Primedica Argus, 905 Sheehy Drive, Horsham, Pennsylvania 19044
Received April 27, 1999; accepted August 17, 1999
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ABSTRACT |
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Key Words: acclimation; fertility; inhalation exposure; male rats; nose-only exposure; sperm motility; stress; testicular atrophy.
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INTRODUCTION |
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Stress produces marked changes in many of the hormones controlled by the adrenal glands (Pare and Glavin, 1986). Stress-induced increases in the release of corticosterone B, noradrenaline, and adrenaline have been demonstrated in the rat following either restraint or inhalation exposure (Bernet et al., 1998
). Significant increases in the incidence of spontaneous testicular degeneration have been observed in 10- to 12-week old Crl:CD®BR VAF/Plus® (Sprague-Dawley) male rats used in inhalation studies, as compared with male rats of the same age and strain used in oral toxicity studies (Lee et al., 1993
). The reported testicular degeneration was postulated to be associated with the stress of restraint, inhalation, or heat associated with confinement in inhalation tubes (Dodd et al., 1999
). In this paper, assessment of mating, histopathology of the male reproductive organs, and sperm density and motility were employed to evaluate adverse effects of confinement stress on male rats.
In acute and subchronic nose-only inhalation studies of the toxicity of trifluoroiodomethane vapor in Fischer 344 rats (Dodd et al., 1997), and of HFC-143a in Sprague-Dawley rats (Brock et al., 1996
), testicular atrophy was noted. The studies were repeated, using whole-body exposure chambers, and no testicular effects were observed (Brock et al., 1996
; Dodd et al., 1999
). The effects observed in the nose-only studies were attributed to restraint (Lee et al., 1993
) and thermal stress (Brock et al., 1996
; Dodd et al., 1999
). In a nose-only study of 1-Nitropyrene (Chan, 1996
), testicular atrophy was observed and was considered a secondary effect resulting from the daily (6 hr/day, 5 days per week for 13 weeks) confinement within the exposure tubes. A lesion of the testes consisting of partial to total loss of the germ cells, diagnosed as atrophy, was observed in all male rats, both control and treated. The extent of atrophy ranged from minimal to moderate, depending on the extent of seminiferous tubule involvement. Hypospermia of the epididymis, characterized by decreased numbers of spermatazoa within the lumens of epididymal tubules, was also associated with testicular atrophy. Because all groups demonstrated the effect, these changes were not considered a chemical-related effect but rather due to heat or pressure resulting from daily confinement within the exposure tubes.
In a previous study (Rothenberg et al, in preparation), procedures for acclimation of pregnant rats to nose-only exposures were developed, and it was demonstrated that if adequate acclimation procedures were followed, satisfactory baseline data were obtained for both Segment II (developmental toxicity) and Segment III (peri/postnatal toxicity) studies. In that study no data were obtained for male rats. The purpose of the present study was to determine whether male rats could be exposed nose-only daily for 2 h without adverse effects induced by the exposure procedures. Nose-only inhalation exposure is required for Segment I (fertility) studies and for the harmonized tripartite ICH guidelines stages A and B of the reproductive cycle (US FDA, 1994). Because previous investigators reported adverse testicular effects (Bernet et al, 1998; Brock et al., 1996
; Dodd et al., 1997
, 1999
; Lee et al., 1993
), particular emphasis was placed on the evaluation of fertility in this study. In addition, care was taken to avoid excessive temperatures in the nose-only exposure tubes, because prolonged exposure to excessive temperatures has been reported to be associated with testicular atrophy (Bedford, 1991
). In contrast, the current data are in accord with those reported by Alexander et al. (1996). No adverse effects of nose-only exposures were observed.
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MATERIALS AND METHODS |
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The Crl:CD®BR VAF/Plus® (Sprague-Dawley) rat was selected as the test system because it is one mammalian species accepted and widely used throughout industry for nonclinical studies; historical data and experience exist at the testing facility (Christian and Voytek, 1982; Christian, 1984
; Lang, 1988
); and proposed future studies will use this species and strain. The duration of exposure was selected on the basis of literature that indicates a possible reduced fertility in male rats confined in nose-only inhalation apparatus (Bailey, 1996
; Bernet et al., 1998
; Brock et al., 1996
.; Dodd et al., 1997
, 1999
; Frame et al., 1992
; Lee et al., 1993
) and that 28 days of exposures will be sufficient to detect adverse effects (Bailey, 1996
; Takayama et al., 1995
).
