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Inhalation toxicity of sulfuryl fluoride in rats and rabbits
FUNDAMENTAL
AND
APPLIED
TOXICOLOGY
I2,540-557
( 1989)
inhalation Toxicity of Sulfuryl Fluoride in Rats and Rabbits D. L. EISENBRANDT Mammalian
AND K. D. NITSCHKE
and Environmental Toxicology Research Laboratory, Health and Environmental Sciences, The Dow Chemical Company, Midland, Michigan 48674
Received June 2, 1988; accepted September 22, 1988 Inhalation Toxicity of Sulfuryl Fluoride in Rats and Rabbits. EISENBRANDT, D. L., AND K. D. (1989). Fundam. Appl. Toxicol. 12,540-557. The inhalation toxicity of the structural fumigant sulfuryl fluoride (S02Fz) was evaluated in rats and rabbits. Exposures for a preliminary 2-week study were 6 hr/day, 5 days/week, to 0, 100,300, or 600 ppm SO,F, . Nine of ten rats at 600 ppm died or were moribund between the second and sixth exposures. Extensive kidney lesions were present in all rats exposed to 600 ppm, whereas only minimal renal changes were noted in rats at 300 ppm. Upper and lower respiratory tissues were inflamed in the single rat that survived the 2-week exposure to 600 ppm. Rabbits exposed to 600 ppm S02F2 were hyperactive and one animal had a convulsion. Exposure to 300 or 600 ppm for 2 weeks resulted in vacuolation and/or malacia in the cerebrum of all rabbits and most of these rabbits also had moderate inflammation of nasal tissues; a few rabbits at 600 ppm had inflammation of the trachea or bronchi. A subsequent I3-week study evaluated rats and rabbits exposed to 0, 30, 100, or 300 ppm S02Fz (337 ppm TWA for rabbits). Rabbits initially were exposed to a high concentration of 600 ppm; however, convulsions were noted in two animals after nine exposures and the concentration subsequently was reduced to 300 ppm. Vacuolation and/or malacia were observed in the cerebrum of all rabbits at the highest concentration; one rabbit exposed to 100 ppm also had cerebral vacuolation. Rabbits at the highest concentration, as well as one rabbit exposed to 100 ppm, had inflammation of the nasal tissues. Rats exposed to 300 ppm SOzF2 for 13 weeks had mottled incisor teeth, minimal renal effects,pulmonary histiocytosis, inflammation of nasal tissues, and cerebral vacuolation. Also, rats exposed to 100 ppm SO,F, for 13 weeks had mottled teeth. Fluoride toxicity was suggested by mottled teeth in rats as well as elevation of serum fluoride levels in rats and rabbits exposed to SOzFz for 13 weeks. Although repeated exposure of rats and rabbits to 100-600 ppm SOzF2 resulted in toxicity of the kidneys (rats only), brain, and respiratory system, no effects were detected in animals exposed to 30 ppm for 13 NITSCHKE,
W&S.
Q 1989 Society of Toxicology.
Sulfuryl fluoride (S02F2; VIKANE)’ is a mmigant that controls drywood termites and other wood-destructive pests. The material typically is applied as a gas in sealed buildings and chloropicrin is introduced prefumigation into the structure as a vacating agent. Fumigation concentrations of S02F2 vary from approximately 1000 to 40,000 ppm and the concentration depends on the fumigation conditions and the target pest. An industrial hygiene study deter-
mined that short-term exposure of applicators to S02F2 varied from 0.9 to 8.7 ppm and the time-weighted-average (TWA) exposures were less than 5 ppm (Vaccaro, unpublished data, The Dow Chemical Co., 1988). The current threshold limit value (TLV) for occupational exposure is 5 ppm, with a short-term exposure limit (STEL) of 10 ppm (ACGIH, 1987). Human fatalities have been associated with accidental overexposure to S02F2 or attributed to suicide (Scheuexman, 1986; Nuckolls et al., 1987).
’ VIRANE gas fumigant, a trademark of The Dow Chemical Company. 0272-0590/89 $3.00 Copyright 0 1989 by the Society ofToxicology. All rights of reproduction in any form reserved.
540
INHALATION
TOXICITY
The LC50 (95% confidence interval) for a 1-hr exposure to S02F2 was 3730 ppm (309045 10 ppm) for male rats and 3020 ppm (2830-3220 ppm) for female rats (Vemot et al., 1977). Truhaut et al., (1973) found that mice were more sensitive than rats to the lethal effects of S0,F2, whereas rabbits were less sensitive. Death was attributed to asphyxiation that was associated with tonic convulsions and blockade of respiratory muscles. Animals that died after exposure to 6000 to 10,000 ppm S02F2 had pulmonary congestion and alveolar hemorrhage. Lesions were not detected in animals that died after exposure to lower concentrations. Concentrations of 4000 ppm S02Fz incapacitated rats within 45 min and resulted in death within several hours of exposure (Nitschke et al., 1986). Exposure to lO,OOO40,000 ppm decreased the time to incapacitation and death occurred within minutes. Nearly all rats convulsed during or after exposure to S02F2 and rats exposed to higher concentrations were cyanotic. Respiration typically ceased for 15-30 set after a convulsion and several rats died immediately after a convulsion. Serum fluoride levels were elevated in these animals and the toxicity of SOzF2 was attributed, at least in part, to fluoride. The current studies were designed to evaluate the subchronic inhalation toxicity of S02Fz in rats and rabbits. MATERIALS
AND
METHODS
Experimental design. The 2.week study consisted of groups of 5 rats/sex and 3 rabbits/sex that were scheduled for exposure to 0, 100,300, or 600 ppm SO,F, for 6 hr/ day, 5 days/week, for nine exposures. Subsequently, groups of 10 rats/sex were scheduled for exposure to 0, 30,100, or 300 ppm SOIF, for 6 hr/day, 5 days/week, for 13 weeks and groups of 7 rabbits/sex were scheduled for exposure to 0, 30, 100, or 600 ppm S02Fz for 6 hr/day, 5 days/week, for 13 weeks. However, clinical effects in rabbits exposed to 600 ppm S02F2 required that the concentration be reduced to 300 ppm after the ninth exposure. Parameters evaluated were clinical observations, body weight, hematology, clinical chemistry, urinalysis, gross pathology, and microscopic pathology.
OF SULFURYL
FLUORIDE
541
Test material. Sulfuryl fluoride, a colorless, odorless gas, was obtained from the Agricultural Products Department, Dow Chemical U.S.A. Analysis ofthe test material (Lot No. TWP 830919408) revealed the sample to be 99.8% SOZF2. Exposure system. Animals were exposed to SOzF2 in 4. l-m3 chambers constructed of stainless steel and glass. Control animals were placed in a chamber of similar design that provided clean, filtered air. Chamber airtlow was maintained at approximately 800 liters/min. SO,F* was metered at a controlled rate into the main-chamber airstream where the gas was diluted to the desired concentration before the airstream entered the chamber. The test atmosphere for the 2-week study was generated by metering SOIF, directly from cylinders into the airstream via flowmeters. The test atmosphere for the 13week study was generated by metering S02F2 from SARAN* bags with an RPG pump (Fluid Metering Inc., Oyster Bay, NY). The air supplied to the chambers was controlled by a system designed to maintain approximately 22°C and 50% relative humidity. A minimummaximum thermometer and a hygrometer were placed in each chamber and values were recorded at the end of each exposure period. The concentration of S02F2 in each exposure chamber was determined once or twice per hour with a MIRAN 1A infrared spectrophotometer (Foxboro/Wilks, South Norwalk, CT) at a wavelength of 11.8 Wm. The infrared spectrophotometer was calibrated with standards made by diluting measured volumes of S02F2 with a measured volume of filtered compressed air. The analytical system was evaluated with at least one standard prior to each day of exposure. The distribution of SOzF2 gas within each chamber was measured from six to eight sample points within the breathing zone and varied by less than 0.5%. Animals. Male and female Fischer 344 rats (6-7 weeks of age, Charles River Breeding Laboratories, Kingston, NY) and male and female New Zealand White rabbits (4-5 months of age, Hazelton Dutchland, Inc., Denver, PA) were acclimated for at least 7 and 14 days, respectively, prior to the initial exposures. Animals were assigned randomly by weight to exposure groups. Rabbits were negative for Encephalitozoon cuniculi titers. Animals on the 2-week study and rats on the 13-week study were fed Certified Purina Chow (Ralston Purina Co., St. Louis, MO) and water ad libitum except during exposures. Rabbits on the 13-week study were fed a maintenance diet (8 oz/day) of the same feed rather than ad libitum. Prior to the study, as well as the 18 hr/day and weekends when animals were not in exposure chambers, the animals were housed in rooms that controlled temperature at 21°C relative humidity at 50%, and a light-dark cycle at 12 hr.
’ SARAN, a trademark of The Dow Chemical Company.
542
EISENBRANDT
Clinical observations. The animals were observed after each exposure and changes in appearance or behavior were noted. Additional daily observations were conducted prior to each exposure and on weekends. Animals were weighed prior to the first, fifth, sixth, and ninth exposures for the 2-week study and weekly for the 13-week study. Clinicalpathology. Blood samples were collected from all animals that survived until study termination. Samples were collected from the posterior orbital sinus of rats and from a marginal ear vein of rabbits just prior to necropsy. Hematologic parameters included hematocrit, hemoglobin, erythrocyte count, red blood cell indices, total leukocyte counts, and platelet counts (ELT-8, Ortho Instruments, Boston, MA). Differential leukocyte counts were evaluated with an ACS-1000 (Honeywell, Inc., Denver, CO). Blood smears also were examined qualitatively. Urine was collected from all rats that survived until study termination. Urine was collected prior to the ninth exposure for the 2-week study and alter 11 weeks of exposure for the 13-week study. Urinary parameters included bilirubin, glucose, ketones, blood, pH, protein, urobilinogen (Chemstrip 7, Biodynamics/Division of BMC, Indianapolis, IN), and specific gravity (American Optical Co., Keene, NH). Urinalysis was not performed on rabbits. Serum samples for clinical chemistry were collected at necropsy from all animals that survived until study termination. Parameters evaluated were urea nitrogen, alanine aminotransferase activity, aspartate aminotransferase activity, alkaline phosphatase activity, glucose, total protein, albumin, and globulin (Centrifichem System 500, Baker Instruments Corp., Allentown, PA). Additional parameters evaluated only for animals on the 13week study included calcium, phosphorus, creatinine, and fluoride. The fluoride was measured with a fluoridespecific electrode (Orion Research, Cambridge, MA). Anatomic pathology. Animals that survived the exposures were necropsied the day after the last exposure to S02F2. Rats were fasted overnight prior to the scheduled necropsy; rabbits were not fasted. Rats on the 2-week study were anesthetized with methoxytlurane, whereas rats on the 13-week study and all rabbits were anesthetized with carbon dioxide; the animals then were euthanized by decapitation. Terminal body weights and weights ofbrain, heart, liver, kidneys, thymus (rats only), and tes tes were recorded. All animals were examined for gross pathological alterations by a veterinary pathologist. Animals that died or were moribund prior to the scheduled sacrifice were necropsied as soon as possible. An extensive set of tissues was collected from each animal and preserved in neutral, phosphate-buffered 10% formalin. The lungs were infused with buffered formalin to their approximate, normal inspiratory volume and the nasal cavity was flushed with formalin via the pharyngeal duct to ensure rapid fixation of tissues.
