Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Short‐term dermal toxicity and mutagenicity of coal coprocessing products in the rat I. Chu , D. C. Villeneuve , V. E. Secours , R. Otson & V. E. Valli To cite this article: I. Chu , D. C. Villeneuve , V. E. Secours , R. Otson & V. E. Valli (1991) Short‐term dermal toxicity and mutagenicity of coal coprocessing products in the rat, Journal of Toxicology and Environmental Health, 33:3, 317-326, DOI: 10.1080/15287399109531530 To link to this article: http://dx.doi.org/10.1080/15287399109531530
Published online: 15 Oct 2009.
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SHORT-TERM DERMAL TOXICITY AND MUTAGENICITY OF COAL COPROCESSING PRODUCTS IN THE RAT I. Chu, D. C. Villeneuve, V. E. Secours, R. Otson
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Bureau of Chemical Hazards, Environmental Health Directorate, Ottawa, Ontario, Canada V. E. Valli Biopath Analysts Ltd., Guelph, Ontario, Canada
The present study was conducted to determine the dermal toxicity of coal coprocessing products and to assess their potential health hazards. Croups of 10 male and 10 female Sprague-Dawley rats were administered dermally coal coprocessing products (light gas oil, LCO; heavy gas oil I, HCOI; heavy gas oil II, HCOII) at 1 g/kg body weight/d for 14 d. The control and positive control groups received normal saline and a coal liquefaction product (CLP) at the same dose level, respectively. Treatment with either the three fractions of coprocessing products or CLP caused decreased growth rate and food consumption in animals of both sexes. Liver enlargement occurred in groups treated with HGOI, HGOII, and CLP. Decreased serum glucose was observed in animals of both sexes treated with the three fractions and CLP. Treatment with HCOI and CLP caused an elevation of hepatic microsomal ethoxyresorufin deethylase activity in the rat of both sexes. The three fractions and CLP caused mild anemia. Mild treatment-related histological changes were observed in the liver, spleen, thyroid, bone marrow, and kidney. All three fractions of coprocessing products were tested for their mutagenicity in five strains of Salmonella typhimurium: TA98, TA100, TA1535, TA1537, and TA1538. HCOI, after metabolic activation, was found to be mutagenic in the strains of TA98, TA100, and TA1538. In contrast, HGOII was mutagenic in the five strains with or without metabolic activation. These data indicate that HCOI and HCOII are more toxic than LGO, and should be subjected to further studies to determine their long-term effects.
INTRODUCTION Diminishing petroleum reserves and sharp price increases in the late 1970s have provided incentives for many industrial nations to develop alternate liquid fuels. In North America, coal coprocessing products are the most promising alternative sources due to vast coal and bitumen reserves in Western Canada and the United States. Coprocessing products are a petroleum-like oily liquid that is produced by hydrogénation The authors thank T. Lee of Alberta Research Council for fractionation; E. Lee of British Columbia Research for performing mutagenicity assays; N. Beament, J. Kelly, K. Kittle, C. J. Powell, B. Reed, A. Viau, and A. Yagminas for technical assistance; and M. Beaudette for data handling. This project is funded in part by the Federal Panel on Energy Research and Development. 317 Journal of Toxicology and Environmental Health, 33:317-326, 1991 Copyright © 1991 by Hemisphere Publishing Corporation
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of a slurry of pulverized coal and bitumen under high temperature and pressure. Previous studies from this laboratory have shown that treatment of rats with coal liquefaction products by dermal or inhalation routes resulted in growth suppression and anemia (Chu et al., 1988a, 1988b, 1989). The purpose of the present study was to explore potential toxic effects of coprocessing products in the rat and to provide information that could be used to assess their potential toxic hazards. The dermal route was selected for the study because it was considered to be a major route of occupational