The purpose of this study was to identify whether conducting nose-only inhalation studies at reduced exposure room temperatures (6470°F) and using Cannon® butt plates with tail coolers prevented testicular atrophy, reduced fertility, and changes in sperm count and concentration. Cannon® (Lab Products, Seaford, DE) butt plates with tail cooler (Fig. 1) are designed to minimize thermal stress by providing a heat-conducting path from the metal plate positioning the rodent inside the Plexiglas® tube to the cool room air. An additional advantage is that the tail is kept cool, so that the rodent can self-regulate its body temperature by changes in blood circulation to the tail, a normal means of achieving homeostasis. To ensure that most of the tail cooler and the rat's tail were outside the tube in the cool room air, the exposure tubes were cut to a length such that not more than 1 inch of the tail cooler was within the tube.
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After acclimation and estrous cycle evaluation, female rats were selected for study on the basis of physical appearance, body weights recorded during the precohabitation period, and evidence of normal estrous cycling. The cohabitation period consisted of a maximum of 14 days. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be at day 0 of presumed gestation and assigned to individual housing. Male rats that did not mate within the first 7 days of cohabitation were assigned an alternate female rat and remained in cohabitation for a maximum of 7 additional days.
The study room was maintained under conditions of negative airflow relative to a hallway and independently supplied with a minimum of ten changes per h of 100% fresh air that had been passed through 99.97% HEPA filters (Airo Clean® room). Room temperature and humidity were monitored constantly throughout the study. Whereas room temperature is usually set at 64-79°F for rodent studies, the targeted room temperature for these inhalation studies was 64-70°F. Actual room temperature range was 64-73°F (18-26°C); relative humidity was 50-82%. Whenever the room temperature exceeded the targeted maximum (70°F), the nose-only chamber was cooled directly using ice packs. The rats were individually housed in stainless steel, wire-bottomed cages, except during the exposures and the cohabitation period. During cohabitation, each pair of rats was housed in the male rat's cage. All cage sizes and housing conditions were in compliance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996). Rats were given ad libitum access to Certified Rodent Diet® #5002 (PMI Nutrition International, St. Louis, MO) in individual feeders. Local water that had been processed by passage through a reverse osmosis membrane (R.O. water) was available to the rats ad libitum from an automatic watering system or from individual water bottles. Chlorine (up to 1 ppm) was added to the processed water as a bacteriostat.
The male rats in Group II were placed in exposure tubes once daily for 28 consecutive days before cohabitation, daily during the cohabitation interval, and daily until sacrificed for sperm evaluation. The duration of exposure was approximately 2 h. The length of the study was approximately 8 weeks. Nose-only exposures were conducted in Intox® inhalation chambers, into which room air was circulated, with ports for 60 rodents (30 per side). The exposure tubes had butt plates with tail coolers. At the completion of the exposure period, the tubes were removed from the chamber, the front ends of the tubes were removed, and the rats were allowed to climb out of the tubes; rats were then returned to their home cages. Each daily exposure period began at approximately the same time. The airflow rate through the exposure chamber (2530 LPM [liters per minute]) was set to give at least 1215 air changes per h. Room air was supplied through a HEPA/AC (activated charcoal) filter. After the initial acclimation period, rats assigned to the control group (Group I) were not placed in the exposure tubes or apparatus described above, but were housed in the same room used for the exposures, and therefore subjected to the same environmental conditions as the Group ll rats.
Male and female rats were observed for viability at least twice daily. Male rats were examined weekly for clinical observations during the acclimation period, daily before exposure, and on the day sacrificed. During daily exposures, the rats were observed continuously for signs of distress. Postexposure observations were recorded daily within approximately 3090 min after the rats were returned to their cages. Rectal body temperature measurements for male rats were made using a rectal probe (Physitemp Instruments Model RET-2) and digital thermometer (Physitemp Instruments Model Bat-10 R LOP). The same equipment was used to record the body temperature of one rat in Group II at approximately 30-min intervals during exposure. The temperature immediately behind the butt plate was also recorded to determine whether excessive testicular temperatures were attained.