AND NITSCHKE Histopathologic examination was completed on an extensive set of tissues for all animals in the control and high-concentration groups. The set of tissues was similar to those listed in guidelines of the U.S. Environmental Protection Agency (1982). The same tissues were examined for rats in the 300 ppm exposure group on the 2-week study because of deaths in the 600 ppm group. Histopathologic examination of animals in other intermediate exposure groups from the 2-week study and all intermediate exposure groups from the 13-week study was less extensive, but included as a minimum kidneys, nasal tissues, trachea, lungs, and oral tissues; also, brain sections were examined for all intermediate groups except the 2-week, 100 ppm rats. Tissues were processed for light microscopy by conventional techniques and stained with hematoxylin and eosin. Special stains for animals from the 2-week study included von Kossa’s stain of lung, kidneys, heart, and stomach as well as phosphotungstic acid-hematoxylin stain of lung and/or kidneys from selected rats in the high-concentration group. Brain sections from selected rabbits on the 2-week study as well as the brain sections from selected rats and rabbits on the 13-week study were evaluated with luxol-fast blueperiodic acid-Schiff-hematoxylin and Sevier-Munger silver stains. Statistical evaluation. Body weights, organ weights, organ weights relative to terminal body weights, selected hematology data (excluding red blood cell indices and differential white blood cell counts), clinical chemistry data, and urinary specific gravity were evaluated by Bartlett’s test for equality of variances. Based on the outcome ofBartlett’s test, exploratory data analysis was performed by a parametric or nonparametric analysis of variance (ANOVA), followed respectively by Dunnett’s test or the Wilcoxon rank-sum test with a Bonferroni correction for multiple comparisons. Because of effectsidentified in the 2-week study, Dunnett tests, and Wilcoxon rank-sum tests for the heart and kidney weights, blood urea nitrogen and total white blood cell counts for rats in the 13week study were evaluated with one-sided tests. Since multiple, interrelated parameters were compared statistically in the same group of animals, the frequency of false-positive errors may have been much greater than the nominal 01levels. Thus, the final toxicologic interpretation of the data also considered whether an orderly dose-response relationship existed and whether the findings appeared to be plausible and consistent with other observations.
RESULTS Two- Week Study Rats and rabbits were exposed to TWA concentrations (mean f SD) of 100 + 6,293
INHALATION
FIG.
TOXICITY
OF SULFURYL
FLUORIDE
543
1. Papillary necrosis in a male rat that died after exposure to 600 ppm SO,F, for 1 week. X50.
-C 17, and 597 f 13 ppm for target concentrations of 100,300, and 600 ppm, respectively. The chamber temperature and relative humidity were comparable for animals exposed to 0,100,300, and 600 ppm SOzF2.
Rats Nine of ten rats exposed to 600 ppm SOzF2 died or became moribund between the second and sixth exposures; one female rat survived until termination. Rats at this exposure concentration were lethargic after the second
exposure and were less active after each subsequent exposure. Convulsions or other neurologic signs were not observed in these animals. These animals also consumed less food after the second and subsequent exposures and body weights were decreased significantly. One female that survived all nine exposures to 600 ppm SOzF2 weighed only 80.3 g as compared to controls that averaged 129.9 g. Rats exposed to 0,100, or 300 ppm S02F2 appeared normal throughout the exposure. Renal effectsat 600 ppm. Rats that died after several exposures to 600 ppm S02F, had severe kidney lesions that included papillary
544
EISENBRANDT
AND NITSCHKE
FIG. 2. Degeneration, necrosis, and regeneration of collecting ducts (C) in the inner medulla of a male rat that died after exposure to 600 ppm SO,F, for 1 week. Interstitial inflammation is associated with the tubular lesion. X 199.
necrosis (Fig. 1). The remainder of the papillary epithelium was moderately hyperplastic. There was a variable amount of subacute inflammation associated with the necrosis, and collecting ducts were dilated as a result of obstruction at the papillae (Fig. 2). Epithelial cells in the descending portion of proximal tubules had decreased amounts of basophilic cytoplasm as well as patchy segments of epithelial cell necrosis and regeneration. Convoluted tubules in the cortex had minimal changes that included an occasional degenerate cell and variable regenerative activity.
The female rat that survived 2 weeks at 600 ppm SOzF2 had moderate microscopic changes in collecting ducts, but no papillary necrosis (Fig. 3). This female also had hyperplasia of the renal papillary epithelium, and the epithelial cells of the descending portion of the proximal tubules were basophilic. Relative kidney weight was increased significantly in this rat (Table 1) and renal function was compromised. Urea nitrogen was elevated significantly (2 13 mg/dl), whereas urinalysis revealed a pH of 5 .O and a 3+ glucose; the glucosuria was secondary to hyperglyce-
INHALATION
TOXICITY
OF SULFURYL
FLUORIDE
545
FIG. 3. Inner medulla of female rat after exposure to 600 ppm S02F2 for 2 weeks. Collecting ducts are prominent due to regeneration (hyperplasia) and degeneration. Chronic interstitial inflammation also is present. X 199.
mia (1483 mg/dl). On the other hand, the specific gravity of the urine (1.020) was within a normal range. Renal e$ets at 300ppm. Five of the ten rats (both male and female) exposed to 300 ppm S02F2 for 2 weeks had minimal hyperplasia of the collecting ducts in the inner renal medulla. Two of these rats also had slight hyperplasia of the papillary epithelium. In addition, three of these rats had slightly basophilic epithelial cells in the descending part of the proximal tubules. Kidney weights were increased about 7% in animals from the 300 ppm exposure
group (Table 1). Urinalysis data and urea nitrogen values were normal in these rats. Otherjindings. Treatment-related effects in the respiratory system were present in the female that survived the 2-week exposure to 600 ppm S02F2. Severe, diffuse inflammation and multifocal ulceration of the nasal mucosa in this rat were accompanied by slight bronchioalveolar inflammation in the lungs. Numerous bacteria were associated with the inflammation in the respiratory system. Myeloid hyperplasia in the bone marrow of this female was attributed to inflam-
546
EISENBRANDT
AND TABLE
NITSCHKE 1
SELEC~EDABSOLUTEANDRELATIVEORGANWEIGHTDATAFORRATSEXPOSED TO SULFVRYL FLUORIDE FOR 2 WEEKS Concentration (mm)
N”
Terminal body weight (id
Kidneys
Heart g
g/l~g
Thymus
g
g/loog
I3
g/l@-)g
Males 0
5
173.76 I!? 9.7
0.617 +- 0.029
0.355 + 0.008
1.452 kO.141
0.834 t 0.048
0.327 I!Z0.018
0.189 +0.015
100
5
167.7 f 6.2
0.637 -t 0.036
0.380* + 0.015
1.443 + 0.076
0.861 + 0.041
0.322 + 0.032
0.192 + 0.019
300
5
172.3 + 6.7
0.614 + 0.016
0.356 + 0.008
1.545 + 0.105
0.896 k 0.041
0.299 + 0.02 1
0.174 -t 0.014
600
0
-c
-
-
-
-
0
5
112.2 k4.7
0.459 + 0.028
1.071 + 0.063
0.955 ?I 0.059
0.322 r 0.027
0.287 20.015
100
5
109.4 + 7.9
0.467 2 0.034
0.427 + 0.017
1.123 * 0.070
1.028 +- 0.065
0.287 -t 0.039
0.262 + 0.023
300
5
107.6 f 5.0
0.471 + 0.034
0.438* + 0.018
1.155 t- 0.056
1.075* f 0.070
0.260* + 0.033
0.241* f 0.027
600
1
67.6*
1.422*
0.031*
0.046*
0.313*
0.463*
0.96 1
a Number of animals. ’ Values are means + SD. ’ -, No data because of dead animals. * Statistically identified difference from control mean by Dunnett’s test, a = 0.05.
mation of the respiratory system as was an elevation in total white blood cell count (Table 2) and neutrophilia (42%). This female also had alterations in organ weights and hematologic and serum chemical parameters that were attributed to severe debilitation, body weight loss, and dehydration. Relative heart weights of females exposed to 300 or 600 ppm SOzF, slightly exceeded historical control values, although the increase for the 600 ppm female reflected primarily decreased body weight (Table 1). Total white blood cell counts were increased slightly in some rats exposed to SOzFz (Table 2); however, only males exposed to 300 ppm and the female exposed to 600 ppm exceeded
historical control values. Other than the female at the highest concentration, correlative findings were not present and reasons for the elevated white blood cell counts and heart weights were not apparent. Four of nine rats that died after exposure to 600 ppm S02F2 had pulmonary edema and/or hemorrhage or fibrin within alveoli. Also, thrombi were present in pulmonary capillaries of three rats. Other histopathologic observations in rats exposed to 600 ppm were considered secondary to renal disease or nonspecific terminal changes. Such observations included visceral congestion, stomach erosions, metastatic mineralization, hypertrophy of the adrenal cortex, as well as necrosis
INHALATION
TOXICITY
TABLE 2 TOTAL WHITE BLOOD CELL COUNTS OF RATS EXPOSED TOSULFURYLFLUORIDEFOR ~WEEKS Concentration (mm) 0 100
Na
Males
515
6.16 Ik 0.9
515
1.4
It 0.4 300
515
8.8*
t- 0.8 600
O/l
-c
Females 5.3
kO.8 6.6*
+ 0.6 1.9*
k 0.7 11.6*
0 Number of animals of each sex (M/F). b Values are mean total white blood cell counts (X 103/ mm3) f SD. ’ -, No data because of dead animals. * Statistically identified difference from control mean by Dunnett’s test. a = 0.05.
and depletion of lymphoid tissue in the thymus, spleen, and lymph nodes. Related decreases in thymic weights were most noticeable in female rats at the higher exposure concentrations (Table 2).
Rabbits One female rabbit exposed to 600 ppm SOzF2 had a convulsion after the fifth exposure; the convulsion resulted in a fractured tibia. Another female exposed to 600 ppm had a fractured vertebra after the sixth exposure, although convulsions were not observed. Both rabbits with fractures were euthanized and the other 600 ppm female survived until termination. Male and female rabbits that survived exposure to 600 ppm were slightly hyperactive compared to controls. Rabbits exposed to 0, 100, or 300 ppm appeared normal throughout the exposure period. There were no significant differences for in-life body weights. Nervous system eficts. Vacuolation was present in the brain of all rabbits that were
OF SULFURYL
FLUORIDE
547
exposed to 300 or 600 ppm SOzF2 (Fig. 4). Lesions were restricted to the globus pallidus and putamen (basal nuclei) as well as the external and internal capsules (myelinated tracts). Malacia was present in the cerebrum of all rabbits exposed to 600 ppm as well as one male and one female exposed to 300 ppm (Fig. 5 and 6). Reactive gliosis and demyelination accompanied the malacia. Respiratory systemeficts. Most rabbits exposed to 300 or 600 ppm SOzF2 had moderate, subacute to chronic inflammation of nasal mucosa with mucopurulent exudate in the nasal cavities. The trachea of some rabbits in the 600 ppm exposure group had minimal acute inflammation. In addition, the single female rabbit that survived the 2-week exposure at 600 ppm had acute inflammation of the bronchi and bronchioles. Other Jindings. Terminal body weights of some rabbits exposed to 300 or 600 ppm SOzF2 were decreased slightly. Some of these rabbits also had decreased liver weights at termination. The weight changes were attributed to altered nutritional status as were altered cytoplasmic homogeneity of liver cells and decreased serum albumin levels in some rabbits. A variable hematopoietic response was associated with the inflammation in the respiratory system of some rabbits. Alterations included lymphoid hyperplasia in the mediastinal lymph nodes and spleen.
