* E. I. DuPont de Nemours and Company, Haskell Laboratory for Toxicology and Industrial Medicine, Newark, Delaware 19714;
Pfizer Inc., Central Research Division, Eastern Point Road, Groton, Connecticut 06340;
Vulcan Chemicals, Birmingham, Alabama 35238; and
Unilever Research, SEAC Toxicology Unit, 45 River Road, Edgewater, New Jersey 07020
Received January 9, 2001; accepted March 20, 2001
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ABSTRACT |
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Key Words: halogenated hydrocarbons; chlorinated alkanes; inhalation; repeated exposure; immunotoxicity; hepatotoxicity.
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INTRODUCTION |
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Given the large toxicology database on chlorinated hydrocarbons, it seemed reasonable to develop occupational exposure guidance levels for HCC-230fa (1,1,1,3,3,3-hexachloropropane) based largely on those data. Indeed, a structureactivity relationship analysis using the database of the other chloropropanes suggested that the target organs for toxicity, and hence the occupational guidance, would be similar for this compound. However, the results of preliminary studies with HCC-230fa indicated otherwise. Therefore, a more extensive toxicological program was undertaken for HCC-230fa.
In the 14-day range-finding inhalation study (DuPont unpublished data, 1996), groups of male Crl:CD®(SD)IGS BR rats were exposed 6 h/day, 5 days/week to 10, 50, or 150 ppm of HCC-230fa vapors. Rats exposed to 150 ppm had severely decreased mean total leukocyte count at the end of the exposure period. In addition, rats in this group had moderately decreased mean platelet count at the end of the exposure period. In rats exposed to 150 ppm, bone marrow atrophy, atrophy of the prostate, and centrilobular hypertrophy in the liver accompanied by individual cell necrosis were observed. In addition, in rats exposed to 50 and 150 ppm, microscopic changes were observed, including lymphoid depletion in the lungs, spleen, thymus, and/or mesenteric lymph node, atrophy of the seminal vesicles, testicular degeneration characterized by loss of spermatogonia, scattered individual germ cell necrosis, and occasional germ cell debris within lumina. In addition, rats from all exposure groups had oligospermia with germ cell debris in the epididymides. No other microscopic effects were seen in the 10-ppm exposure group.
After a 14-day recovery period, the same microscopic changes that were present previously continued to be observed in the lungs, spleen, mesenteric lymph node, seminal vesicles, testes, and epididymides. Lesions in the testes and epididymides were more severe in the 150-ppm exposure animals at 14 days of recovery than after the final exposure, indicating continued progression of the testicular lesion. The testes after 14 days of recovery were essentially devoid of germ cell epithelium.
Given the results of the 14-day study, it was clear that additional data for longer exposure periods were needed to assess the toxicity of this compound and develop dose response data for adverse effects. Therefore, the objectives of this study were 2-fold. First, there was a need to develop data in order to establish appropriate occupational guidance for industrial operations. Second, the toxicity of HCC-230fa was compared to other chlorinated propane analogs to better understand the relationship of structure and toxicity.
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MATERIALS AND METHODS |
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Animal husbandry.
A total of 250 male and 86 female (nulliparous and nonpregnant) Crl:CD®(SD)IGS BR rats was received from Charles River Breeding Laboratories (Raleigh, North Carolina, USA). The rats were approximately 5 weeks of age on the day of arrival and were quarantined for 6 days and housed an additional 3 days prior to testing. Rats were weighed and observed for clinical signs of disease during the quarantine and pretest periods. Rats were housed singly in suspended, stainless steel, wire-mesh cages in a Bioclean® room (laminar flow air circulation pattern). The position of cages on racks was systematically rotated every 2 weeks. Animal rooms were maintained on a timer-controlled, 12-h light/12-h dark cycle. Environmental conditions of the rooms were targeted to be within a temperature range of 23 ± 2°C and a relative humidity range of 50 ± 10%. Except during exposures, Purina Certified Rodent Chow® #5002 and tap water were available ad libitum. Animals were humanely cared for and sacrificed according to principles provided by the Society of Toxicology and described in Guiding Principles in the Use of Animals in Toxicology.
Atmosphere generation and analysis conditions.