exposure. MATERIALS A N D METHODS A crude coal coprocessing product was obtained from Canada Centre for Mineral and Energy Technology (CANMET); this product was subsequently fractionated according to the boiling range into naphtha, LGO, HGOI, and HGOII at Alberta Research Council, Devon, Alberta. Because of its high volatility, naphtha was subjected only to mutagenicity assays but not dermal studies. A medium-boiling coal liquefaction product (CLP, 154-378°C) was made using the solvent refined process (SRCII) from a pilot plant of Sandwell Technologies (Mississauga, Ont.). This test substance was used as a positive control in the present study because its chemical characteristics and toxic effects were reported in our previous studies (Chu et al., 1988a, 1989). Chemical analysis was performed using solvent extraction methods with pentane, acid and base; data are presented in Table 1. Animal Treatment Male and female Sprague-Dawley rats weighing approximately 350 and 200 g, respectively, were purchased from Charles River Laboratories (St. Constante, Que.). The animals were acclimatized to laboratory conditions (temperature 22 ± 2°C, humidity 40-60%) for 7 d before treatment. To prepare the site for dermal application, the hair at the interscapular region was clipped 24 h prior to the treatment and clipped as required TABLE 1. Boiling Point Ranges and Composition of Coal Coprocessing Products
Boiling point range (°C) Aliphatic hydrocarbons Aromatic hydrocarbons Polar compounds Acids Base
HGOI
HCOII
243-409 63.8 16.6 14.7
387-521 53.2 28.6 15.3 0.46 0.13
1.8
0.63
Note. Compositions are based on w/w %.
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during the study. Each of the test materials was applied to an 8-cm2 area on groups of 10 male and 10 female rats at 1.0 g/kg body weight/d, 7 d/wk for 2 wk. Control groups were treated with normal saline (1.0 ml) in a similar manner. The positive control groups received the CLP at the same dose levels as coprocessing products. The animals were provided with food (Purina Chow, Ralston Purina) and water ad libitum and maintained on a 12-h light/dark cycle. Body weights and food consumption were determined weekly. Clinical observations were made daily. At termination, all animals were anaesthetized with pentobarbital followed by exsanguination via the abdominal aorta. Gross examination was performed on each animal at the time of necropsy. The brain, heart, liver, spleen, and kidney were excised and weighed. Two milliliters of the blood was immediately transferred into vacutainers containing EDTA and used to measure the following hématologie parameters: hemoglobin, packed cell volume (PCV), red blood cell counts, mean corpuscular volume, mean corpuscular hemoglobin concentration, platelet counts, and total (Baker 9000 cell counter) and differential leukocyte counts. The rest of the blood was emptied into serum separation test tubes. After the blood clot formed the tubes were then centrifuged at 3600 rpm (Sorvall model GLC-1) and the serum was analyzed using a Technicon microanalyzer (model 12/60) for inorganic phosphate, alkaline phosphatase (SAP), aspartate aminotransferase (AST), total protein, calcium, cholesterol, glucose, uric acid, and lactic dehydrogenase. A sample of liver was excised and homogenized for the determination of microsomal aniline hydroxylase (AH; Fouts, 1963), aminopyrine demethylase (APDM; Cochin and Axelrod, 1959) and ethoxyresorufin deethylase activities (ER; Burke and Mayer, 1974). A selection of tissues were taken and fixed in 10% buffered formalin (pH 7.4) for routine histological examination. Tissues examined histologically included brain, pituitary, liver, adrenal, thyroid, parathyroid, thymus, lungs, trachea, bronchi, thoracic aorta, esophagus, gastric cardia, fundus and pylorus, duodenum, pancreas, colon, kidney, spleen, bone marrow, mesenteric and mediastinal lymph nodes, testes or ovaries, epididymis, skin, skeletal muscle, and heart. Data were analyzed using one-way analysis of variance and Duncan's multiple range test (Nie et al., 1977) to identify the groups that were significantly different (p < .05). Mutagemcity Assay Five strains of Salmonella typhimurium—TA98, TA100, TA1535, TA1537, and TA1538—were obtained from Dr. B. Ames of the University of California, Berkeley. The liver S9 activation system from Aroclor 1254-induced male Sprague-Dawley rats was purchased from Molecular Toxicology Inc., Annapolis, Md. The mutagenicity assays were carried out according