Thirteen- WeekStudy Rats were exposed to TWA concentrations (mean f SD) of 29.8 -t 1.5, 100 a 5, or 297 + 12 ppm for target concentrations of 30, 100, or 300 ppm SOzF2, respectively. Rabbits were exposed to concentrations of 29.8 + 1.5, 100 + 5, or 337 st 98 ppm for target concentrations of 30, 100, or 300 ppm S02F2, respectively. The higher analytical concentration for rabbits (337 ppm) was due to nine exposures at 600 ppm at the beginning of the study. The chamber temperature and relative humidity were comparable for animals in control and treated exposure groups.
548
EISENBRANDT
AND NITSCHKE
FIG. 4. Vacuolation in myelinated tracts (arrows) and neuropil (arrowheads) in the cerebrum of a female rabbit exposed to 300 ppm SOzFz for 2 weeks. X64.
FIG. 5. Malacia and gliosis in the cerebrum of a male rabbit exposed to 600 ppm SOzFz for 2 weeks. The external capsule (arrowhead) and other myelinated tracts (arrows) have decreased myelin, vacuolation, and proliferation of oligodendrogha. X64.
INHALATION
TOXICITY
OF SULFURYL
FLUORIDE
549
FIG. 6. Malacia in the external capsule (EC) of a female rabbit exposed to 600 ppm S02F, for 2 wf :eks. Pyknotic nuclei (arrows) and glial proliferation are present. X 158.
Rats Exposure to 300 ppm SOzF* decreased the body weight of male rats after 45 days on test and females after 24 days on test (Fig. 7). Also, rats exposed to either 100 or 300 ppm S02Fz had mottled upper and lower incisor teeth. Nervous systemeffects.The brain of all rats exposed to 300 ppm SO,F, for 13 weeks had minimal microscopic vacuolation (Fig. 8). The vacuolation was restricted to the area of the caudate-putamen nuclei and was more prominent in the white fiber tracts of the internal capsule than in the adjacent neuropil. Special stains did not characterize the vacuoles or reveal any additional effects. Respiratory system eficts. Multiple, pale foci were present on the pleural surfaces of the lungs of most rats exposed to 300 ppm. Histopathologic examination revealed that the pale foci corresponded to subpleural histiocytosis (Fig. 9). Furthermore, there was minimal subacute inflammation of the nasal
mucosa in all female and most male rats exposed to 300 ppm S02F2. A few males had more severe inflammation of the respiratory and olfactory mucosa, which was associated with mucopurulent exudate in the nasal passages as well as degeneration and reactive changes in the mucosa (Fig. 10). Renal eficts. Kidneys of male rats exposed to 300 ppm SO2FZ had a slight decrease in microscopic protein droplets (a,,,-globulin) which normally are present in the convoluted tubules; the change was considered secondary to a decrease in body weight. Most female rats exposed to 300 ppm S02FZ had very slight hyperplasia of the renal collecting ducts which was most apparent in the outer portion of the inner zone of the medulla. Urinary specific gravity was decreased in male and female rats exposed to 300 ppm S02FZ for 13 weeks, although other urinary parameters were not affected (Table 3). Otherfindings. Rats exposed to the highest concentration of S02F2 had slight elevations of serum fluoride levels; however, the differ-
550
EISENBRANDT
AND NITSCHKE EEbML’= RATS
350-I
0
FIG.
20
40
60
DAYS
ON TEST
80
100
0
20
40
60
DAYS
ON TEST
80
100
7. Body weights of male and female rats; 13-week inhalation study of S02F2.
ences from control values were not statistitally significant (Table 4). There were no consistent toxicologic effects of treatment on hematology or clinical chemistry parameters. Many of the absolute and relative organ
weights of rats exposed to 300 ppm S02F2 were statistically identified as different from control values; however, the changes were associated with decreased body weights in these animals.
FIG. 8. Vacuolation in the fibers of the internal capsule and the caudate-putamen nuclei of a rat exposed to 300 ppm S02Fz for I 3 weeks. X 19 I.
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TOXICITY
OF SULFURYL
FLUORIDE
FIG. 9. Subpleural histiocytosis in lungs of a rat exposed to 300 ppm S02F2 for 13 weeks. X 19 1.
FIG. 10. Nasal tissue from a rat exposed to 300 ppm S02Fz for 13 weeks. A few males had severe, diffuse inflammation of the nasal mucosa. Note the inflammation ofthe olfactory mucosa (arrowhead) with mucopundent exudate in the nasal passages (arrows). X 19 1.
551
552
EISENBRANDT TABLE 3
URINARYSPECIRCGRAVITYOFRATSEXPOSED TOSULFLJRYLFLUORIDEFORI 3 WEEKS Concentration (wm)
N”
Males
0
IO/10
1.063’ f 0.007
1.049 * 0.015
30
IO/IO
1.061 f 0.010
1.049 f 0.007
100
lO/lO
1.062 + 0.006
1.050 + 0.017
300
1019
1.050* kO.013
1.040 kO.014
Females
’ Number of animals of each sex (M/F). b Values are means + SD. * Statistically identified difference from control mean by Dunnett’s test, a = 0.05.
Rabbits Convulsions were observed in one male and one female rabbit following the ninth exposure to the initial concentration of 600 ppm S02F2. Head bobbing was observed prior to and after a tonic convulsion by the male. Deep breathing, salivation, and rapid eye blinking accompanied convulsions and head bobbing in the female. A second female rabbit was euthanized atier the eighth exposure to 600 ppm because of posterior paralysis which was attributed to a fractured vertebra. As a consequence of the clinical effects noted at 600 ppm, the highest exposure concentration was reduced to 300 ppm for the remainder of the 13-week study. There were no clinical signs in rabbits exposed to 300 ppm for the subsequent 11 weeks and no clinical signs were detected in rabbits exposed to 30 or 100 ppm SO,F, for the entire 13-week study. Visual inspection revealed decreased food consumption for rabbits exposed to the highest concentration of S02F2 ; the decrease was noted after the ninth exposure and continued for approximately 4 weeks. Body weights
AND NITSCHKE
(Fig. 11) of males exposed to the highest concentration of S0,F2 were decreased significantly from Days 11 to 60 and remained below control values throughout the study. Body weights of females at the highest concentration also were decreased consistently. Minimal decreases in body weights were recorded for both sexes exposed to 100 ppm. Nervous systemeficts. Lesions in the brain of rabbits on the 13-week study were similar to those in the 2-week study. Microscopic changes were present in the area of the putamen, globus pallidus, internal capsule, and external capsule of the cerebrum. Three males and one female exposed to 337 ppm S02F2 had severe malacia, whereas only slight vacuolation was present in three male and five female rabbits. The vacuolation in some females was accompanied by gliosis and endothelial cell hypertrophy. A single female rabbit in the 100 ppm concentration group had moderate vacuolation of the cerebrum. Respiratory system eficts. Treatment-related microscopic changes were observed in the nasal tissues of all male and female rabbits exposed to 337 ppm S02Fz and one male exposed to 100 ppm. The changes consisted of variable degrees of inflammation in the nasal mucosa and purulent exudate in the nasal passages. Hypertrophy and hyperplasia of the respiratory epithelium (goblet cells and pseudostratified epithelial cells) were observed primarily on the nasal turbinates. Slight to moderate degeneration of olfactory epithelium was associated with the inflammation in two males and three females exposed to 337 ppm SOzF2. Other jindings. Serum fluoride levels of male and female rabbits exposed to 30, 100, or 337 ppm SO,F, were significantly increased over control values (Table 4). Total white blood cell counts for male rabbits exposed to 337 ppm S02F2 (9900 f 2400/mm3) were significantly increased over controls (7600 + 1000/mm3), although differential white blood cell counts were normal. Other hematologic parameters were unaffected by
INHALATION
TOXICITY
OF SULFURYL
553
FLUORIDE
TABLE 4 SERUM FLUORIDE LEVELS IN RATS AND RABBITS EXPOSED TO SULFURYL FLUORIDE
FOR 13 WEEKS Rabbits
Rats Concentration (mm)
Males
Females
Males
Females
0
Mean” SD N
0.996 r 0.788 9
0.607 k 0.449 10
0.065’ + 0.014 7
0.560 + 0.026 7
30
Mean SD N
0.715 k 0.288 10
0.738 f 0.630 10
0.170t f 0.069 7
0.697* + 0.070 7
100
Mean SD N
0.881 t 0.66 1 10
0.575 + 0.198 10
0.408t t 0.035 7
0.829* + 0.088 7
3001337
Mean SD N
1.154 f 0.574 10
1.366 AZ0.959 10
0.62lt -+ 0.060 7
1.003* + 0.045 6
n Values are mean fluoride levels (&ml) f SD for the indicated number of animals (N). ‘Six of the control male rabbits and one of the 30 ppm male rabbits had no fluoride detected (values less than the limit of 0.06 &ml). Therefore, the detection limit of 0.06 &ml was used to calculate the mean and SD for these animals as well as for statistical comparisons. * Statistically identified difference from control mean by Dunnett’s test, OL= 0.05. t Statistically identified difference from control mean by Wilcoxon’s test, a = 0.05.
treatment. Slight decreases in total serum protein, albumin, and globulin in males exposed to 30 or 337 ppm (but not males exposed to 100 ppm) were of questionable toxicologic significance, although the decreases at the higher concentration may have been associated with the lower nutritional status of the animals. Other clinical chemistry parameters for treated animals were normal. Liver weights of female rabbits exposed to 337 ppm SO,F, were significantly decreased as compared to controls (Table 5). Liver weights of males at 337 ppm as well as liver weights for both sexes at 100 ppm also were decreased somewhat. The decrease in liver weight values was most likely secondary to decreased food consumption and body weight. DISCUSSION Clinical observations in animals exposed to high concentrations of S02F2 (Truhaut d
al., 1973; Nitschke et al., 1986) are similar to those of acute fluoride toxicity (Drill, 1954; Taylor et al., 1961; Goodman et al., 1980). Elevation of serum fluoride levels in rats exposed to 4000 or 10,000 ppm S02F2 also suggests that fluoride is important in the acute toxicity of the compound (Nitschke et al., 1986). In addition, elevation of plasma fluoride levels has been reported in human deaths that were attributed to suicide or accidental overexposure to S02F2 (Scheuerman, 1986; Nuckolls et al., 1987). Fluoride is a potent inhibitor of intermediary metabolism in mammals and particularly inhibits enzymes involved with energy metabolism (Hodge and Smith, 1965; Matthews, 1970; Wiseman, 1970). Elevation of serum fluoride levels in rats and rabbits exposed to S02F2 for 13 weeks in the current studies suggests that fluoride is important in the toxicity of the compound. Mottled incisor teeth in rats exposed to 100
554
EISENBRANDT
AND NITSCHKE
MA1 F RABBITS 4600 4400 4200 -
4200
4000 -
4000
3800 -
3800
3600 -
3600
3400
3400
3200
3200
z 2 0
0
20
40
DAYS
60
50
0
100
20
ON TEST
337PPM
40
60
DAYS
ON TEST
80
100
FIG. 11. Body weights of male and female rabbits; 13-week inhalation study of SO,?F,.
or 300 ppm S02F2 for 13 weeks are indicative of enamel defects in developing teeth and are compatible with dental fluorosis (Drill, 1954; Taylor et al., 196 1). Fluoride ion as well as stress may have contributed to several indirect effects in rats exposed to higher concentrations of SO,F, in our 2-week study. Hypertrophy of the adre-
nal cortex, hyperglycemia, and necrosis of lymphoid tissue were observed in rats exposed to 600 ppm SOzF,. Suketa et al. (1985) have reported that single doses (35 mg/kg, ip) of sodium fluoride (NaF) elevate serum glucase in rats. Also, fluoride enhances glucose6-phosphatase activity in the liver and kidney and stimulates adrenocortical function (ele-
TABLE 5 ABSOLUTE
AND RELATIVE
LIVER WEIGHTS
FOR RABBITS EXPOSED TO SULFURYL FLUORIDE MR 13 WEEKS Males
Concentration @pm)
g
Females g/I~g
is
idl~g
0
Mean“ SD N
130.3 f 19.5 7
3.141 + 0.354 7
143.4 k 25.3 7
3.153 + 0.533 7
30
Mean SD N
129.3 _t 22.3 7
3.128 f 0.466 I
137.7 f 19.7 7
3.000 + 0.430 7
100
Mean SD N
99.4* -c 22.5 7
2.598 + 0.601 7
113.6 * 33.1 7
2.565 k 0.63 I 7
337
Mean SD N
110.9 + 18.9
2.871 2 0.405
7
7
103.6* k 21.9 6
2.399* + 0.435 6
DValues are means + SD for the indicated number of animals (NJ. * Statistically identified difference from control mean by Dunnett’s test, a = 0.05.