Vapor atmospheres of HCC-230fa were generated by metering the liquid test substance into a heated Instatherm flask (146183°C) with a Harvard Apparatus Model 22 Syringe Infusion Pump. Nitrogen, introduced into the flask, carried the HCC-230fa vapor through heated, nonreactive stainless steel lines (91140°C) into an air stream that was directed into the top of the exposure chamber. The chamber concentration of HCC-230fa was controlled by varying the amount of HCC-230fa evaporated in the chamber air stream. Nitrogen and air streams were passed through the control chamber at approximately the same flow rates as those used in the exposure chambers. In addition, the nitrogen was passed through a heated Instatherm flask (107147°C). Homogeneous distribution of the compound within the chamber was determined prior to initiating the study. The exposure chambers (New York University style) used on this study were constructed of stainless steel and glass and had a nominal internal volume of 1.4 m3. The chambers were operated in a one-pass, flow-through mode with air flow rates adequate to provide sufficient oxygen for rats and enable adequate distribution of HCC-230fa in the chambers.
Chamber airflow was set to achieve at least 12 air changes/h within the exposure chamber. Chamber temperature was targeted to 23 ± 1°C. Chamber relative humidity was targeted at 50 ± 10%. Chamber oxygen concentration was targeted to at least 19%. Air flow, temperature, and relative humidity were monitored continually with a Lander Control Systems Toxicology Monitoring System and were recorded at 15-min intervals during each exposure. Percent oxygen was measured with a Biosystems Model 3100R oxygen monitor and recorded two times during each exposure.
The atmospheric concentration of HCC-230fa was determined by gas chromatography at approximately 45-min intervals during each 6-h exposure. Vapor samples were drawn by vacuum pump from two representative areas of the chamber where animals were exposed. Chamber atmosphere samples were directly injected into a Hewlett Packard Model 5880 gas chromatograph equipped with a flame ionization detector for determination of HCC-230fa concentration. All samples were chromatographed isothermally at 135°C on a 30-m Alltech ATTM-35 column. The atmospheric concentration of HCC-230fa was determined from a standard curve derived from vapor standards. The vapor standards were prepared prior to each exposure by injecting known volumes of liquid HCC-230fa into Tedlar® bags that contained known volumes of air.
Study design.
Rats were exposed to the test substance in stainless steel wire mesh cages (sexes separate). Position of the cages within the chamber was rotated daily. Eight groups of 30 male rats each and eight groups of 10 female rats each (two groups per exposure concentration) were exposed to concentrations of HCC-230fa targeted to 0 (controls), 0.5, 2.5, or 25 ppm. These exposure concentrations were selected based on the results of a 14-day study. Four groups of male and four groups of female rats were used for standard toxicological evaluations. The remaining four groups of male rats were used for immunotoxicological and sperm assessment evaluations, and the remaining four groups of female rats were used for immunotoxicological evaluation. Animal exposures were whole body for 6 h/day, 5 days/ week over an approximate 90-day interval.
Prior to exposure, all rats were weighed and individually observed for clinical signs. During each exposure, rats visible from the front of the exposure chamber for each exposure concentration were observed for mortality approximately every hour. In addition, group clinical signs of toxicity and an acoustic startle response to a sharp rap on the chamber were determined approximately every 2 h for rats visible from the front of the exposure chamber. All rats designated for standard toxicological evaluations were examined after exposures for abnormal behavior and appearance.
The amount of food consumed by each rat designated for standard toxicological evaluations was determined weekly during the study. From these determinations and body weight data, mean individual food consumption and food efficiency were calculated.
Blood was collected for clinical pathology evaluation from 10 rats per group designated for standard toxicological evaluations on test days 50 and 96. At each sampling time, after an overnight fast, blood was taken from the orbital sinus of each rat under light carbon dioxide anesthesia. The hematological parameters measured or calculated consisted of the number of erythrocytes, leukocytes, and platelets; hemoglobin concentration and hematocrit; mean corpuscular volume; mean corpuscular hemoglobin; mean corpuscular hemoglobin concentration; and relative numbers of neutrophils, band neutrophils, lymphocytes, atypical lymphocytes, monocytes, eosinophils, and basophils. Absolute values for various types of leukocytes were calculated from the leukocytic data. Blood cell counts, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration were determined on a Serono Baker System 9000® hematology analyzer. Differential cell counts were determined on a Hematrak® Automated Differential System cell counter. Reticulocyte smears were prepared at each sampling time.