Body weights for male rats were recorded weekly during acclimation, daily during the exposure period, and on the day sacrificed. Feed consumption values for male rats were recorded weekly during the exposure period, except during cohabitation. Body weights for female rats were recorded weekly before and during the cohabitation period and on days 0, 6, and 13 of presumed gestation. Mating was evaluated daily during the cohabitation period and confirmed by observation of spermatozoa in a vaginal smear and/or a copulatory plug in situ.
Male rats were sacrificed by carbon dioxide asphyxiation after completion of the cohabitation period, and a gross necropsy of the thoracic, abdominal, and pelvic viscera was performed. Individual weights were recorded for the right testis, left testis, left epididymis (whole and cauda), right epididymis, seminal vesicles (with and without fluid), and prostate. A portion of the left cauda epididymis was used for evaluation of cauda epididymal sperm density (concentration) and motility by using computer-assisted sperm analysis (CASA). Motility was evaluated by the Hamilton Thorne IVOS by collection of a sample from the left cauda epididymis through a swim-out method. A homogenate was prepared for evaluation by the Hamilton Thorne IVOS to determine sperm concentration (sperm per gram of tissue weight). The remaining portion of the left epididymis, the right epididymis, prostate, and seminal vesicles were fixed in neutral buffered 10% formalin. The testes were fixed in Bouin's solution for 4896 h and then retained in neutral buffered 10% formalin. Representative samples of preserved tissues were routinely processed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for microscopic evaluation.
Female rats were sacrificed by carbon dioxide asphyxiation on gestation day 13. Pregnancy status was determined; the carcass was discarded without further evaluation. Uteri from rats that appeared nonpregnant were stained with 10% ammonium sulfide to confirm absence of implantation sites (Salewski, 1964).
Statistical evaluations.
All adult and pup incidence data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution (Snedecor and Cochran, 1967). Parameters involving continuous data were analyzed using Bartlett's Test of Homogeneity of Variances (Sokal and Rohlf, 1969a
) and the Analysis of Variance (Snedecor and Cochran, 1967
), when appropriate (i.e., Bartlett's Test was not significant [p > 0.05]). Body weights, body weight changes, and feed consumption data were among variables analyzed as continuous data. If the Analysis of Variance was significant (p
0.05), Dunnett's Test (Dunnett, 1955
), was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate (i.e., Bartlett's Test was significant [p
0.05]), the Kruskal-Wallis Test (Sokal and Rohlf, 1969b
) was used, when 75% or fewer ties were present. The Fisher's Exact Test (Siegel, 1956
) was used when more than 75% ties were present. In cases where the Kruskal-Wallis Test was statistically significant (p
0.05), Dunn's Method of Multiple Comparisons (Dunn, 1964
) was used to identify the statistical significance of the individual groups.
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RESULTS |
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Absolute weights of the male reproductive organs and terminal body weights are presented in Table 1. Terminal body weights were significantly decreased (p
0.01) for the rats placed in exposure tubes. This 24% decrease in body weight for the treated rats, compared to the untreated group, was considered related to the additional handling and stress associated with placement in exposure tubes, the additional time away from the feed containers, and the additional exercise done by rats when returned to their cages after the stimulus of handling. Absolute weight of the left and right epididymides, left cauda epididymis, seminal vesicles with fluid, and prostate were significantly decreased (p
0.05 or p
0.01) for the rats placed in exposure tubes. However, relative organ weights to terminal body weight ratios (Table 2
) of the left and right epididymis, left cauda epididymides, left and right testes, and seminal vesicles with and without fluid were significantly increased (p
0.01) for the rats placed in exposure tubes. This decrease in absolute reproductive organ weight and increase in relative reproductive organ weight for the treated rats, compared to the untreated group, reflected the previously described reduction in body weight for the treated group.
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There were no statistically significant differences in cauda epididymal total sperm count and density (concentration), which were unaffected by placement of the rats in exposure tubes.
Histopathology
The type and incidence of the histomorphologic observations in the reproductive tissues for the male rats are presented in Table 9. The microscopic changes encountered were considered to have occurred spontaneously and to be unrelated to treatment. No microscopic changes were observed in the testes of any male rat of either group. The only finding observed in rats that had been placed in exposure tubes was an increase in necrotic cells in the lumen of the epididymal tubules of 3 of these 20 rats. There was no microscopic change in the testes that could be correlated with the increased amount of sloughed necrotic germ cells, which were probably spermatids. As this was a unilateral phenomenon, it was considered unrelated to placement of the rats in the exposure tubes.