INHALATION
TOXICITY
vation of serum and urinary 17-hydroxycorticosterone concentrations). Elevation of plasma glucocorticoids is known to induce lymphoid necrosis in rats (Munck and Crabtree, 198 1). The current studies revealed that SOZF2 was nephrotoxic in rats, but not rabbits. Fluoride ion may be significant in the nephrotoxicity of S02F2; several studies have documented the renal toxicity of fluoride ion. Rochester strain rats treated with 8-45 mg/ kg NaF iv or ip developed necrosis, regeneration, and dilatation of tubules in the middle or inner third of the renal cortex (Taylor et al., 1961). Although papillary necrosis was not detected, the tubular changes were similar to those in the S02F2 studies as were decreased urinary specific gravity and increased sugar excretion. Chronic dietary administration of NaF (4- 15 mg/day) to hooded rats resulted in polydypsia, hyperglycemia, polyuria, glycosuria, and chronic nephrosis (Bond and Murray, 1952). Dilute urine with low specific gravity suggested that fluoride ion altered the function of collecting ducts. Fluoride nephrotoxicity in Fischer 344 rats resulted in vasopressin-resistant polyuric renal failure which resembled nephrogenic diabetes insipidus (Rush and Willis, 1982). Accumulation of fluoride in the medulla inhibited glycolysis and thus reduced energy stores needed for solute transport and production of cyclic adenosine monophosphate (CAMP). Significant distal nephron physiologic changes also have been reported with methoxyflurane which is metabolized to inorganic fluoride in humans and animals (Mazze et al., 1973). The nephrotoxicity induced by higher concentrations of SOzF2 in rats may have resulted from metabolic changes similar to those induced by NaF. Fluoride ion may have caused metabolic deficiencies in epithelial cells of the collecting ducts which resulted in compensatory hypertrophy and hyperplasia. Excessive exposure possibly then overwhelmed the compensatory response and resulted in degeneration and cell death. However, papillary necrosis can result from either
OF SULFURYL
FLUORIDE
555
direct toxic effects on collecting duct epithelial cells or papillary &hernia (Sabatini, 1984). Degeneration and necrosis of proximal tubules in rats exposed to 600 ppm S02F2 may have been a primary toxic effect on the epithelial cells although such changes can result from papillary necrosis (Murray et al., 1972) or &hernia (Dobyan et al., 1977). Daily inhalation exposure of rats and rabbits to S02F2 resulted in treatment-related neurotoxicity. Convulsions and hyperactivity were observed in some rabbits exposed to 600 ppm and focal malacia and/or vacuolation were identified in the cerebrum of rabbits exposed to 100,300, or 600 ppm S02F2. Neurotoxicity was not detected in rats during the 2-week study; however, the subsequent 13-week study revealed microscopic vacuolation in the brains of rats exposed to 300 ppm S02FZ. A parallel 13-week study revealed neurophysiologic changes in rats exposed to 100 or 300 ppm (Mattsson et al., 1988). However, auditory brainstem responses and brain histology were normal 2 months postexposure which indicated that the brain effects in rats were reversible. Although the mechanism of neurotoxicity is unknown, possibly S02F2 or fluoride ion has a direct effect on the metabolism of nervous tissue and the focal nature of the lesions may be related to regional differences in metabolism of the brain. Pulmonary edema and hemorrhage were present in some of the rats on the 2-week study that died after exposure to 600 ppm S02F2. Although these pulmonary effects may have been nonspecific terminal changes, similar pulmonary lesions have been reported previously in animals and humans that died after exposure to high concentrations of SOZFZ (Truhaut et al., 1973; Nitschke et al., 1986; Scheuerman, 1986; Nuckolls et al., 1987). Inflammation of the respiratory mucosa also was associated with exposure of rats and rabbits to the higher concentrations of S02F2 for 2 or 13 weeks. The inflammation was primarily in nasal tissues, but lower airways were affected in a few animals after exposure to 600 ppm for 2 weeks. Furthermore,
556
EISENBRANDT
rats treated for 13 weeks at 300 ppm had pulmonary alveolar histiocytosis. The respiratory inflammation associated with exposure to SOZF2 may be due to irritation although the mechanism is not known. The current studies have identified several differences in the toxicity of S02F2 following subchronic exposures as compared to acute exposure. Acute exposure of rats to high concentrations (2000 to 40,000 ppm) of S02F2 resulted in convulsions, pulmonary edema, respiratory arrest, and death (Truhaut et al., 1973; Nitschke et al., 1986). Exposure of rats to 600 ppm S02F2 in our 2-week study also was fatal; however, in contrast to the acute studies, death was attributed primarily to renal papillary necrosis. Renal toxicity was not present in rabbits and has not been reported in cases of human overexposure. Although acute overexposure of animals and humans to S02F2 resulted in pulmonary edema, the typical finding in animals that survived the 2week or 13-week exposure was inflammation in the respiratory system which suggests irritation. Both acute and subchronic exposure to SOZF2 are associated with neurotoxicity. Convulsions in rats (Nitschke et al., 1986) and seizures in humans (Nuckolls et al., 1987) have been reported with acute exposure to high concentrations of S02F,. The subchronic neurotoxicity of SO,F, at concentrations of 100-600 ppm was characterized by convulsions in rabbits, pathologic changes in the brain of rats and rabbits, and electrophysiologic alterations in rats (Mattsson et al., 1988). However, no neurologic or other effects have been detected in animals exposed to 30 ppm for 13 weeks. Similarly, examination of workers exposed to occupational levels of S02FZ has not demonstrated significant differences from controls on a broad range of psychological and neurological tests (Anger et al., 1986). Therefore, work practices of fumigators appear to be adequate to protect them from possible neurotoxic consequences of SO,F, overexposure.
AND NITSCHKE
ACKNOWLEDGMENTS The authors thank D. A. Dittenber, L. E. Frauson, J. E. Phillips, S. A. Pugh, C. M. Streeter, E. L. Wolfe, and M. A. Zimmer for their contributions to the studies.
REFERENCES ACGIH (1987). Threshold Limit Values for Chemical Substances in the Work Environment. American Conference of Governmental industrial Hygienists, Washington, DC. ANGER, W. K., MOODY, L., BURG, J., BRIGHTWELL, W. S., TAYLOR, B. J., Russo, J. M., DICKERSON, N., SETZER, J. V., JOHNSON, B. L., AND HICU, K. (1986). Neurobehavioral evaluation of soil and structural fumigators using methyl bromide and sulfuryl fluoride. Neurotoxicology 7, 137-I 56. BOND, A. M., AND MURRAY, M. M. (1952). Kidney function and structure in chronic fluorosis. Brit. J. Exp. Pathol. 33, 168- 176. DOBYAN, D. C., NAGLE, R. B., AND BUL,GER, R. E. (1977). Acute tubular necrosis in the rat kidney following sustained hypotension, physiologic and morphologic observations. Lab. Invest. 37,4 1I-422. DRILL, V. A. (1954). Pharmacology in Medicine. McGraw-Hill, New York. GOODMAN, A. G., GOODMAN, L. S., AND GILMAN, A. ( 1980). The Pharmacological Basis of Therapeutics, 6th ed. MacMillan, New York. HODGE, H. C., AND SMITH, F. A. (1965). Fluorine Chemistry (J. H. Simons, Ed.), Vol. 4, pp. 176-183. Academic Press, New York. MATTHEWS, J. (1970). Changes in cell function due to inorganic fluoride. In Handbook of Experimental Pharmacology, Pharmacology of Fluorides (F. A. Smith, Ed.), pp. 98-143. Springer-Verlag, New York. MA~SSON, J. L., ALBEE, R. R., EISENBRANDT, D. L., AND CHANG, L. W. (1988). Subchronic neurotoxicity in rats of the structural fumigant, sulfuryl fluoride. Neurotoxicof. Teratoi. 10, 127-l 33, MAZZE, R. I., COUSINS,M. J., AND KOSIK, J. C. (1973). Strain differences in metabolism and susceptibility to the nephrotoxic effects of methoxyflurane in rats. J. Pharmacol. Exp. Ther. 184,48 l-488. MUNCK, A., AND CRABTREE, G. R. (198 I). Glucocorticoid-induced lymphocyte death. In Cell Death in Biology and Pathology (I. D. Bowen and R. A. Lockshin, Eds.), pp. 329-359. Chapman & Hall, New York. MURRAY, G., WYLLIE, R. G., HILL, G. S., RAMSDEN, P. W., AND HEFTINSTALL, R. H. (1972). Experimental papillary necrosis of the kidney. Amer. J. Pathol. 67, 285-298. NITSCHKE, K. D., ALBEE, R. R., MAT-TX~ON, J. L., AND MILLER, R. R. (I 986). Incapacitation and treatment
INHALATION
TOXICITY
of rats exposed to a lethal dose of sulfuryl fluoride. Fundam. Appl. Toxicol. 7,664-610. NUCKOLLS, J. G., SMITH, D. C., OXLEY, D. W., HACKLER, R. L., TRIPATHI, R. K., A~STRONG, C. W., AND MILLER, G. B. (1987). Fatalities resulting from sulfuryl fluoride exposure alter home fumigation-Virginia. J. Amer. Med. Assoc. 258, 20412044. RUSH, G. F., AND WILLIS, L. P. (1982). Renal tubular effects of sodium fluoride. J. Pharmacol. Exp. Ther. 223,275-279. SABATINI, S. (1984). Pathophysiology of drug-induced papillary necrosis. Fundam. Appl. Toxicol. 4, 909921. SCHEUEFWAN, E. H. (1986). Suicide by exposure to sulfury1 fluoride. J. Foren. Sci. 31,1154-I 158. SUKETA, Y., ASAO, Y., KANAMOTO, Y., SAKASHITA, T., AND OKADA, S. (1985). Changes in adrenal function as a possible mechanism for elevation of serum glucose by a single large dose of fluoride. Toxicol. Appl. Pharmacol. 80,199-205.
OF SULFURYL
FLUORIDE
557
J. M., SCOTT, J. K., MAYNARD, E. A., SMYIITH, F. A., AND HODGE, H. C. ( 196 1). Toxic effects of fluoride on the rat kidney. Toxicol. Appl. Pharmacol. 3, 278-3 14. TRUHAUT, R., BOUDENE, Cl., AND CLUET, J.-L. (1973). Toxicite de quelques derives gazeux fluores et oxyfluores du soufre. Arch. Mal. Prof Med. Trav. Secur. Sot. 34,581-591. U.S. Environmental Protection Agency (1982). Pesticide Assessment Guidehnes, Subdivision F, Hazard EvaIuation: Human and Domestic Animals, pp. 98-100. U.S. Environmental Protection Agency, Washington, DC.