The serum chemistry parameters measured or calculated were activities of alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, sorbitol dehydrogenase, and concentrations of glucose, urea nitrogen, calcium, phosphate, bilirubin, cholesterol, creatinine, total protein, albumin, globulin, sodium, potassium, and chloride. Clinical chemical analysis was conducted using a Boehringer Mannheim/Hitachi 717 clinical chemistry analyzer with appropriate reagents.
One day prior to each bleeding time, an overnight urine specimen was collected from each rat to measure volume, osmolality, urobilinogen, and pH, and also to determine the presence of hemoglobin or occult blood, glucose, protein, bilirubin, and acetoacetic acid. Osmolality was determined on a Precision System Multi-OsmetteTM 2430 osmometer. Urine biochemical constituents were measured with Ames MultistixTM urine chemistry dipsticks on a ClinitekTM 200 urine chemistry analyzer. Urine appearance (color and transparency) was recorded and the sediment from each specimen was microscopically examined.
On the day after the last exposure, 10 rats per group designated for standard toxicological evaluations were sacrificed by carbon dioxide anesthesia and exsanguination and necropsied. These rats were designated 0-day recovery animals. The order of sacrifice for the pathological evaluation was random among all treatment groups. All remaining male rats were allowed to recover for either 1 or 3 months. At the end of the 1-month recovery period, 10 male rats per group designated for standard toxicological evaluations were sacrificed for pathologic examination. In addition, 10 male rats per group designated for sperm assessment and immunotoxicological evaluations were sacrificed for evaluation. Approximately 2 weeks prior to the 3-month recovery sacrifice, all remaining male rats designated for standard toxicological evaluations were bled for hematological evaluation. These rats were subsequently sacrificed for pathological evaluation at the end of the 3-month recovery period. The remaining male rats designated for sperm assessment and immunotoxicological evaluation were sacrificed without evaluation prior to the 3-month recovery period.
The liver, kidneys, lungs, brain, adrenal glands, testes, and ovaries were weighed at necropsy. Each rat was given a complete gross examination, and representative samples of the following tissues were saved: liver, kidneys, lungs, duodenum, heart, spleen, brain (cerebrum, midbrain, cerebellum, medulla/pons), spinal cord, stomach, jejunum, ileum, pancreas, cecum, colon, rectum, mesenteric and mandibular lymph nodes, thymus, adrenal glands, sciatic nerve, thyroid gland, parathyroid glands, trachea, esophagus, pharynx/larynx, eyes, prostate, seminal vesicles, urinary bladder, testes epididymides, mammary glands, ovaries, uterus, vagina, sternum (with bone marrow), nose, salivary glands, exorbital lacrimal glands, pituitary gland, skeletal muscle, and skin. All tissues were fixed in 10% neutral buffered formalin except for eyes, testes, and epididymides, which were fixed in Bouin's solution.
All tissues collected from rats sacrificed at the end of the exposure period were processed, embedded in paraffin, cut at a nominal thickness of 5 µm, stained with hematoxylin and eosin (H&E), and examined microscopically. Nose, pharynx/larynx, liver, kidneys, lungs, prostate, testes, epididymides, and seminal vesicles from 1-month and 3-month recovery rats in the 0- and 25-ppm groups were also processed to slides and examined with a light microscope. Kidney was identified as a possible target organ and was processed to slides in the 0.50- and 2.5-ppm groups
Immunotoxicological evaluation.
Ninety-two days after study initiation, five male and five female rats per dose group designated for immunotoxicological evaluation were injected intravenously (iv) in a lateral tail vein with 1 ml of 2 x 108 sheep red blood cells (SRBC)/ml. Ninety-three days after study initiation, the remaining five male and five female rats per dose group designated for immunotoxicological evaluation were injected iv in the lateral tail vein with 1 ml of 2 x 108 SRBC/ml. On the day after the last exposure (6 days after SRBC injection), the animals were euthanized by carbon dioxide anesthesia and exsanguination. Blood was collected from each rat, and serum was obtained and stored frozen (0°C). The primary humoral immune response was evaluated by examining individual animal serums for SRBC-specific IgM levels using an enzyme-linked immunosorbent assay (ELISA) (Temple et al., 1993). Data were acquired using an MR 5000 96-well microplate reader (Dynatech Laboratories, Chantilly, VA) and analyzed using the Revelation Software (Version 2.0) (Dynatech Laboratories). Sera pooled from male rats injected with SRBC and dosed with the known immunosuppressive agent cyclophosphamide (2.5 or 5 mg/kg, ip) were conducted with the study samples as a positive control. The data were expressed as the highest dilution (i.e., titer) to give an absorbance value of 0.5 (e.g., 1:500 = 500).