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to the procedures of Ames et al. (1975) and are described briefly as follows: Fractions of coprocessing products and positive controls (sodium azide, 9-aminoacridine, 2-nitrofluorene, and 2-aminoanthracene) were each dissolved in dimethyl sulfoxide to prepare appropriately diluted solutions of test chemicals. The solution (0.1 ml), inoculum (S. typhimurium), and phosphate buffer pH 7.4 (0.5 ml) were added to 2 ml histidinedeficient top agar at 45°C and mixed thoroughly. The mixture was poured onto minimal basal agar and incubated at 37°C for 48 h. After incubation, the numbers of colonies were counted by an automatic colony counter (Artek model 880). Each sample was screened at various concentrations with the five strains in the presence and absence of the S9 activation system. RESULTS Clinical Observation Animals of both sexes treated with HGOI, LCO, and CLP had skin lesions at the site of application, which consisted of dermal thickening, sclerosis and focal ulcérations. No skin lesions were seen in HGOIItreated animals, and no other signs of toxicity were observed. Growth Rate and Food Consumption Growth suppression and decreased food consumption were observed in animals of both sexes treated with LGO, HGOI, HGOII, and CLP CTable 2). Treatment with HGOI and HGOII appeared to cause a greater growth suppression in both sexes than LGO and CLP. Gross Observation Examination of visceral organs at necropsy showed that the spleens of HGOII- and CLP-treated groups displayed rough surface and white spots. Organ Weight Increased liver weights were observed in groups of both sexes treated with HGOI, HGOII, and CLP (Table 2). Biochemical Changes Decreased serum glucose was observed in animals of both sexes treated with HGOI and HGOII, and in female rats treated with CLP (Table 3). Increased serum alkaline phosphatase occurred in HGOI- and CLPtreated groups. Hepatic ER activities were increased in animals of both sexes receiving HGOI and CLP. Serum cholesterol levels were elevated in
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TABLE 2. Body Weights, Food Consumption, and Liver Weights of Rats Treated with Coal Coprocessing Products Initial weight (g)
n3
Treatment
Food intake (g/rat/d)
Liver weight
37 30" 18" 33" 18"
26 ± 3 21 ± 2 " 19 ± 1" 17 ± 4" 21 ± 1"
14.6 13.1 16.2 20 18.3
+ ± ± ± ±
2.0" 2.8" 1.6"
11 14" 13" 20" 13"
20 17 16 13 16
9.0 9.4 12.7 12.5 13.5
± ± ± ± ±
0.75 0.87 1.6" 2.4" 1.5"
Final weight (g)
(g)
Male Control HGOI HGOII CLP
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347 347 350 350 351
10 10 10 10 10
LGO
± ± ± ± ±
400 332 301 321 331
13 13 14 16 16
+ ± ± ± ±
2.8 1.8
Female Control
204 207 202 207 204
10 10 10 10 10
LGO
HGOI HGOII CLP
± ± ± ± ±
241 221 203 201 223
11 10 10 8 11
± ± ± ± ±
± ± ± ± ±
2 2" 2" 1" 2"
a
Number of animals; data represent means ± SD. "Significantly different from controls (p < .05).
TABLE 3. Biochemical Changes of Rats Administered Dermally Coal Coprocessing Products
Treatment
Glucose (mg/dl)
Alkaline phosphatase (mlU/ml)
156 157 122 119 145
144 162 295 123 212
Cholesterol (mg/dl)
Hepatic ethoxyresorufin deethylase activity (nmol/mg protein/h)
51 45 54 110 61
+ ± + ± ±
15 17 8 25" 20
0.58 0.64 0.90 0.45 1.0
± + ± ± ±
0.17 0.17 0.29" 0.06 0.16"
116 86 100 198 116
± ± ± ± ±
24 14 6 27" 33
0.47 0.52 0.83 0.32 0.76
± ± + ± ±
0.08 0.07 0.22" 0.08 0.18"
Male Control LGO
HGOI HCOII CLP
10 10 10 10 10
+ ± ± ± ±
20 47 15" 21" 14
± + ± ± ±
36 42 (9) 56" 32 51"
Female Control LGO
HCOI HCOII CLP a
10 10 10 10 10
210 183 151 128 176
± + + ± ±
21 39 (9) 21" 22" 28"
154 138 276 218 179
+ ± ± ± ±
48 39 92" 88 56
Unless otherwise given in parentheses; data denote means ± SD of 10 animals. "Significantly different from controls (p < .05).
