7.4 f
1.3 0.9 0.9 1.0
7.5 f 0.9 8.1 + 1.2 7.5 f 1.1 7.4-+ 1.1
43.3 43.7 46.3 43.4
+ 6.3 I!I4.8 zk4.1 + 4.9
47.8 zk 5.7 51.7?5.2* 46.3 + 4.7 47.6 t 4.6
44.9 46.4 47.7 45.5
+ + f +
50.6 54.8 49.2 48.8
28, females
49.6 47.1 48.3 48.6
Pup body weight (g)-Day
5.3 + 0.6 5.1 t- 0.4 5.0 I!z0.5 5.0 f 0.5
4’
7.1 f 1.2 7.3 + 1.1 1.3 f 0.6 1.5 + 0.9
Pup body weight (g)-Day 10 30 90
1
5.2 f 0.5 5.1 kO.5 5.1 kO.4 5.2 k 0.6
Pup body weight (g)-Day 10 30 90
f 2b
f2a
+ f k +
6.2 5.7 4.3 3.9
28, males
51.7 f 6.5 49.8 f 5.2 51.0 + 4.7 5l.Ok5.5
6.8 4.9 4.4 6.6
+ + k +
5.7 5.4* 5.0 5.8
’ Means f SD of litter means. * After culling. * Statistically different from control value by the Dunnett’s test (alpha = 0.05). nates were recorded during the lactation period. To reduce the variation in the growth of the pups, litters with more than eight pups were reduced by random selection on Day 4 postpartum to eight pups (four male and four female if possible). In litters with fewer than four pups of a particular sex, additional pups of the opposite sex were retained to achieve a litter size of eight. Litters with fewer than eight pups were not culled. Any weanlings not held for the next generation or for pathologic examination were sacrificed without further examinations. Gross necropsy and histopathology on adult rats. All adults from the f0 and f, generations were subjected to a complete gross necropsy examination. The adults were fasted overnight, weighed, anesthetized with methoxyflurane, and sacrificed by decapitation. Eyes were examined in situ using a glass slide technique with fluorescent illumination. An extensive list of tissues were collected
and preserved in neutral, phosphate-buffered 10% formalin. The lungs were infused with neutral, phosphatebuffered 10% formalin to their approximate normal inspiratory volume. The nasal cavity was flushed with formalin via the pharyngeal duct to ensure rapid fixation of the tissue. Histologic examination of reproductive tissues (cervix, coagulating glands, epididymides, ovaries, oviducts, prostate, seminal vesicles, testes, uterus, and vagina) and apparent target tissues (lacrimal/Harderian glands, larynx, liver, lungs, nasal, stomach, trachea, and bladder) was performed on the 0- and 90ppm-exposure groups. Based upon the findings in the 90-ppm-exposure group, nasal tissues and stomachs from rats in the lo- and 30-ppm-exposure groups were also examined. Gross necropsy on weanling rats. At the time of weaning, 10 pups/sex/exposure level from the fib and fib litters
1,fDICHLOROPROPENE
137
REPRODUCTION
TABLE 4 LITTER SIZE AT BIRTH AMONG RATS EXPOSED TO DCPT Generation DCPT (mm)
Number of litters
f ,a
f lb
fh
f ztl
10+4 10+4 1252 11+3
9+4 11+2 11+3 10+3
10*4 9+3 10*4 lo-+4
5446 52~48 49:51 51:49
41:53 50:50 5644 41:53
55:45 47:53 51:49 51:49
Number of live pups 0 10 30 90
22-25 22-25 19-23 25-27
10&2” 10+3 11+2 11+2 Pup sex ratio (%)
0 10 30 90
22-25 22-25 19-23 25-21
45:55b 49:51 54:46 49:51
Note. No value significantly decreased in comparison to the control value by Dunnett’s test. a Mean + SD. b Male:female.
were randomly selected for a complete gross necropsy examination. The sacrifice and collection of tissues were conducted as described for adults. Statistical evaluation. Body weights and body weight gains were evaluated by Bartlett’s test for equality of variances, a = 0.01 (Winer, 197 1). Based upon the outcome of Bartlett’s test, parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA), a = 0.10, was performed. Cohabitation time required to produce a sperm-positive vaginal smear, gestation length, and litter size were analyzed by a nonparametric ANOVA. If the ANOVA was significant Dunnett’s test, (Y = 0.05, two-sided (Winer, 1971), or Wilcoxon’s rank-sum test, a = 0.05, two-sided (Hollander and Wolfe, 1973) with Bonferroni’s correction (Miller, 1966), was performed. The fertility indices (mating, conception, and gestation) were analyzed by the Fischer exact probability test, (Y= 0.05, one-sided (Siegel, 1956). Evaluation of the neonatal sex ratio was performed by the binomial distribution test, (Y= 0.05, twosided (Steel and Torrie, 1960). Neonatal survival indices were analyzed using the litter as the experimental unit by the censored Wilcoxon test, (Y = 0.05, one-sided, as modified by Haseman and Hoe1 (1974). Because numerous measurements were statistically compared in the same group of animals, the overall falsepositive rate (type I errors) was much greater than the cited (Ylevels would suggest. Thus, the final interpretation of numerical data considered statistical analyses along with other factors such as dose-response relation-
ships and whether the results were significant in the light of other biologic and pathologic findings.