After an approximately 1-month recovery period, five male rats per dose group designated for immunotoxicological evaluation were injected iv on test day 128 and the remaining five per dose group on test day 129 with SRBC as described above. Six days later, the animals were euthanized, serum was collected, and the SRBC-specific humoral immune response was evaluated as described above.
Sperm assessment evaluation.
Ten males per group designated for sperm assessment evaluation were euthanized by carbon dioxide anesthesia and exsanguination. The right epididymis was removed and weighed. The right cauda epididymis was excised and placed in 32°C phosphate-buffered saline with 10 mg/ml bovine serum albumin, pricked with a needle to facilitate the release of sperm, and placed in an incubator at 32°C for 5 min. After incubation, an aliquot was placed in a sample chamber and placed under a microscope containing a camera linked to a video cassette recorder. At least 10 fields per sample were videotaped for approximately 10 s per field. The videotapes were later analyzed, and the percentage of motile cells among at least 200 cells examined per animal was determined.
After removal of an aliquot of sampling for videotaping, the right cauda epididymis was minced and further incubated. An aliquot was stained with eosin, and smears were prepared on microscope slides. Caudal epididymal sperm were examined to determine the frequency of morphologically abnormal sperm, expressed as percentage of normal cells among at least 200 cells examined per animal. The specific types of abnormal morphology were not categorized.
The left epididymis and left testis, following removal, were flash-frozen in liquid nitrogen and stored at 65 to 85°C until analyzed. After thawing, the cauda epididymis was excised, weighed, and homogenized. After thawing, the testis was weighed and homogenized. Sperm count per cauda epididymis and per gram cauda epididymis, and spermatid count per testis and per gram testis were determined.
Statistical analyses.
Descriptive statistics including mean, standard deviation, and standard error of the mean were used to summarize experimental data (Zar, 1984). Incidences of clinical observations were evaluated by the Cochran-Armitage test for trend (Armitage, 1955
; Cochran, 1954
). Mean body weights, body weight gains, food consumption, and mean absolute and relative (to body weight and to brain weight) organ weights were statistically analyzed by a one-way analysis of variance (Freund and Walpole, 1980
; Montgomery, 1984
). Pairwise comparisons between test and control groups (sexes separate) were made with the Dunnett's test (Hochberg and Tamhane, 1987a
).
For clinical pathology evaluations, a one-way analysis of variance and Bartlett's test were performed for each sampling time (Milliken and Johnson, 1984; Montgomery, 1984
). Dunnett's test was used to compare means from the control group and each of the test groups. When the results of Bartlett's test were significant, the Kruskal-Wallis test (Gibbons, 1976
; Hollander and Wolfe, 1973
) was employed and the Mann-Whitney U test (Hollander and Wolfe, 1973
; Lehmann, 1975
) was used to compare means from the control groups and each of the exposed groups.
For immunotoxicology evaluations, SRBC-specific serum IgM antibody titer data were transformed to Log2 to obtain normality or homogenous variances. For monotone data, a two-sided Jonckheere's trend test was used in a stepdown manner (Hochberg and Tamhane, 1987b; Marcus et al., 1976
). For nonmonotone data, the responses for each end point were initially checked for normality and homogeneity of variances using the Shapiro-Wilk (Shapiro and Wilk, 1965
) and Levene's tests (Levene, 1960
), respectively. If either test failed, then Tukey's outlier detection rule (Emerson and Strenio, 1983
) was followed to identify and eliminate possible outliers from the data. If the data satisfied normality and homogeneity requirements, i.e., there were no outliers, then Dunnett's test (Dunnett, 1955
) was used to compare the treatment groups to the control, both using the full dataset and the outlier-cleaned data. If the conclusions of the Dunnett's tests were the same, then those results were accepted. Otherwise, the nonparametric Dunn's test (Dunn, 1964
) was used to compare the treatment groups to the control group on the full dataset.
All sperm parameters were statistically analyzed using Jonckheere's test for trend (Jonckheere, 1954). Except for the Bartlett's test (p < 0.005), all significance was judged at p < 0.05.