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HGOII groups. No apparent differences in biochemical changes between males and females were observed. Hepatic aniline hydroxylase and aminopyrine A/-demethylase activities were not affected by treatment.
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Hematological Changes Hematological data are presented in Table 4. Treatment with LGO, HGOI, and CLP resulted in decreased erythrocyte counts and hemoglobin and hematocrit values in both sexes. Increased lymphocytes and neutrophils were also observed in these groups. HGOII had no effects on erythrocyte and leukocyte counts. Histopathology Treatment with coprocessing products caused mild changes in liver, thyroid, thymus, bone marrow, kidney, and skin of animals of both sexes, and the changes were similar to those produced by CLP. Skin was a major site of action. Crust formation, epidermal inflammation, and parakeratosis were observed. Superficial dermis changes consisted of focal edema and occasionally of sclerosis. Changes in the liver were proliferative in nature and consisted of cytoplasmic vacuolation, nuclear vesiculation, and increased cytoplasmic density. There were degenerative changes in the thyroid characterized by angular collapse of follicles, increased epithelial height, and focal areas of papillary proliferation. The thymus was a target organ with cortical atrophy of varying degree. Changes in the kidney were characterized by the presence in the proxiTABLE 4. Hematological Changes of Rats Treated with Coal Coprocessing Products Hemoglobin (g/dl)
Treatment
Hematocrit (%)
Erythrocytes (x 106/Ml)
Neutrophils
Lymphocytes
3 (x 10 /itl)
(x
IO 3 /MD
Male Control LGO
HGOI HGOII CLP
10 9 10 10 10
15.0 13.7 12.5 14.4 12.2
± ± ± ± ±
1.4 1.2b 0.9b 1.0 0.6b
41 37 34 39 32
± ± ± ± ±
4.5 3.4b 2.3b 2.9 1.4b
10 10 10 10 10
14.2 13.0 11.1 14.3 11.4
± ± ± ± ±
2.5 3.1b 7.8b 1.6 0.6b
40 35 31 40 32
± ± ± ± ±
1.1 1.2b 2.1 b 4.1 1.8b
7.6 6.8 6.2 7.4 6.1
± ± ± ± ±
0.85 0.72b 0.48b 0.57 0.31b
1.1 7.2 15 2.9 12
± ± ± ± ±
0.6 3.2b 4.4b 4.1 4.1 b
6.2 9.9 13 6.5 11
± ± ± ± ±
1.5 2.5b 4.9b 2.1 5.0b
± ± ± ± ±
0.26 0.21b 0.51b 0.90 0.366
0.8 2.5 12 0.9 4.2
± ± ± ± ±
0.5 1.4b 4.6b 0.5 3.3b
3.7 5.5 8.7 6.2 5.4
± ± ± ± ±
0.9 1.4 2.4b 2.6b 2.4
Female Control LGO
HGOI HGOII CLP
"Number of animals. Significantly different from controls (p < .05).
7.0 6.3 5.5 7.7 6.0
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TABLE 5. Mutagenicity of HCOI Salmonella typhimuríum revertants/plate
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A
Control, 0 10 50 100 250 500
17 20 21 19 18 9
B
± ± ± ± ± ±
2 1 3 1 2 1
19 25 51 90 28 12
TA1538
TA100
TA98 HGOI (/ig/plate)
B
A
± ± ± ± ± ±
3 3 13a 4a 10 2
144 162 202 203 32 30
± ± ± ± ± ±
22 28 36 20a 12 19
142 177 375 541 151
± ± ± ± ±
26 25 61 a 78a 13
40 ± 8
A
B
13 + 5 11 + 3 8 ± 1 10 ± 3 13 ± 2 8 ± 1
12 15 44 80 122 37
± ± ± ± ± ±
6 4 3a 14a 4a 6a
Note. Data represent mean ± SD obtained from three determinations; A, without metabolic activation; B, with S9 metabolic activation. Mutagenicity data of positive controls are given in Table 6. a Significantly increased over the control (p < .05).