TAYLOR,
VERNOT, KEAD,
E. H., MACEWEN,
J. D., HAUN,
C. C., KIN-
E. R. (1977). Acute toxicity and skin corrosion data for some organic and inorganic compounds and aqueous solutions. Toxicol. Appl. Pharmacol. 42,4 17423. WISEMAN, A. (1970). Effect of inorganic fluoride on enzymes. In Handbook of Experimental Pharmacology, Pharmacology ofFluorides (F. A. Smith, Ed.), pp. 4897. Springer-Verlag, New York.
AND
APPLIED
TOXICOLOGY
I2,540-557
( 1989)
inhalation Toxicity of Sulfuryl Fluoride in Rats and Rabbits D. L. EISENBRANDT Mammalian
AND K. D. NITSCHKE
and Environmental Toxicology Research Laboratory, Health and Environmental Sciences, The Dow Chemical Company, Midland, Michigan 48674
Received June 2, 1988; accepted September 22, 1988 Inhalation Toxicity of Sulfuryl Fluoride in Rats and Rabbits. EISENBRANDT, D. L., AND K. D. (1989). Fundam. Appl. Toxicol. 12,540-557. The inhalation toxicity of the structural fumigant sulfuryl fluoride (S02Fz) was evaluated in rats and rabbits. Exposures for a preliminary 2-week study were 6 hr/day, 5 days/week, to 0, 100,300, or 600 ppm SO,F, . Nine of ten rats at 600 ppm died or were moribund between the second and sixth exposures. Extensive kidney lesions were present in all rats exposed to 600 ppm, whereas only minimal renal changes were noted in rats at 300 ppm. Upper and lower respiratory tissues were inflamed in the single rat that survived the 2-week exposure to 600 ppm. Rabbits exposed to 600 ppm S02F2 were hyperactive and one animal had a convulsion. Exposure to 300 or 600 ppm for 2 weeks resulted in vacuolation and/or malacia in the cerebrum of all rabbits and most of these rabbits also had moderate inflammation of nasal tissues; a few rabbits at 600 ppm had inflammation of the trachea or bronchi. A subsequent I3-week study evaluated rats and rabbits exposed to 0, 30, 100, or 300 ppm S02Fz (337 ppm TWA for rabbits). Rabbits initially were exposed to a high concentration of 600 ppm; however, convulsions were noted in two animals after nine exposures and the concentration subsequently was reduced to 300 ppm. Vacuolation and/or malacia were observed in the cerebrum of all rabbits at the highest concentration; one rabbit exposed to 100 ppm also had cerebral vacuolation. Rabbits at the highest concentration, as well as one rabbit exposed to 100 ppm, had inflammation of the nasal tissues. Rats exposed to 300 ppm SOzF2 for 13 weeks had mottled incisor teeth, minimal renal effects,pulmonary histiocytosis, inflammation of nasal tissues, and cerebral vacuolation. Also, rats exposed to 100 ppm SO,F, for 13 weeks had mottled teeth. Fluoride toxicity was suggested by mottled teeth in rats as well as elevation of serum fluoride levels in rats and rabbits exposed to SOzFz for 13 weeks. Although repeated exposure of rats and rabbits to 100-600 ppm SOzF2 resulted in toxicity of the kidneys (rats only), brain, and respiratory system, no effects were detected in animals exposed to 30 ppm for 13 NITSCHKE,
W&S.
Q 1989 Society of Toxicology.
Sulfuryl fluoride (S02F2; VIKANE)’ is a mmigant that controls drywood termites and other wood-destructive pests. The material typically is applied as a gas in sealed buildings and chloropicrin is introduced prefumigation into the structure as a vacating agent. Fumigation concentrations of S02F2 vary from approximately 1000 to 40,000 ppm and the concentration depends on the fumigation conditions and the target pest. An industrial hygiene study deter-
mined that short-term exposure of applicators to S02F2 varied from 0.9 to 8.7 ppm and the time-weighted-average (TWA) exposures were less than 5 ppm (Vaccaro, unpublished data, The Dow Chemical Co., 1988). The current threshold limit value (TLV) for occupational exposure is 5 ppm, with a short-term exposure limit (STEL) of 10 ppm (ACGIH, 1987). Human fatalities have been associated with accidental overexposure to S02F2 or attributed to suicide (Scheuexman, 1986; Nuckolls et al., 1987).
’ VIRANE gas fumigant, a trademark of The Dow Chemical Company. 0272-0590/89 $3.00 Copyright 0 1989 by the Society ofToxicology. All rights of reproduction in any form reserved.
540
INHALATION
TOXICITY
The LC50 (95% confidence interval) for a 1-hr exposure to S02F2 was 3730 ppm (309045 10 ppm) for male rats and 3020 ppm (2830-3220 ppm) for female rats (Vemot et al., 1977). Truhaut et al., (1973) found that mice were more sensitive than rats to the lethal effects of S0,F2, whereas rabbits were less sensitive. Death was attributed to asphyxiation that was associated with tonic convulsions and blockade of respiratory muscles. Animals that died after exposure to 6000 to 10,000 ppm S02F2 had pulmonary congestion and alveolar hemorrhage. Lesions were not detected in animals that died after exposure to lower concentrations. Concentrations of 4000 ppm S02Fz incapacitated rats within 45 min and resulted in death within several hours of exposure (Nitschke et al., 1986). Exposure to lO,OOO40,000 ppm decreased the time to incapacitation and death occurred within minutes. Nearly all rats convulsed during or after exposure to S02F2 and rats exposed to higher concentrations were cyanotic. Respiration typically ceased for 15-30 set after a convulsion and several rats died immediately after a convulsion. Serum fluoride levels were elevated in these animals and the toxicity of SOzF2 was attributed, at least in part, to fluoride. The current studies were designed to evaluate the subchronic inhalation toxicity of S02Fz in rats and rabbits. MATERIALS
AND
METHODS
Experimental design. The 2.week study consisted of groups of 5 rats/sex and 3 rabbits/sex that were scheduled for exposure to 0, 100,300, or 600 ppm SO,F, for 6 hr/ day, 5 days/week, for nine exposures. Subsequently, groups of 10 rats/sex were scheduled for exposure to 0, 30,100, or 300 ppm SOIF, for 6 hr/day, 5 days/week, for 13 weeks and groups of 7 rabbits/sex were scheduled for exposure to 0, 30, 100, or 600 ppm S02Fz for 6 hr/day, 5 days/week, for 13 weeks. However, clinical effects in rabbits exposed to 600 ppm S02F2 required that the concentration be reduced to 300 ppm after the ninth exposure. Parameters evaluated were clinical observations, body weight, hematology, clinical chemistry, urinalysis, gross pathology, and microscopic pathology.
OF SULFURYL
FLUORIDE
541
Test material. Sulfuryl fluoride, a colorless, odorless gas, was obtained from the Agricultural Products Department, Dow Chemical U.S.A. Analysis ofthe test material (Lot No. TWP 830919408) revealed the sample to be 99.8% SOZF2. Exposure system. Animals were exposed to SOzF2 in 4. l-m3 chambers constructed of stainless steel and glass. Control animals were placed in a chamber of similar design that provided clean, filtered air. Chamber airtlow was maintained at approximately 800 liters/min. SO,F* was metered at a controlled rate into the main-chamber airstream where the gas was diluted to the desired concentration before the airstream entered the chamber. The test atmosphere for the 2-week study was generated by metering SOIF, directly from cylinders into the airstream via flowmeters. The test atmosphere for the 13week study was generated by metering S02F2 from SARAN* bags with an RPG pump (Fluid Metering Inc., Oyster Bay, NY). The air supplied to the chambers was controlled by a system designed to maintain approximately 22°C and 50% relative humidity. A minimummaximum thermometer and a hygrometer were placed in each chamber and values were recorded at the end of each exposure period. The concentration of S02F2 in each exposure chamber was determined once or twice per hour with a MIRAN 1A infrared spectrophotometer (Foxboro/Wilks, South Norwalk, CT) at a wavelength of 11.8 Wm. The infrared spectrophotometer was calibrated with standards made by diluting measured volumes of S02F2 with a measured volume of filtered compressed air. The analytical system was evaluated with at least one standard prior to each day of exposure. The distribution of SOzF2 gas within each chamber was measured from six to eight sample points within the breathing zone and varied by less than 0.5%. Animals. Male and female Fischer 344 rats (6-7 weeks of age, Charles River Breeding Laboratories, Kingston, NY) and male and female New Zealand White rabbits (4-5 months of age, Hazelton Dutchland, Inc., Denver, PA) were acclimated for at least 7 and 14 days, respectively, prior to the initial exposures. Animals were assigned randomly by weight to exposure groups. Rabbits were negative for Encephalitozoon cuniculi titers. Animals on the 2-week study and rats on the 13-week study were fed Certified Purina Chow (Ralston Purina Co., St. Louis, MO) and water ad libitum except during exposures. Rabbits on the 13-week study were fed a maintenance diet (8 oz/day) of the same feed rather than ad libitum. Prior to the study, as well as the 18 hr/day and weekends when animals were not in exposure chambers, the animals were housed in rooms that controlled temperature at 21°C relative humidity at 50%, and a light-dark cycle at 12 hr.
’ SARAN, a trademark of The Dow Chemical Company.
542
EISENBRANDT
Clinical observations. The animals were observed after each exposure and changes in appearance or behavior were noted. Additional daily observations were conducted prior to each exposure and on weekends. Animals were weighed prior to the first, fifth, sixth, and ninth exposures for the 2-week study and weekly for the 13-week study. Clinicalpathology. Blood samples were collected from all animals that survived until study termination. Samples were collected from the posterior orbital sinus of rats and from a marginal ear vein of rabbits just prior to necropsy. Hematologic parameters included hematocrit, hemoglobin, erythrocyte count, red blood cell indices, total leukocyte counts, and platelet counts (ELT-8, Ortho Instruments, Boston, MA). Differential leukocyte counts were evaluated with an ACS-1000 (Honeywell, Inc., Denver, CO). Blood smears also were examined qualitatively. Urine was collected from all rats that survived until study termination. Urine was collected prior to the ninth exposure for the 2-week study and alter 11 weeks of exposure for the 13-week study. Urinary parameters included bilirubin, glucose, ketones, blood, pH, protein, urobilinogen (Chemstrip 7, Biodynamics/Division of BMC, Indianapolis, IN), and specific gravity (American Optical Co., Keene, NH). Urinalysis was not performed on rabbits. Serum samples for clinical chemistry were collected at necropsy from all animals that survived until study termination. Parameters evaluated were urea nitrogen, alanine aminotransferase activity, aspartate aminotransferase activity, alkaline phosphatase activity, glucose, total protein, albumin, and globulin (Centrifichem System 500, Baker Instruments Corp., Allentown, PA). Additional parameters evaluated only for animals on the 13week study included calcium, phosphorus, creatinine, and fluoride. The fluoride was measured with a fluoridespecific electrode (Orion Research, Cambridge, MA). Anatomic pathology. Animals that survived the exposures were necropsied the day after the last exposure to S02F2. Rats were fasted overnight prior to the scheduled necropsy; rabbits were not fasted. Rats on the 2-week study were anesthetized with methoxytlurane, whereas rats on the 13-week study and all rabbits were anesthetized with carbon dioxide; the animals then were euthanized by decapitation. Terminal body weights and weights ofbrain, heart, liver, kidneys, thymus (rats only), and tes tes were recorded. All animals were examined for gross pathological alterations by a veterinary pathologist. Animals that died or were moribund prior to the scheduled sacrifice were necropsied as soon as possible. An extensive set of tissues was collected from each animal and preserved in neutral, phosphate-buffered 10% formalin. The lungs were infused with buffered formalin to their approximate, normal inspiratory volume and the nasal cavity was flushed with formalin via the pharyngeal duct to ensure rapid fixation of tissues.