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RESULTS |
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Male rats in the 25-ppm exposure group had statistically significantly lower mean body weights and mean body weight gains throughout the exposure phase of the study (Fig. 1). At the conclusion of the exposure period, the body weights for the 25-ppm group were about 7% lower than body weights of control rats. Male rats in the 0.5-ppm exposure group also had significantly lower body weights periodically during the exposure phase of the study. However, these lower weights were not considered related to the administration of the compound because of the lack of a dose response observed with the 2.5-ppm exposure group. No statistically significant differences in mean body weights or mean body weight gains were found in any test group of male rats during the recovery period.
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No clinical signs of toxicity attributable to HCC-230fa exposure were observed in this study. Clinical signs observed such as discharge of the eye or nose, stained fur, wet perineum, and alopecia occurred sporadically in all groups including the controls. These observations were considered incidental findings and typical of rats subjected to inhalation exposures.
Male rats in the 25-ppm group had minimal decreases in white blood cell (WBC) and lymphocyte counts at the 50- and 96-day sampling times (Table 1). In the 25-ppm female group, mean WBC and lymphocyte counts were generally lower than controls, although not statistically significant. In males, there was no evidence that the leukocytic changes persisted, as the mean values for WBC and lymphocyte count were similar to controls after approximately a 3-month recovery period. Females were not evaluated for recovery. No other significant hematological changes were observed.
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The mean relative liver weights in males and females and mean relative kidney weight in female rats were significantly higher in the 25-ppm groups than in controls at the end of the exposure phase of the study (Table 2). These effects were considered to be secondary to lower mean final body weights in the 25-ppm groups. After a 1-month recovery, the mean relative kidney weight of male rats was statistically higher in the 25-ppm group than in the controls due to lower mean final body weight in the 25-ppm group. Although not statistically different (except for the low-concentration group), the testicular weights of rats in the HCC-230fa-treated groups were slightly lower than control rats and exhibited a doseresponse relationship. At the 1- and 3-month recovery necropsy, however, there were no differences in testicular weights among the treated rats. There were no other compound-related differences in organ weights observed during the study.
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In rats sacrificed at the end of the exposure, minimal hepatocellular hypertrophy in 4 of 10 males in the 25-ppm group was observed and was characterized by a slight increase in the cytoplasmic volume of hepatocytes with an increased cytoplasmic eosinophilia. A minimal chronic progressive nephropathy in 4 of 10 males in the 25-ppm group was also observed and was considered to be compound related. (Table 3). Chronic progressive nephropathy is a common age-related spontaneous renal disease in rats, but increases in the incidence or severity of spontaneous disease are often considered compound related. Minimal chronic progressive nephropathy was recognized by basophilic tubules and proteinaceous material within the tubular lumina. As the disease progresses in severity, it can ultimately result in widespread fibrosis and destruction of the tubular and glomerular elements. No other compound-related microscopic changes were observed in the exposed rats, including the bone marrow, testes, and myocardium.
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Exposure of male and female rats to HCC-230fa did not significantly alter the SRBC-specific primary IgM antibody response (data not shown). Likewise, the SRBC-specific primary IgM antibody response was not significantly altered in the 1-month recovery male rats. As expected, the known immunosuppressant agent, cyclophosphamide, decreased the humoral response to SRBC.
At the conclusion of the exposure period, a statistically significant decrease in epididymal sperm number per cauda epididymis, percent motile sperm, and percent normal sperm morphology was observed at 25 ppm (Table 4). After a 1-month recovery period, a statistically significant decrease in percent normal sperm morphology was noted compared with controls at 25 ppm (Table 5
). In addition, a nonsignificant decrease (15%) was noted in epididymal sperm per cauda epididymis. All other sperm parameters were comparable to controls following a 1-month recovery period.
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DISCUSSION |
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Pathological effects in the myocardium have been observed for some of the isomers of the chlorinated propanes 1,2,3-trichloropropanes and 1,1,1,3-tetrachloropropane. Similar myocardial pathology has been reported for 1,1,1,3,3-pentafluoropropane (Rusch et al., 1999). These data would suggest that halogenation at the end carbons might be necessary to elicit a myocardial effect. However, myocardial effects have not been observed for 1,1,2,2,3-pentachloropropane and other chlorinated propanes (Johannsen et al., 1988a
; Kolesar et al., 1995
). In addition, no effects in the heart were observed in the current study with HCC-230fa. Clearly, this mode of action and the relationship with degree of halogenation and carbon chain length needs to be further explored.