RESULTS
Chamber conditions. The average analytical concentrations of DCPT present in the chambers during exposures of the f0 and f, generations were 10, 30, or 89 ppm and 10, 30, or 90 ppm, respectively. Mean chamber temperature and relative humidity ranged from 2 1 to 23°C and 44 to 60%, respectively. Clinical observations. No treatment-related effects on general appearance or demeanor were observed in any rats during the exposure periods. A total of four adult rats, one f0 and one f, 10 ppm male, one fi 10 ppm female, and one f, 90 ppm female, died during the test period. The cause of death of these animals was not related to exposure to DCPT. Body weights.The mean body weights offs and f, adult male rats exposed to 90 ppm DCPT were statistically decreased through-
138
BRESLIN ET AL.
FIG. 3. Photomicrographs of the nasal turbinate from control adult f, male (A) and fra adult f, male exposed to 90 ppm DCPT (B). The respiratory mucosa (arrows) of the control rat is one two cd ooidal to column nar cells thick. The hyperplastic mucosa (arrows) of the treated rat is three: to five :ll layersi thick and has a pseudostratified appearance. H & E, 440X.
1,3-DICHLOROPROPENE
REPRODUCTION
139
FIG. 3-Continued.
out most of the treatment periods (Fig, 1). sure periods, with occasional statistically The mean body weights off,, and fi adult fe- identified decreases occurring during the premale rats in the 90-ppm-exposure group were mating, gestation, and lactation periods (Fig. also decreased throughout most of the expo- 2). No significant effects on body weights
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were observed in adult males or females exposed to 10 or 30 ppm DCPT at any time during the study. Reproductive data. No adverse treatmentrelated effects on the mating, conception, or gestation indices (Table l), cohabitation time required for mating (Table l), pup survival indices (Table 2), pup body weights (Table 3), or litter size (Table 4) were observed at any exposure level in the fi,, fib, fZa, or fZblitters. During the first 24 hr postpartum, five pups in the IO-ppm-exposure group from the fib litters died (one pup from each of five litters), resulting in a statistically identified decrease in the Day 1 (data not presented) and Day 4 (Table 2) pup survival indices. This effect was considered spontaneous in view of the lack of an effect at this dose level in the fr,, fZa, or fZb litters or in the 30- or 90-ppm-exposure groups. Similarly, no adverse treatment-related effects on gestation length, pup sex ratio (Table 4), or external alterations of pups were observed in this study (alteration data not presented). Gross and histopathology pathology. No adverse treatment-related histopathologic effects were observed on male or female reproductive tissues. However, adult male and female rats from the 90-ppm-exposure group showed treatment-related effects on the nasal mucosa. Lesions in the respiratory epithelium occurred in the nasal turbinate section taken at the level of the first ruga palatina (Fig. 3). This alteration, involving a small percentage of the respiratory mucosa, consisted of disorganization of the respiratory epithelium at the tip of the dorsal concha; the respiratory hyperplasia was limited to a slight, uniform thickening which occasionally produced a pseudostratified appearance. Histopathologic changes in the olfactory epi-
ET AL.
thelium (Fig. 4) characterized by focal degenerative processes in the neuronal layer and loss of sustentacular cell cytoplasm were also observed in adult male and female rats in the 90-ppm-exposure group. An increased incidence of active and/or healing ulcers was also observed in adult f0 and fi female rats in the 90-ppm-exposure group. These ulcers were considered a result of stress secondary to exposure to DCPT and not a direct effect of the test material. There were no gross or histopathologic changes observed in the adult rats from the lo- or 30-ppm-exposure groups. In addition, no gross pathologic changes were observed in pups from the fib or fZb litters at any exposure level.
DISCUSSION Inhalation of DCPT vapors produced parental toxicity in both f0 and fi adult rats. Male and female f0 and f, adults from the 90ppm-exposure group exhibited significant decreases in body weight during the treatment period. The body weight effect was most pronounced in males. The decreased body weights in males and females at 90 ppm were consistent with previous reports of decreased body weight and/or body weight gain in males and females exposed to vapors of DCPT at concentrations of 60 ppm and higher (Hanley et al., 1987; Stott et al., 1988a,b). Treatment-related effects of the nasal mucosa were also observed in f0 and f, adult rats in the 90-ppm group. These effects included a slight, focal hyperplasia of the respiratory epithelium of the tip of the dorsal concha and/or focal degenerative changes in the olfactory epithelium. The increased inci-
FIG. 4. Photomicrograph of olfactory mucosa, dorsomedial portion of nasal cavity at the level of the second palatal ridge, from a control adult f, female (A) and an adult f, female exposed to 90 ppm DCPT (B). The mucosa of the control rat comprises columns of six to eight neurons and a uniform layer of sustentacular cell cytoplasm adjacent to the surface (arrow). Focal degeneration of the mucosa (between arrows) in the treated rat is characterized by disorganization and thinning of the neuronal layer and loss of sustentacular cell cytoplasm. S, nasal septum. H & E, 270X.