AND NITSCHKE Histopathologic examination was completed on an extensive set of tissues for all animals in the control and high-concentration groups. The set of tissues was similar to those listed in guidelines of the U.S. Environmental Protection Agency (1982). The same tissues were examined for rats in the 300 ppm exposure group on the 2-week study because of deaths in the 600 ppm group. Histopathologic examination of animals in other intermediate exposure groups from the 2-week study and all intermediate exposure groups from the 13-week study was less extensive, but included as a minimum kidneys, nasal tissues, trachea, lungs, and oral tissues; also, brain sections were examined for all intermediate groups except the 2-week, 100 ppm rats. Tissues were processed for light microscopy by conventional techniques and stained with hematoxylin and eosin. Special stains for animals from the 2-week study included von Kossa’s stain of lung, kidneys, heart, and stomach as well as phosphotungstic acid-hematoxylin stain of lung and/or kidneys from selected rats in the high-concentration group. Brain sections from selected rabbits on the 2-week study as well as the brain sections from selected rats and rabbits on the 13-week study were evaluated with luxol-fast blueperiodic acid-Schiff-hematoxylin and Sevier-Munger silver stains. Statistical evaluation. Body weights, organ weights, organ weights relative to terminal body weights, selected hematology data (excluding red blood cell indices and differential white blood cell counts), clinical chemistry data, and urinary specific gravity were evaluated by Bartlett’s test for equality of variances. Based on the outcome ofBartlett’s test, exploratory data analysis was performed by a parametric or nonparametric analysis of variance (ANOVA), followed respectively by Dunnett’s test or the Wilcoxon rank-sum test with a Bonferroni correction for multiple comparisons. Because of effectsidentified in the 2-week study, Dunnett tests, and Wilcoxon rank-sum tests for the heart and kidney weights, blood urea nitrogen and total white blood cell counts for rats in the 13week study were evaluated with one-sided tests. Since multiple, interrelated parameters were compared statistically in the same group of animals, the frequency of false-positive errors may have been much greater than the nominal 01levels. Thus, the final toxicologic interpretation of the data also considered whether an orderly dose-response relationship existed and whether the findings appeared to be plausible and consistent with other observations.
RESULTS Two- Week Study Rats and rabbits were exposed to TWA concentrations (mean f SD) of 100 + 6,293
INHALATION
FIG.
TOXICITY
OF SULFURYL
FLUORIDE
543
1. Papillary necrosis in a male rat that died after exposure to 600 ppm SO,F, for 1 week. X50.
-C 17, and 597 f 13 ppm for target concentrations of 100,300, and 600 ppm, respectively. The chamber temperature and relative humidity were comparable for animals exposed to 0,100,300, and 600 ppm SOzF2.
Rats Nine of ten rats exposed to 600 ppm SOzF2 died or became moribund between the second and sixth exposures; one female rat survived until termination. Rats at this exposure concentration were lethargic after the second
exposure and were less active after each subsequent exposure. Convulsions or other neurologic signs were not observed in these animals. These animals also consumed less food after the second and subsequent exposures and body weights were decreased significantly. One female that survived all nine exposures to 600 ppm SOzF2 weighed only 80.3 g as compared to controls that averaged 129.9 g. Rats exposed to 0,100, or 300 ppm S02F2 appeared normal throughout the exposure. Renal effectsat 600 ppm. Rats that died after several exposures to 600 ppm S02F, had severe kidney lesions that included papillary
544
EISENBRANDT
AND NITSCHKE
FIG. 2. Degeneration, necrosis, and regeneration of collecting ducts (C) in the inner medulla of a male rat that died after exposure to 600 ppm SO,F, for 1 week. Interstitial inflammation is associated with the tubular lesion. X 199.
necrosis (Fig. 1). The remainder of the papillary epithelium was moderately hyperplastic. There was a variable amount of subacute inflammation associated with the necrosis, and collecting ducts were dilated as a result of obstruction at the papillae (Fig. 2). Epithelial cells in the descending portion of proximal tubules had decreased amounts of basophilic cytoplasm as well as patchy segments of epithelial cell necrosis and regeneration. Convoluted tubules in the cortex had minimal changes that included an occasional degenerate cell and variable regenerative activity.
The female rat that survived 2 weeks at 600 ppm SOzF2 had moderate microscopic changes in collecting ducts, but no papillary necrosis (Fig. 3). This female also had hyperplasia of the renal papillary epithelium, and the epithelial cells of the descending portion of the proximal tubules were basophilic. Relative kidney weight was increased significantly in this rat (Table 1) and renal function was compromised. Urea nitrogen was elevated significantly (2 13 mg/dl), whereas urinalysis revealed a pH of 5 .O and a 3+ glucose; the glucosuria was secondary to hyperglyce-
INHALATION
TOXICITY
OF SULFURYL
FLUORIDE
545
FIG. 3. Inner medulla of female rat after exposure to 600 ppm S02F2 for 2 weeks. Collecting ducts are prominent due to regeneration (hyperplasia) and degeneration. Chronic interstitial inflammation also is present. X 199.
mia (1483 mg/dl). On the other hand, the specific gravity of the urine (1.020) was within a normal range. Renal e$ets at 300ppm. Five of the ten rats (both male and female) exposed to 300 ppm S02F2 for 2 weeks had minimal hyperplasia of the collecting ducts in the inner renal medulla. Two of these rats also had slight hyperplasia of the papillary epithelium. In addition, three of these rats had slightly basophilic epithelial cells in the descending part of the proximal tubules. Kidney weights were increased about 7% in animals from the 300 ppm exposure
group (Table 1). Urinalysis data and urea nitrogen values were normal in these rats. Otherjindings. Treatment-related effects in the respiratory system were present in the female that survived the 2-week exposure to 600 ppm S02F2. Severe, diffuse inflammation and multifocal ulceration of the nasal mucosa in this rat were accompanied by slight bronchioalveolar inflammation in the lungs. Numerous bacteria were associated with the inflammation in the respiratory system. Myeloid hyperplasia in the bone marrow of this female was attributed to inflam-
546
EISENBRANDT
AND TABLE
NITSCHKE 1
SELEC~EDABSOLUTEANDRELATIVEORGANWEIGHTDATAFORRATSEXPOSED TO SULFVRYL FLUORIDE FOR 2 WEEKS Concentration (mm)
N”
Terminal body weight (id
Kidneys
Heart g
g/l~g
Thymus
g
g/loog
I3
g/l@-)g
Males 0
5
173.76 I!? 9.7
0.617 +- 0.029
0.355 + 0.008
1.452 kO.141
0.834 t 0.048
0.327 I!Z0.018
0.189 +0.015
100
5
167.7 f 6.2
0.637 -t 0.036
0.380* + 0.015
1.443 + 0.076
0.861 + 0.041
0.322 + 0.032
0.192 + 0.019
300
5
172.3 + 6.7
0.614 + 0.016
0.356 + 0.008
1.545 + 0.105
0.896 k 0.041
0.299 + 0.02 1
0.174 -t 0.014
600
0
-c
-
-
-
-
0
5
112.2 k4.7
0.459 + 0.028
1.071 + 0.063
0.955 ?I 0.059
0.322 r 0.027
0.287 20.015
100
5
109.4 + 7.9
0.467 2 0.034
0.427 + 0.017
1.123 * 0.070
1.028 +- 0.065
0.287 -t 0.039
0.262 + 0.023
300
5
107.6 f 5.0
0.471 + 0.034
0.438* + 0.018
1.155 t- 0.056
1.075* f 0.070
0.260* + 0.033
0.241* f 0.027
600
1
67.6*
1.422*
0.031*
0.046*
0.313*
0.463*
0.96 1
a Number of animals. ’ Values are means + SD. ’ -, No data because of dead animals. * Statistically identified difference from control mean by Dunnett’s test, a = 0.05.
mation of the respiratory system as was an elevation in total white blood cell count (Table 2) and neutrophilia (42%). This female also had alterations in organ weights and hematologic and serum chemical parameters that were attributed to severe debilitation, body weight loss, and dehydration. Relative heart weights of females exposed to 300 or 600 ppm SOzF, slightly exceeded historical control values, although the increase for the 600 ppm female reflected primarily decreased body weight (Table 1). Total white blood cell counts were increased slightly in some rats exposed to SOzFz (Table 2); however, only males exposed to 300 ppm and the female exposed to 600 ppm exceeded
historical control values. Other than the female at the highest concentration, correlative findings were not present and reasons for the elevated white blood cell counts and heart weights were not apparent. Four of nine rats that died after exposure to 600 ppm S02F2 had pulmonary edema and/or hemorrhage or fibrin within alveoli. Also, thrombi were present in pulmonary capillaries of three rats. Other histopathologic observations in rats exposed to 600 ppm were considered secondary to renal disease or nonspecific terminal changes. Such observations included visceral congestion, stomach erosions, metastatic mineralization, hypertrophy of the adrenal cortex, as well as necrosis
INHALATION
TOXICITY
TABLE 2 TOTAL WHITE BLOOD CELL COUNTS OF RATS EXPOSED TOSULFURYLFLUORIDEFOR ~WEEKS Concentration (mm) 0 100
Na
Males
515
6.16 Ik 0.9
515
1.4
It 0.4 300
515
8.8*
t- 0.8 600
O/l
-c
Females 5.3
kO.8 6.6*
+ 0.6 1.9*
k 0.7 11.6*
0 Number of animals of each sex (M/F). b Values are mean total white blood cell counts (X 103/ mm3) f SD. ’ -, No data because of dead animals. * Statistically identified difference from control mean by Dunnett’s test. a = 0.05.
and depletion of lymphoid tissue in the thymus, spleen, and lymph nodes. Related decreases in thymic weights were most noticeable in female rats at the higher exposure concentrations (Table 2).
Rabbits One female rabbit exposed to 600 ppm SOzF2 had a convulsion after the fifth exposure; the convulsion resulted in a fractured tibia. Another female exposed to 600 ppm had a fractured vertebra after the sixth exposure, although convulsions were not observed. Both rabbits with fractures were euthanized and the other 600 ppm female survived until termination. Male and female rabbits that survived exposure to 600 ppm were slightly hyperactive compared to controls. Rabbits exposed to 0, 100, or 300 ppm appeared normal throughout the exposure period. There were no significant differences for in-life body weights. Nervous system eficts. Vacuolation was present in the brain of all rabbits that were
OF SULFURYL
FLUORIDE
547
exposed to 300 or 600 ppm SOzF2 (Fig. 4). Lesions were restricted to the globus pallidus and putamen (basal nuclei) as well as the external and internal capsules (myelinated tracts). Malacia was present in the cerebrum of all rabbits exposed to 600 ppm as well as one male and one female exposed to 300 ppm (Fig. 5 and 6). Reactive gliosis and demyelination accompanied the malacia. Respiratory systemeficts. Most rabbits exposed to 300 or 600 ppm SOzF2 had moderate, subacute to chronic inflammation of nasal mucosa with mucopurulent exudate in the nasal cavities. The trachea of some rabbits in the 600 ppm exposure group had minimal acute inflammation. In addition, the single female rabbit that survived the 2-week exposure at 600 ppm had acute inflammation of the bronchi and bronchioles. Other Jindings. Terminal body weights of some rabbits exposed to 300 or 600 ppm SOzF2 were decreased slightly. Some of these rabbits also had decreased liver weights at termination. The weight changes were attributed to altered nutritional status as were altered cytoplasmic homogeneity of liver cells and decreased serum albumin levels in some rabbits. A variable hematopoietic response was associated with the inflammation in the respiratory system of some rabbits. Alterations included lymphoid hyperplasia in the mediastinal lymph nodes and spleen.