There is extensive literature on the metabolism of the chlorinated alkanes, and it is this research that has generated a greater understanding into the mechanisms of liver toxicity for this class of compounds. The hepatocellular hypertrophy observed in the current study suggested that HCC-230fa undergoes some metabolism. Indeed, the increase in hypertrophy observed in the high-dose group might reflect an adaptive response to metabolism of the compound. Metabolic studies with other chlorinated propanes have been published (Timchalk et al., 1991; Mahmood et al., 1991
), and the data indicate that these compounds undergo limited metabolism compared to the shorter-chain chlorinated hydrocarbons. Recent but unpublished data indicate that HCC-230fa undergoes some metabolism with less than 50% elimination within 24 h following inhalation exposure (Sumner et al., 2000; Sumner et al., in preparation). In those studies, female rats retained the compound to a greater extent than males, an effect largely attributed to a greater accumulation of radioactivity in body fat. The differences in toxicity between sexes observed in the current study might be explained on the basis of the retention of the compound in fat, i.e., males tend to be more susceptible to the toxicity of the compound because less accumulates in fatty tissue.
A mild chronic progressive nephropathy was observed with HCC-230fa at the end of the exposure period. The incidence and severity of the lesion increased by the end of the 1-month recovery period. Terrill et al. (1991) reported a similar pathology with 1,3-dichloropropane, whereas Johannsen et al. (1988a) reported that 1,1,2,2,3-pentachloropropane produced a moderate tubular degeneration. However, histopathological alterations of the kidneys were not observed with either tetrachloropropane or trichloropropane. Although hepatotoxicity is considered a biological effect of chlorinated organic solvents, kidney effects of haloalkanes and haloalkenes have not been commonly associated with this class of chemicals (Plaa and Larson, 1965). However, like those for hepatotoxicity, the kidney effects from these compounds are more likely a result of local metabolism of the compounds to reactive intermediates.
Male and female rats in the 25-ppm group had minimal decreases in total leukocyte counts at the 50- and 96-day sampling times, with males showing a statistically significant decline. These changes, reflected in lymphocyte counts, were likely compound related, but the magnitude of the changes was marginal for biological adversity, especially in females. These leukocytic changes were not accompanied by histopathologic changes in bone marrow or lymph nodes. However, a statistically significant depression in the proportion of polychromatic erythrocytes among total erythrocytes, indicative of bone marrow cytotoxicity, was observed in male rats exposed at 25 ppm. Changes in leukocyte counts are not generally observed with administration of the chlorinated alkanes, although both increases and decreases have been observed with chlorinated propanes or propenes (Haut et al., 1996; Johannsen et al., 1988a
). In spite of the decrease in leukocyte counts, the immune status of the animals did not appear to be compromised, as the primary humoral immune response to SRBC was not altered.
At the conclusion of the exposure period, a statistically significant decrease in epididymal sperm number per cauda epididymis, percent motile sperm, and percent normal sperm morphology were observed only at 25 ppm. The decrease observed in the percent motility at the 25-ppm concentration was believed to be biologically significant. However, the biological significance of the decreases observed in the epididymal sperm number and percent normal sperm morphology is unclear. For both parameters the concurrent control group was at the high end of our laboratory's historical control data. In addition, the values at the 25 ppm concentration were at the low end of the historical control range. For the epididymal sperm number per cauda epididymis (x106), the control was 219.2; the 25-ppm group value was 194.6 with a historical control range of 174.5229.1. For the percent normal sperm morphology, the control was 98% and the 25-ppm group was 96%, with a historical control range of 96.198.1%. These changes were observed in the absence of any histopathological alterations in the testes and epididymides. The lack of histopathological effects on the testes and epididymides were surprising, based on the results of the 14-day range-finding study. Additional work may be needed to address this apparent discrepancy in the reproductive tissue effects from these two studies following HCC-230fa exposure.
After a 1-month recovery period, a statistically significant decrease in percent normal sperm morphology was noted compared with controls (97.8 vs 98.3%) at 25 ppm. However, this decrease was not believed to be biologically significant. The value, 97.8%, was well within the historical control range for our laboratory. The apparent nonsignificant decrease in epididymal sperm number per cauda epididymis (15%) was not considered biologically significant. The decrease appears to be due to two animals indicated by the large standard deviation. In one of these two animals, the epididymis was devoid of sperm, whereas in the other rat, a sperm count of approximately 100 (106 per cauda epididymis) was recorded. Other sperm cell parameters in these two rats were also reduced. If the results of these two animals are considered statistical outliers, the mean values become quite similar to control, i.e., 257.4 for controls versus 259 (106 per cauda epididymis) for rats in the 25-ppm group. However, the potential for the decrease to be due to exposure to HCC-230fa cannot be totally ruled out without additional data.