mal tubules of hyaline droplets, which occurred more frequently in the treated groups than the control. Morphological alterations in the spleen were characterized by follicular atrophy with reduced blood in splenic interstitium and increased levels of extramedullary hematopoiesis. Hyperplastic changes were observed in the bone marrow with increased proportions of granulocytes and dyserythropoiesis. In general, histological changes were mild in nature and were more severe in males than in females. Mutagenicity Of the four coprocessing fractions tested, naphtha and LGO were devoid of any mutagenic activity with or without metabolic activation. HGOI was weakly mutagenic in 5. typhimurium TA98, TA100, and TA1538, only after S9 metabolic activation (Table 5). In contrast, HGOI I was mutagenic in all five strains of S. typhimurium with and without metabolic activation. In the presence of rat liver S9 fraction, the mutagenic activity of HGOII was greatly enhanced (Table 6). Compared to the known mutagen 2-aminoanthracene and others, both HGOI and HGOII were still considered to be weak mutagens. As shown in Table 6, HGOII has a higher degree of aromaticity than HGOI, and it is more mutagenic than the latter. Thus these two parameters of the coal liquid appear to be related. Chemicals such as those distillable in the low-boiling LGO fraction have no mutagenicity. DISCUSSION Topical application of coprocessing products resulted in growth suppression, increased liver weights, increased hepatic microsomal activity,
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TABLE 6. Mutagenicity of HGOII Salmonella typhimurium revertants/plate TA98 Test material (ftg/plate)
w
Control HGOII 100 500 1000 2500 5000 2-Aminoanthracene 2 Sodium azide 10 9-Aminoacridine 100 2-Nitrofluorene 50
A
TA100
B 19 ± 2 17 24 26 48 12
± ± ± ± ±
1 3' 7 7' 7
18 ± 2
A 19 ± 3
155 396 488 644 425
± ± ± ± ±
10' 47a 60' 89a 86a
1417 ± 613'
403 457 558 279 72
A
B
144 ± 22
± ± ± ± ±
17a 60a 73' 17' 17
145 ± 10
146 ± 26 570 1109 1418 985 120
± ± ± ± ±
90' 82a 83 a 100a 14
1953 ± 131' •
1203 ± 137'
TA1537
TA1535
18 ± 1 94 138 98 83 13
A
B
± ± ± ± ±
7a 12' 10' 8' 2'
15 ± 3
17 ± 5 131 355 612 390 55
± ± ± ± ±
11' 67a 82 a 29 a 11'
314 ± 102'
± ± ± ± ±
1 6 1 3 1
9 ± 3
A 7 ± 2
239 508 644 762 14
± ± ± ± ±
30a 39' 84' 58' 3'
1243 ± 154a
B
13 ± 5 19 15 21 20 19
± ± ± ± ±
1 2 6 2 2
12 ± 6 540 740 865 1090 428
± ± ± ± ±
89a 56' 110' 57' 59a
16 ± 4
1163 ± 62a
—
1065 ± 150a
774 ± 119' 1515 ± 253 a
— 1091 ± 129;
B
7 ± 3 8 12 7 5 2
TA1538
—
—
—
—
—
—
Note. Data denote means ± SD obtained from three determinations; A, without metabolic activation; B, with metabolic activation system. 'Significantly increased over the control (p < .05).