Thirteen- WeekStudy Rats were exposed to TWA concentrations (mean f SD) of 29.8 -t 1.5, 100 a 5, or 297 + 12 ppm for target concentrations of 30, 100, or 300 ppm SOzF2, respectively. Rabbits were exposed to concentrations of 29.8 + 1.5, 100 + 5, or 337 st 98 ppm for target concentrations of 30, 100, or 300 ppm S02F2, respectively. The higher analytical concentration for rabbits (337 ppm) was due to nine exposures at 600 ppm at the beginning of the study. The chamber temperature and relative humidity were comparable for animals in control and treated exposure groups.
548
EISENBRANDT
AND NITSCHKE
FIG. 4. Vacuolation in myelinated tracts (arrows) and neuropil (arrowheads) in the cerebrum of a female rabbit exposed to 300 ppm SOzFz for 2 weeks. X64.
FIG. 5. Malacia and gliosis in the cerebrum of a male rabbit exposed to 600 ppm SOzFz for 2 weeks. The external capsule (arrowhead) and other myelinated tracts (arrows) have decreased myelin, vacuolation, and proliferation of oligodendrogha. X64.
INHALATION
TOXICITY
OF SULFURYL
FLUORIDE
549
FIG. 6. Malacia in the external capsule (EC) of a female rabbit exposed to 600 ppm S02F, for 2 wf :eks. Pyknotic nuclei (arrows) and glial proliferation are present. X 158.
Rats Exposure to 300 ppm SOzF* decreased the body weight of male rats after 45 days on test and females after 24 days on test (Fig. 7). Also, rats exposed to either 100 or 300 ppm S02Fz had mottled upper and lower incisor teeth. Nervous systemeffects.The brain of all rats exposed to 300 ppm SO,F, for 13 weeks had minimal microscopic vacuolation (Fig. 8). The vacuolation was restricted to the area of the caudate-putamen nuclei and was more prominent in the white fiber tracts of the internal capsule than in the adjacent neuropil. Special stains did not characterize the vacuoles or reveal any additional effects. Respiratory system eficts. Multiple, pale foci were present on the pleural surfaces of the lungs of most rats exposed to 300 ppm. Histopathologic examination revealed that the pale foci corresponded to subpleural histiocytosis (Fig. 9). Furthermore, there was minimal subacute inflammation of the nasal
mucosa in all female and most male rats exposed to 300 ppm S02F2. A few males had more severe inflammation of the respiratory and olfactory mucosa, which was associated with mucopurulent exudate in the nasal passages as well as degeneration and reactive changes in the mucosa (Fig. 10). Renal eficts. Kidneys of male rats exposed to 300 ppm SO2FZ had a slight decrease in microscopic protein droplets (a,,,-globulin) which normally are present in the convoluted tubules; the change was considered secondary to a decrease in body weight. Most female rats exposed to 300 ppm S02FZ had very slight hyperplasia of the renal collecting ducts which was most apparent in the outer portion of the inner zone of the medulla. Urinary specific gravity was decreased in male and female rats exposed to 300 ppm S02FZ for 13 weeks, although other urinary parameters were not affected (Table 3). Otherfindings. Rats exposed to the highest concentration of S02F2 had slight elevations of serum fluoride levels; however, the differ-
550
EISENBRANDT
AND NITSCHKE EEbML’= RATS
350-I
0
FIG.
20
40
60
DAYS
ON TEST
80
100
0
20
40
60
DAYS
ON TEST
80
100
7. Body weights of male and female rats; 13-week inhalation study of S02F2.
ences from control values were not statistitally significant (Table 4). There were no consistent toxicologic effects of treatment on hematology or clinical chemistry parameters. Many of the absolute and relative organ
weights of rats exposed to 300 ppm S02F2 were statistically identified as different from control values; however, the changes were associated with decreased body weights in these animals.
FIG. 8. Vacuolation in the fibers of the internal capsule and the caudate-putamen nuclei of a rat exposed to 300 ppm S02Fz for I 3 weeks. X 19 I.
INHALATION
TOXICITY
OF SULFURYL
FLUORIDE
FIG. 9. Subpleural histiocytosis in lungs of a rat exposed to 300 ppm S02F2 for 13 weeks. X 19 1.
FIG. 10. Nasal tissue from a rat exposed to 300 ppm S02Fz for 13 weeks. A few males had severe, diffuse inflammation of the nasal mucosa. Note the inflammation ofthe olfactory mucosa (arrowhead) with mucopundent exudate in the nasal passages (arrows). X 19 1.
551
552
EISENBRANDT TABLE 3
URINARYSPECIRCGRAVITYOFRATSEXPOSED TOSULFLJRYLFLUORIDEFORI 3 WEEKS Concentration (wm)
N”
Males
0
IO/10
1.063’ f 0.007
1.049 * 0.015
30
IO/IO
1.061 f 0.010
1.049 f 0.007
100
lO/lO
1.062 + 0.006
1.050 + 0.017
300
1019
1.050* kO.013
1.040 kO.014
Females
’ Number of animals of each sex (M/F). b Values are means + SD. * Statistically identified difference from control mean by Dunnett’s test, a = 0.05.
Rabbits Convulsions were observed in one male and one female rabbit following the ninth exposure to the initial concentration of 600 ppm S02F2. Head bobbing was observed prior to and after a tonic convulsion by the male. Deep breathing, salivation, and rapid eye blinking accompanied convulsions and head bobbing in the female. A second female rabbit was euthanized atier the eighth exposure to 600 ppm because of posterior paralysis which was attributed to a fractured vertebra. As a consequence of the clinical effects noted at 600 ppm, the highest exposure concentration was reduced to 300 ppm for the remainder of the 13-week study. There were no clinical signs in rabbits exposed to 300 ppm for the subsequent 11 weeks and no clinical signs were detected in rabbits exposed to 30 or 100 ppm SO,F, for the entire 13-week study. Visual inspection revealed decreased food consumption for rabbits exposed to the highest concentration of S02F2 ; the decrease was noted after the ninth exposure and continued for approximately 4 weeks. Body weights
AND NITSCHKE
(Fig. 11) of males exposed to the highest concentration of S0,F2 were decreased significantly from Days 11 to 60 and remained below control values throughout the study. Body weights of females at the highest concentration also were decreased consistently. Minimal decreases in body weights were recorded for both sexes exposed to 100 ppm. Nervous systemeficts. Lesions in the brain of rabbits on the 13-week study were similar to those in the 2-week study. Microscopic changes were present in the area of the putamen, globus pallidus, internal capsule, and external capsule of the cerebrum. Three males and one female exposed to 337 ppm S02F2 had severe malacia, whereas only slight vacuolation was present in three male and five female rabbits. The vacuolation in some females was accompanied by gliosis and endothelial cell hypertrophy. A single female rabbit in the 100 ppm concentration group had moderate vacuolation of the cerebrum. Respiratory system eficts. Treatment-related microscopic changes were observed in the nasal tissues of all male and female rabbits exposed to 337 ppm S02Fz and one male exposed to 100 ppm. The changes consisted of variable degrees of inflammation in the nasal mucosa and purulent exudate in the nasal passages. Hypertrophy and hyperplasia of the respiratory epithelium (goblet cells and pseudostratified epithelial cells) were observed primarily on the nasal turbinates. Slight to moderate degeneration of olfactory epithelium was associated with the inflammation in two males and three females exposed to 337 ppm SOzF2. Other jindings. Serum fluoride levels of male and female rabbits exposed to 30, 100, or 337 ppm SO,F, were significantly increased over control values (Table 4). Total white blood cell counts for male rabbits exposed to 337 ppm S02F2 (9900 f 2400/mm3) were significantly increased over controls (7600 + 1000/mm3), although differential white blood cell counts were normal. Other hematologic parameters were unaffected by
INHALATION
TOXICITY
OF SULFURYL
553
FLUORIDE
TABLE 4 SERUM FLUORIDE LEVELS IN RATS AND RABBITS EXPOSED TO SULFURYL FLUORIDE
FOR 13 WEEKS Rabbits
Rats Concentration (mm)
Males
Females
Males
Females
0
Mean” SD N
0.996 r 0.788 9
0.607 k 0.449 10
0.065’ + 0.014 7
0.560 + 0.026 7
30
Mean SD N
0.715 k 0.288 10
0.738 f 0.630 10
0.170t f 0.069 7
0.697* + 0.070 7
100
Mean SD N
0.881 t 0.66 1 10
0.575 + 0.198 10
0.408t t 0.035 7
0.829* + 0.088 7
3001337
Mean SD N
1.154 f 0.574 10
1.366 AZ0.959 10
0.62lt -+ 0.060 7
1.003* + 0.045 6
n Values are mean fluoride levels (&ml) f SD for the indicated number of animals (N). ‘Six of the control male rabbits and one of the 30 ppm male rabbits had no fluoride detected (values less than the limit of 0.06 &ml). Therefore, the detection limit of 0.06 &ml was used to calculate the mean and SD for these animals as well as for statistical comparisons. * Statistically identified difference from control mean by Dunnett’s test, OL= 0.05. t Statistically identified difference from control mean by Wilcoxon’s test, a = 0.05.
treatment. Slight decreases in total serum protein, albumin, and globulin in males exposed to 30 or 337 ppm (but not males exposed to 100 ppm) were of questionable toxicologic significance, although the decreases at the higher concentration may have been associated with the lower nutritional status of the animals. Other clinical chemistry parameters for treated animals were normal. Liver weights of female rabbits exposed to 337 ppm SO,F, were significantly decreased as compared to controls (Table 5). Liver weights of males at 337 ppm as well as liver weights for both sexes at 100 ppm also were decreased somewhat. The decrease in liver weight values was most likely secondary to decreased food consumption and body weight. DISCUSSION Clinical observations in animals exposed to high concentrations of S02F2 (Truhaut d
al., 1973; Nitschke et al., 1986) are similar to those of acute fluoride toxicity (Drill, 1954; Taylor et al., 1961; Goodman et al., 1980). Elevation of serum fluoride levels in rats exposed to 4000 or 10,000 ppm S02F2 also suggests that fluoride is important in the acute toxicity of the compound (Nitschke et al., 1986). In addition, elevation of plasma fluoride levels has been reported in human deaths that were attributed to suicide or accidental overexposure to S02F2 (Scheuerman, 1986; Nuckolls et al., 1987). Fluoride is a potent inhibitor of intermediary metabolism in mammals and particularly inhibits enzymes involved with energy metabolism (Hodge and Smith, 1965; Matthews, 1970; Wiseman, 1970). Elevation of serum fluoride levels in rats and rabbits exposed to S02F2 for 13 weeks in the current studies suggests that fluoride is important in the toxicity of the compound. Mottled incisor teeth in rats exposed to 100
554
EISENBRANDT
AND NITSCHKE
MA1 F RABBITS 4600 4400 4200 -
4200
4000 -
4000
3800 -
3800
3600 -
3600
3400
3400
3200
3200
z 2 0
0
20
40
DAYS
60
50
0
100
20
ON TEST
337PPM
40
60
DAYS
ON TEST
80
100
FIG. 11. Body weights of male and female rabbits; 13-week inhalation study of SO,?F,.
or 300 ppm S02F2 for 13 weeks are indicative of enamel defects in developing teeth and are compatible with dental fluorosis (Drill, 1954; Taylor et al., 196 1). Fluoride ion as well as stress may have contributed to several indirect effects in rats exposed to higher concentrations of SO,F, in our 2-week study. Hypertrophy of the adre-
nal cortex, hyperglycemia, and necrosis of lymphoid tissue were observed in rats exposed to 600 ppm SOzF,. Suketa et al. (1985) have reported that single doses (35 mg/kg, ip) of sodium fluoride (NaF) elevate serum glucase in rats. Also, fluoride enhances glucose6-phosphatase activity in the liver and kidney and stimulates adrenocortical function (ele-
TABLE 5 ABSOLUTE
AND RELATIVE
LIVER WEIGHTS
FOR RABBITS EXPOSED TO SULFURYL FLUORIDE MR 13 WEEKS Males
Concentration @pm)
g
Females g/I~g
is
idl~g
0
Mean“ SD N
130.3 f 19.5 7
3.141 + 0.354 7
143.4 k 25.3 7
3.153 + 0.533 7
30
Mean SD N
129.3 _t 22.3 7
3.128 f 0.466 I
137.7 f 19.7 7
3.000 + 0.430 7
100
Mean SD N
99.4* -c 22.5 7
2.598 + 0.601 7
113.6 * 33.1 7
2.565 k 0.63 I 7
337
Mean SD N
110.9 + 18.9
2.871 2 0.405
7
7
103.6* k 21.9 6
2.399* + 0.435 6
DValues are means + SD for the indicated number of animals (NJ. * Statistically identified difference from control mean by Dunnett’s test, a = 0.05.