In the 2-week range-finding study, groups of male rats were exposed to design concentrations of 0, 10, 50, or 150 ppm for 6 h/day for 9 days. Rats exposed to 150 ppm HCC-230fa had decreased mean total leukocyte count, characterized by decreased neutrophil, lymphocyte, and monocyte counts at the end of the exposure period. In addition, rats in this group had decreased mean platelet count at the end of the exposure period. These changes in leukocyte and platelet counts are consistent with injury to pluripotential stem cells in the bone marrow. Oligospermia with germ cell debris in the epididymides was observed in exposed rats in all groups. Other microscopic changes observed in rats exposed to 50 ppm and 150 ppm were lymphoid depletion in the lungs, spleen, thymus, and/or mesenteric lymph node, atrophy of the seminal vesicles, and degeneration/atrophy of the testes. Rats exposed to 150 ppm also had bone marrow atrophy, centrilobular hypertrophy in the liver accompanied by individual cell necrosis, and testicular degeneration characterized by loss of spermatogonia, scattered individual germ cell necrosis, and occasional germ cell debris within the lumina. After a 14-day recovery period, the same pathologic changes that were present previously continued to be observed in the lungs, spleen, mesenteric lymph node, seminal vesicles, testes, and epididymides. Lesions in the testes and epididymides were more severe in the 150-ppm exposed animals at 14 days of recovery than after the ninth exposure, indicating progression of the testicular lesion. Testes of rats exposed to 150 ppm after 14 days of recovery were essentially devoid of germ cell epithelium. All other lesions noted after the ninth exposure were decreased in severity and incidence following the 14-day recovery period, indicating that recovery was present but incomplete.
Numerous reproduction and developmental toxicity studies have been undertaken for the halogenated hydrocarbons, with dibromochloropropane (DBCP) being a prototypic compound for inducing reproductive effects (Whorton and Foliart, 1983). In most of the studies conducted with chlorinated propanes, no effects on reproductive performance were observed in the experimental animals (Johannsen et al., 1988a
,b
; Johannsen et al., 1991
; Torkelson, 1994
). With DBCP, significant testicular lesions with concurrent effects on sperm have been reported (Kluwe, 1981
; Warren et al., 1984
). In the current studies, the effects of HCC-230fa on testicular function were not remarkable. The effects on reproductive function of these slight changes in sperm measures in the current study are uncertain. Based on the 2-week range-finding study, however, doses of 50 ppm or greater might be expected to affect the reproductive capability of male rats. Significant malformations were observed in a developmental toxicity study conducted with HCC-230fa (Munley et. al., in preparation). Reproductive measures in that developmental study were unaffected by HCC-230fa, although that study was limited in terms of understanding the reproductive effects of this compound. These data would suggest that the compound might not affect rodent reproductive capability at exposure concentrations of 2.5 ppm and below. Clearly, additional studies would be needed to fully characterize the reproductive effects of this compound.
From a mechanistic point of view, the results from the 2-week study suggested that HCC-230fa was acting very much like an antimetabolite, i.e., inducing effects on rapidly dividing cells. The compound does induce chromosomal aberrations in mammalian cells (McGown et al., in preparation). Similar results were observed with DBCP, inducing clastogenic effects in mammalian cells. In long-term bioassays, DBCP was found to induce tumors (adenomas and carcinomas) in the kidney and liver. Given the results of the studies with HCC-230fa, one might therefore predict that HCC-230fa would be potentially carcinogenic in longer-term bioassays. However, additional studies would be required to test this hypothesis.
In summary, the subchronic inhalation exposure of rats to HCC-230fa resulted in effects generally common with other chlorinated hydrocarbon solvents, e.g., hepatotoxic changes as well as nephrotoxicity. Effects on white blood cell counts, without an affect on immune function, and slight decreases in sperm measures were also observed. These effects, taken in conjunction with the effects observed on the testes in the 14-day study, suggest that this compound may be acting like an antimetabolite, a material that affects rapidly dividing cells. Additional work may be needed, however, to clarify this potential mechanism.
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ACKNOWLEDGMENTS |
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NOTES |
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Presented at the 39th annual meeting of the Society of Toxicology, March 2000, Philadelphia, PA.
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