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anemia, and mild histological changes in liver, thyroid, kidney, spleen, bone marrow, and thymus. Some of these effects were also observed in rats treated with CLP. CLP was used as a positive control and its toxic effects were well characterized in our previous studies (Chu et al., 1988a, 1988b, 1989). Among the three coprocessing products tested, HGOI appeared to have more toxicological properties in common with CLP than the other two fractions since both HGOI and CLP caused elevations of liver ER and SAP activities. The increased ER activity was consistent with the histological changes in the liver such as proliferative changes in this organ. An examination of physicochemical characteristics of the fractions revealed the similarity in boiling ranges for CLP (154-378°C) and HGOI (243-409°C). Thus, it would appear that the hydrocarbon components in the coal liquids distillable at these boiling ranges were responsible for the changes observed. Rat kidney is known to have high levels of SAP (Clampitt and Hart, 1978); increased serum levels may reflect the histopathological changes observed in the kidneys of rats treated with these liquid fuels. Elevated serum cholesterol levels were observed in HGOII-treated groups and might be related to the effects of the test material on fatty acid metabolism. Decreased serum glucose was noted in the HGOI and HGOII groups, and this effect may be a result of decreased food intake and poor health status rather than specific organ damages. Hematological changes were perhaps the most prominent toxic effects that occurred following administration of these liquid fuels. Studies from our laboratory in rats treated with HGOII for 3 mo showed increased serum iron and total iron-binding capacity, suggesting that the observed anemia may be a result of defective heme synthesis. In general, some biochemical and/or hematological changes observed here may represent an adjustment in the physiological state of the animals suffering decreased food intake and weight loss. HGOI and HGOII were also found to be more toxic than LGO in the mutagenicity assays. The fact that HGOI and HGOII were more active after metabolic activation also suggests the presence in these fractions of mutagenic PAHs components that require metabolic activation into biologically active intermediates. Compared to known mutagens such as 9aminoanthracene and other positive controls, the coprocessing products are still considered to be weak mutagens. In conclusion, treatment with coal coprocessing products resulted in toxicological changes including growth suppression, increased liver weight, increased hepatic ER activity, hematological aberrations, and histological changes in liver, thyroid, kidney, bone marrow, and thymus. These effects are considered to be in the same order of magnitude to those observed for CLP. Based on the data presented above, HGOI and HGOII appear to be more toxic than LGO, and are selected for longerterm studies.
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REFERENCES Ames, B. M., McCann, J., and Yamasaki, E. 1975. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsomes mutagenicity test. Mutat. Res. 31:347-364. Burke, M. D., and Mayer, R. T. 1974. Ethoxyresorufin: Direct fluorimetric assay of a microsomal Odealkylation which is preferentially inducible by 3-methylcholanthrene. Drug. Metab. Dispos. 2:583-588. Chu, I., Villeneuve, D. C , Côté, M., Valli, V. E., and Otson, R. 1988a. The dermal toxicity of a medium-boiling (154-378°C) coal liquefaction product in the rat (Part I). J. Toxicol. Environ. Health 23:193-206. Chu, I., Villeneuve, D. C , Côté, M., Secours, V., Otson, R., and Valli, V. E. 1989b. Dermal toxicity of a high-boiling (bp 250-450°C) coal liquefaction product in the rat (Part II). J. Toxicol. Environ. Health 25:509-525. Chu, I., Rinehart, W., Hoffman, G., Villeneuve, D. C , Otson, R., and Valli, V. E. 1989. Subacute inhalation toxicity of a medium-boiling coal liquefaction product (154-378°C) in the rat (Part III). J. Toxicol. Environ. Health 28:195-204.