INHALATION
TOXICITY
vation of serum and urinary 17-hydroxycorticosterone concentrations). Elevation of plasma glucocorticoids is known to induce lymphoid necrosis in rats (Munck and Crabtree, 198 1). The current studies revealed that SOZF2 was nephrotoxic in rats, but not rabbits. Fluoride ion may be significant in the nephrotoxicity of S02F2; several studies have documented the renal toxicity of fluoride ion. Rochester strain rats treated with 8-45 mg/ kg NaF iv or ip developed necrosis, regeneration, and dilatation of tubules in the middle or inner third of the renal cortex (Taylor et al., 1961). Although papillary necrosis was not detected, the tubular changes were similar to those in the S02F2 studies as were decreased urinary specific gravity and increased sugar excretion. Chronic dietary administration of NaF (4- 15 mg/day) to hooded rats resulted in polydypsia, hyperglycemia, polyuria, glycosuria, and chronic nephrosis (Bond and Murray, 1952). Dilute urine with low specific gravity suggested that fluoride ion altered the function of collecting ducts. Fluoride nephrotoxicity in Fischer 344 rats resulted in vasopressin-resistant polyuric renal failure which resembled nephrogenic diabetes insipidus (Rush and Willis, 1982). Accumulation of fluoride in the medulla inhibited glycolysis and thus reduced energy stores needed for solute transport and production of cyclic adenosine monophosphate (CAMP). Significant distal nephron physiologic changes also have been reported with methoxyflurane which is metabolized to inorganic fluoride in humans and animals (Mazze et al., 1973). The nephrotoxicity induced by higher concentrations of SOzF2 in rats may have resulted from metabolic changes similar to those induced by NaF. Fluoride ion may have caused metabolic deficiencies in epithelial cells of the collecting ducts which resulted in compensatory hypertrophy and hyperplasia. Excessive exposure possibly then overwhelmed the compensatory response and resulted in degeneration and cell death. However, papillary necrosis can result from either
OF SULFURYL
FLUORIDE
555
direct toxic effects on collecting duct epithelial cells or papillary &hernia (Sabatini, 1984). Degeneration and necrosis of proximal tubules in rats exposed to 600 ppm S02F2 may have been a primary toxic effect on the epithelial cells although such changes can result from papillary necrosis (Murray et al., 1972) or &hernia (Dobyan et al., 1977). Daily inhalation exposure of rats and rabbits to S02F2 resulted in treatment-related neurotoxicity. Convulsions and hyperactivity were observed in some rabbits exposed to 600 ppm and focal malacia and/or vacuolation were identified in the cerebrum of rabbits exposed to 100,300, or 600 ppm S02F2. Neurotoxicity was not detected in rats during the 2-week study; however, the subsequent 13-week study revealed microscopic vacuolation in the brains of rats exposed to 300 ppm S02FZ. A parallel 13-week study revealed neurophysiologic changes in rats exposed to 100 or 300 ppm (Mattsson et al., 1988). However, auditory brainstem responses and brain histology were normal 2 months postexposure which indicated that the brain effects in rats were reversible. Although the mechanism of neurotoxicity is unknown, possibly S02F2 or fluoride ion has a direct effect on the metabolism of nervous tissue and the focal nature of the lesions may be related to regional differences in metabolism of the brain. Pulmonary edema and hemorrhage were present in some of the rats on the 2-week study that died after exposure to 600 ppm S02F2. Although these pulmonary effects may have been nonspecific terminal changes, similar pulmonary lesions have been reported previously in animals and humans that died after exposure to high concentrations of SOZFZ (Truhaut et al., 1973; Nitschke et al., 1986; Scheuerman, 1986; Nuckolls et al., 1987). Inflammation of the respiratory mucosa also was associated with exposure of rats and rabbits to the higher concentrations of S02F2 for 2 or 13 weeks. The inflammation was primarily in nasal tissues, but lower airways were affected in a few animals after exposure to 600 ppm for 2 weeks. Furthermore,
556
EISENBRANDT
rats treated for 13 weeks at 300 ppm had pulmonary alveolar histiocytosis. The respiratory inflammation associated with exposure to SOZF2 may be due to irritation although the mechanism is not known. The current studies have identified several differences in the toxicity of S02F2 following subchronic exposures as compared to acute exposure. Acute exposure of rats to high concentrations (2000 to 40,000 ppm) of S02F2 resulted in convulsions, pulmonary edema, respiratory arrest, and death (Truhaut et al., 1973; Nitschke et al., 1986). Exposure of rats to 600 ppm S02F2 in our 2-week study also was fatal; however, in contrast to the acute studies, death was attributed primarily to renal papillary necrosis. Renal toxicity was not present in rabbits and has not been reported in cases of human overexposure. Although acute overexposure of animals and humans to S02F2 resulted in pulmonary edema, the typical finding in animals that survived the 2week or 13-week exposure was inflammation in the respiratory system which suggests irritation. Both acute and subchronic exposure to SOZF2 are associated with neurotoxicity. Convulsions in rats (Nitschke et al., 1986) and seizures in humans (Nuckolls et al., 1987) have been reported with acute exposure to high concentrations of S02F,. The subchronic neurotoxicity of SO,F, at concentrations of 100-600 ppm was characterized by convulsions in rabbits, pathologic changes in the brain of rats and rabbits, and electrophysiologic alterations in rats (Mattsson et al., 1988). However, no neurologic or other effects have been detected in animals exposed to 30 ppm for 13 weeks. Similarly, examination of workers exposed to occupational levels of S02FZ has not demonstrated significant differences from controls on a broad range of psychological and neurological tests (Anger et al., 1986). Therefore, work practices of fumigators appear to be adequate to protect them from possible neurotoxic consequences of SO,F, overexposure.
AND NITSCHKE
ACKNOWLEDGMENTS The authors thank D. A. Dittenber, L. E. Frauson, J. E. Phillips, S. A. Pugh, C. M. Streeter, E. L. Wolfe, and M. A. Zimmer for their contributions to the studies.
REFERENCES ACGIH (1987). Threshold Limit Values for Chemical Substances in the Work Environment. American Conference of Governmental industrial Hygienists, Washington, DC. ANGER, W. K., MOODY, L., BURG, J., BRIGHTWELL, W. S., TAYLOR, B. J., Russo, J. M., DICKERSON, N., SETZER, J. V., JOHNSON, B. L., AND HICU, K. (1986). Neurobehavioral evaluation of soil and structural fumigators using methyl bromide and sulfuryl fluoride. Neurotoxicology 7, 137-I 56. BOND, A. M., AND MURRAY, M. M. (1952). Kidney function and structure in chronic fluorosis. Brit. J. Exp. Pathol. 33, 168- 176. DOBYAN, D. C., NAGLE, R. B., AND BUL,GER, R. E. (1977). Acute tubular necrosis in the rat kidney following sustained hypotension, physiologic and morphologic observations. Lab. Invest. 37,4 1I-422. DRILL, V. A. (1954). Pharmacology in Medicine. McGraw-Hill, New York. GOODMAN, A. G., GOODMAN, L. S., AND GILMAN, A. ( 1980). The Pharmacological Basis of Therapeutics, 6th ed. MacMillan, New York. HODGE, H. C., AND SMITH, F. A. (1965). Fluorine Chemistry (J. H. Simons, Ed.), Vol. 4, pp. 176-183. Academic Press, New York. MATTHEWS, J. (1970). Changes in cell function due to inorganic fluoride. In Handbook of Experimental Pharmacology, Pharmacology of Fluorides (F. A. Smith, Ed.), pp. 98-143. Springer-Verlag, New York. MA~SSON, J. L., ALBEE, R. R., EISENBRANDT, D. L., AND CHANG, L. W. (1988). Subchronic neurotoxicity in rats of the structural fumigant, sulfuryl fluoride. Neurotoxicof. Teratoi. 10, 127-l 33, MAZZE, R. I., COUSINS,M. J., AND KOSIK, J. C. (1973). Strain differences in metabolism and susceptibility to the nephrotoxic effects of methoxyflurane in rats. J. Pharmacol. Exp. Ther. 184,48 l-488. MUNCK, A., AND CRABTREE, G. R. (198 I). Glucocorticoid-induced lymphocyte death. In Cell Death in Biology and Pathology (I. D. Bowen and R. A. Lockshin, Eds.), pp. 329-359. Chapman & Hall, New York. MURRAY, G., WYLLIE, R. G., HILL, G. S., RAMSDEN, P. W., AND HEFTINSTALL, R. H. (1972). Experimental papillary necrosis of the kidney. Amer. J. Pathol. 67, 285-298. NITSCHKE, K. D., ALBEE, R. R., MAT-TX~ON, J. L., AND MILLER, R. R. (I 986). Incapacitation and treatment
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TOXICITY
of rats exposed to a lethal dose of sulfuryl fluoride. Fundam. Appl. Toxicol. 7,664-610. NUCKOLLS, J. G., SMITH, D. C., OXLEY, D. W., HACKLER, R. L., TRIPATHI, R. K., A~STRONG, C. W., AND MILLER, G. B. (1987). Fatalities resulting from sulfuryl fluoride exposure alter home fumigation-Virginia. J. Amer. Med. Assoc. 258, 20412044. RUSH, G. F., AND WILLIS, L. P. (1982). Renal tubular effects of sodium fluoride. J. Pharmacol. Exp. Ther. 223,275-279. SABATINI, S. (1984). Pathophysiology of drug-induced papillary necrosis. Fundam. Appl. Toxicol. 4, 909921. SCHEUEFWAN, E. H. (1986). Suicide by exposure to sulfury1 fluoride. J. Foren. Sci. 31,1154-I 158. SUKETA, Y., ASAO, Y., KANAMOTO, Y., SAKASHITA, T., AND OKADA, S. (1985). Changes in adrenal function as a possible mechanism for elevation of serum glucose by a single large dose of fluoride. Toxicol. Appl. Pharmacol. 80,199-205.
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E. R. (1977). Acute toxicity and skin corrosion data for some organic and inorganic compounds and aqueous solutions. Toxicol. Appl. Pharmacol. 42,4 17423. WISEMAN, A. (1970). Effect of inorganic fluoride on enzymes. In Handbook of Experimental Pharmacology, Pharmacology ofFluorides (F. A. Smith, Ed.), pp. 4897. Springer-Verlag, New York.