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DISCUSSION |
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The control rats weighed 24% more than the rats exposed nose-only. If the organ weights (Table 1) for both groups had been identical, the relative reproductive organ weights (Table 2
) would have been 24% greater for the rats exposed nose-only. Thus the different weight gains resulted in the nose-only exposed male rats having significantly reduced absolute but increased relative reproductive organ weights as compared to the control group values.
There was a small but statistically significant decrease (p 0.05) in sperm count for the rats placed in exposure tubes, compared to the control rats. No statistically significant or dose-dependent differences occurred in the number or percentage of motile sperm, the number of nonmotile sperm, or the percentage of motile sperm. Cauda epididymal sperm count and density (concentration) were unaffected by placement of the rats in exposure tubes. Mating and fertility indices were similar for the two groups and there were no significant differences.
There were no microscopic changes that could be clearly correlated to an adverse effect on the reproductive tissues in the male rats placed in exposure tubes used in inhalation toxicity studies. The only finding observed at an increased incidence in the rats placed in the exposure tubes was increased necrotic germ cells in the epididymal tubules of 3 of 20 rats. No microscopic change in the testes could be correlated with this finding. Occasionally, increased numbers of these germ cells are observed in young male rats as an incidental finding (Russell et al., 1990). No treatment-related microscopic changes were observed in the testes, seminal vesicles, or prostate of the rats placed in the exposure tubes.
In contrast with the studies previously reported (Bernet et al, 1998; Brock et al., 1996
; Chan, 1996
; Dodd et al., 1997
, 1999
; Lee et al., 1993
), no adverse effects were observed as a result of 2-h nose-only exposure of male Sprague Dawley rats. The adverse effects of nose-only exposure in the studies reported by Dodd and Brock were attributed to heat stress (Brock et al., 1996
; Dodd, 1999) or to heat and pressure (Chan, 1996
). Temperature was not considered a major factor in the studies reported by Bernet or Lee, nor were the conditions of exposure discussed (Bernet et al, 1998
; Lee et al., 1993
). In studies on the effects of chronic immobilization stress on reproductive functions, no deleterious effect was observed in adult mice and rats (McGrady et al., 1983; Murthy et al., 1988
). Our study design was based on the hypothesis that it is essential to adequately acclimate rats prior to exposure and also to maintain exposure conditions that ensure avoidance of excessive testicular temperatures.
No attempt was made to include positive controls by duplicating conditions reported to produce adverse effects. Adding a group of rats that were acclimated but that were exposed in a room at 7579°F; a group that was exposed in a cool room but without acclimation; and a group that was exposed in a cool room after acclimation but without use of tail coolers might have clearly determined which of the steps taken was essential to obtaining satisfactory data. Adding these additional groups was not considered an ethical use of animals. Thus the data presented are not sufficient to determine whether both cooling and adequate acclimation are essential, or whether cooling alone is sufficient. We have found it preferable, as a humane approach to animal handling, to always acclimate animals to exposure systems. The data reviewed suggest the hypothesis that all studies in which rats are nose-only exposed for longer than 4 h demonstrate across-group testicular atrophy, independent of the method of acclimation, the strain of rat used, or the exposure conditions employed. No attempt was made in the studies reported herein to confirm or refute this hypothesis.
To confirm that a satisfactory baseline has been obtained, we recommend that nose-only exposures always include an air control group, to ensure that any unexpected adverse effects of handling or room conditions are recognized and included in data interpretation. For most studies, the need for an air control group in nose-only exposures is compensated for by the advantages that oral exposure by grooming is avoided, and that the amount of test material required is far less than that required for whole-body exposures. The data presented demonstrate, as previously observed (Alexander et al., 1996), that male rodents can be exposed via nose-only inhalation without major adverse reproductive effects.
This study was not designed to determine whether adequate acclimation and room cooling are both essential to conduct of reproductive studies on male rats, or whether satisfactory results can be obtained provided the testes remain cool throughout exposures. The data do demonstrate that restraint of male rats in nose-only exposure tubes under well-controlled conditions, following a properly designed acclimation regime, does not result in stress-related effects on male reproductive organs or in any major differences in male reproductive performance compared to untreated control male rats.
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ACKNOWLEDGMENTS |
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NOTES |
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