Clampitt, R. B., and Hart, R. J. 1978. The tissue activities of some diagnostic enzymes in ten mammalian species. J. Comp. Pathol. 88:607-621. Cochin, J., and Axelrod, J. 1959. Biochemical and pharmacological changes in the rat following chronic administration of morphine, nalorphine and normorphine. J. Pharmacol. Exp. Ther. 125:105-110. Fouts, J. R. 1963. Factors influencing the metabolism of drugs in liver microsomes. N. Y. Acad. Sci. 104:875-880. Nie, N. H., Hull, C. H., Jenkins, J. G., Steinbrenner, K., and Bent, O. H. (1977). Statistical Programs for the Social Sciences. Chicago: SPSS, Inc. Received August 28, 1990 Accepted February 15, 1991
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Short‐term dermal toxicity and mutagenicity of coal coprocessing products in the rat I. Chu , D. C. Villeneuve , V. E. Secours , R. Otson & V. E. Valli To cite this article: I. Chu , D. C. Villeneuve , V. E. Secours , R. Otson & V. E. Valli (1991) Short‐term dermal toxicity and mutagenicity of coal coprocessing products in the rat, Journal of Toxicology and Environmental Health, 33:3, 317-326, DOI: 10.1080/15287399109531530 To link to this article: http://dx.doi.org/10.1080/15287399109531530
Published online: 15 Oct 2009.
Submit your article to this journal
View related articles
Citing articles: 2 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh20 Download by: [University of Florida]
Date: 07 November 2015, At: 01:45
SHORT-TERM DERMAL TOXICITY AND MUTAGENICITY OF COAL COPROCESSING PRODUCTS IN THE RAT I. Chu, D. C. Villeneuve, V. E. Secours, R. Otson
Downloaded by [University of Florida] at 01:45 07 November 2015
Bureau of Chemical Hazards, Environmental Health Directorate, Ottawa, Ontario, Canada V. E. Valli Biopath Analysts Ltd., Guelph, Ontario, Canada
The present study was conducted to determine the dermal toxicity of coal coprocessing products and to assess their potential health hazards. Croups of 10 male and 10 female Sprague-Dawley rats were administered dermally coal coprocessing products (light gas oil, LCO; heavy gas oil I, HCOI; heavy gas oil II, HCOII) at 1 g/kg body weight/d for 14 d. The control and positive control groups received normal saline and a coal liquefaction product (CLP) at the same dose level, respectively. Treatment with either the three fractions of coprocessing products or CLP caused decreased growth rate and food consumption in animals of both sexes. Liver enlargement occurred in groups treated with HGOI, HGOII, and CLP. Decreased serum glucose was observed in animals of both sexes treated with the three fractions and CLP. Treatment with HCOI and CLP caused an elevation of hepatic microsomal ethoxyresorufin deethylase activity in the rat of both sexes. The three fractions and CLP caused mild anemia. Mild treatment-related histological changes were observed in the liver, spleen, thyroid, bone marrow, and kidney. All three fractions of coprocessing products were tested for their mutagenicity in five strains of Salmonella typhimurium: TA98, TA100, TA1535, TA1537, and TA1538. HCOI, after metabolic activation, was found to be mutagenic in the strains of TA98, TA100, and TA1538. In contrast, HGOII was mutagenic in the five strains with or without metabolic activation. These data indicate that HCOI and HCOII are more toxic than LGO, and should be subjected to further studies to determine their long-term effects.
INTRODUCTION Diminishing petroleum reserves and sharp price increases in the late 1970s have provided incentives for many industrial nations to develop alternate liquid fuels. In North America, coal coprocessing products are the most promising alternative sources due to vast coal and bitumen reserves in Western Canada and the United States. Coprocessing products are a petroleum-like oily liquid that is produced by hydrogénation The authors thank T. Lee of Alberta Research Council for fractionation; E. Lee of British Columbia Research for performing mutagenicity assays; N. Beament, J. Kelly, K. Kittle, C. J. Powell, B. Reed, A. Viau, and A. Yagminas for technical assistance; and M. Beaudette for data handling. This project is funded in part by the Federal Panel on Energy Research and Development. 317 Journal of Toxicology and Environmental Health, 33:317-326, 1991 Copyright © 1991 by Hemisphere Publishing Corporation