1,3-DICHLOROPROPENE
REPRODUCTION
141
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ET AL.
1 ,fDICHLOROPROPENE
dence of stomach ulcers observed in female rats from the 90-ppm treatment group was attributed to the combined stresses of pregnancy, lactation, and chemical exposure, and was not considered a direct result of DCPT exposure. This interpretation is supported by the results reported by Stott et al. (1988a,b) in which no increase in gastric lesions was observed in male or female Fischer 344 rats following inhalation exposure to 150 ppm DCPT for 90 days or to 60 ppm DCPT for 2 years. In addition, gastric ulcerations are reported to commonly occur in the rat (Burek, 1978; Goodman et al., 1979), the pathogenesis of which may be mucosal damage from the endogenous products of stress (Brodie and Valitski, 1963; Senay and Levine, 1967). No adverse treatment-related effects on body weight or gross or histopathology were observed in adults from the lo- or 30-ppm exposure group. Although exposure to 90 ppm DCPT resulted in parental toxicity, no adverse treatment-related effects were observed on reproductive parameters or neonatal growth or survival at any exposure concentration tested. The statistically significant decrease in Day 1 and 4 pup survival observed in the fib litters at 10 ppm did not occur in previous or subsequent litters and was not observed in the 30- or 90-ppm exposure groups and thus was not considered a result of exposure to DCPT. In conclusion, inhalation exposure of male and female Fischer 344 rats to 10, 30, or 90 ppm DCPT for two generations did not adversely affect reproduction or neonatal growth or survival. Hence, the reproductive no-observed-effect level was 90 ppm. Although no reproductive effects were observed, exposure to 90 ppm DCPT resulted in parental toxicity as indicated by significant
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decreases in body weights and histopathologic effects on the nasal mucosa of adult male and female rats. REFERENCES BRODIE, D. A., AND VATLITSKI, L. S. (1963). Production ofgastric hemorrhage in rats by multiple stresses.Proc. Sot. Exp. Biol. Med. 133,998-1001. BUREK, J. D. (1978). Age-associated pathology. In Puthology of Aging Rats, Chap. 4, pp. 29-167. CRC Press, West Palm Beach, FL. GOODMAN, D. G., WARD, J. M., SQUIRE, R. A., CHU, K. C., AND LINHART, M. S. (1979). Neoplastic and nonneoplastic lesions in aging F344 rats. Toxicol. Appl. Pharmacol. 48,237-248. HANLEY, T. R., JOHN-GREEN, J. A., YOUNG, J. T., CALHOUN, L. L., AND Uo, K. S. ( 1987). Evaluation of the effects of inhalation exposure to 1,3-dichloropropene on fetal development in rats and rabbits. Fundam. Appl. Toxicol. 8,562-570. HASEMAN, J. K., AND HOEL, D. G. (1974). Tables of Gehan’s generalized Wilcoxon test with fixed point censoring. J. Statist. Comput. Simul. 3, 117-135. HOLLANDER, M., AND WOLFE, D. A. (1973). Nonparametric Statistical Methods. Wiley, New York. MILLER, R. G., JR. (1966). Simultaneous Statistical Inference. McGraw-Hill, New York. MILLER, R. R., LETTS, R. L., POTTS, W. J., AND McHENNA, M. J. (1980). Improved methodology for generating controlled test atmospheres. Amer. Ind. Hyg. Assoc. J. 41,844-846. STRAY,E. C., AND LEE, R. J. (1967). Synergism between cold and r&mint for rapid production of stressulcers in rats.Proc.Soc. Exp. Biol.Med. 124,1221-1223. SIU;EL, S. (1956). Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, New York. STEEL, R. G. D., AND TORRIE, J. H. (1960). Principles and Procedures of Statistics. McGraw-Hill, New York. STOTT, W. T., JOHNSON, K. A., LOMAX, L. G., AND CALHOUN, L. L. (1988a). Chronic toxicity and oncogenicity of inhaled technical grade 1,3dichloropropene (DCP) in rats and mice. Toxicologist 8( l), 177. STOTT, W. T., YOUNG, J. T., CALHOUN, L. L., AND BATTJES, J. E. (1988b). Subchronic toxicity of inhaled technical grade 1,3dichloropropene in rats and mice. Fundam. Appl. Toxicol., in press. WINER, B. J. (197 1). Statistical Principles in Experimental Design, 2nd ed. McGraw-Hill, New York.