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of a slurry of pulverized coal and bitumen under high temperature and pressure. Previous studies from this laboratory have shown that treatment of rats with coal liquefaction products by dermal or inhalation routes resulted in growth suppression and anemia (Chu et al., 1988a, 1988b, 1989). The purpose of the present study was to explore potential toxic effects of coprocessing products in the rat and to provide information that could be used to assess their potential toxic hazards. The dermal route was selected for the study because it was considered to be a major route of occupational exposure. MATERIALS A N D METHODS A crude coal coprocessing product was obtained from Canada Centre for Mineral and Energy Technology (CANMET); this product was subsequently fractionated according to the boiling range into naphtha, LGO, HGOI, and HGOII at Alberta Research Council, Devon, Alberta. Because of its high volatility, naphtha was subjected only to mutagenicity assays but not dermal studies. A medium-boiling coal liquefaction product (CLP, 154-378°C) was made using the solvent refined process (SRCII) from a pilot plant of Sandwell Technologies (Mississauga, Ont.). This test substance was used as a positive control in the present study because its chemical characteristics and toxic effects were reported in our previous studies (Chu et al., 1988a, 1989). Chemical analysis was performed using solvent extraction methods with pentane, acid and base; data are presented in Table 1. Animal Treatment Male and female Sprague-Dawley rats weighing approximately 350 and 200 g, respectively, were purchased from Charles River Laboratories (St. Constante, Que.). The animals were acclimatized to laboratory conditions (temperature 22 ± 2°C, humidity 40-60%) for 7 d before treatment. To prepare the site for dermal application, the hair at the interscapular region was clipped 24 h prior to the treatment and clipped as required TABLE 1. Boiling Point Ranges and Composition of Coal Coprocessing Products
Boiling point range (°C) Aliphatic hydrocarbons Aromatic hydrocarbons Polar compounds Acids Base
HGOI
HCOII
243-409 63.8 16.6 14.7
387-521 53.2 28.6 15.3 0.46 0.13
1.8
0.63
Note. Compositions are based on w/w %.
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during the study. Each of the test materials was applied to an 8-cm2 area on groups of 10 male and 10 female rats at 1.0 g/kg body weight/d, 7 d/wk for 2 wk. Control groups were treated with normal saline (1.0 ml) in a similar manner. The positive control groups received the CLP at the same dose levels as coprocessing products. The animals were provided with food (Purina Chow, Ralston Purina) and water ad libitum and maintained on a 12-h light/dark cycle. Body weights and food consumption were determined weekly. Clinical observations were made daily. At termination, all animals were anaesthetized with pentobarbital followed by exsanguination via the abdominal aorta. Gross examination was performed on each animal at the time of necropsy. The brain, heart, liver, spleen, and kidney were excised and weighed. Two milliliters of the blood was immediately transferred into vacutainers containing EDTA and used to measure the following hématologie parameters: hemoglobin, packed cell volume (PCV), red blood cell counts, mean corpuscular volume, mean corpuscular hemoglobin concentration, platelet counts, and total (Baker 9000 cell counter) and differential leukocyte counts. The rest of the blood was emptied into serum separation test tubes. After the blood clot formed the tubes were then centrifuged at 3600 rpm (Sorvall model GLC-1) and the serum was analyzed using a Technicon microanalyzer (model 12/60) for inorganic phosphate, alkaline phosphatase (SAP), aspartate aminotransferase (AST), total protein, calcium, cholesterol, glucose, uric acid, and lactic dehydrogenase. A sample of liver was excised and homogenized for the determination of microsomal aniline hydroxylase (AH; Fouts, 1963), aminopyrine demethylase (APDM; Cochin and Axelrod, 1959) and ethoxyresorufin deethylase activities (ER; Burke and Mayer, 1974). A selection of tissues were taken and fixed in 10% buffered formalin (pH 7.4) for routine histological examination. Tissues examined histologically included brain, pituitary, liver, adrenal, thyroid, parathyroid, thymus, lungs, trachea, bronchi, thoracic aorta, esophagus, gastric cardia, fundus and pylorus, duodenum, pancreas, colon, kidney, spleen, bone marrow, mesenteric and mediastinal lymph nodes, testes or ovaries, epididymis, skin, skeletal muscle, and heart. Data were analyzed using one-way analysis of variance and Duncan's multiple range test (Nie et al., 1977) to identify the groups that were significantly different (p < .05). Mutagemcity Assay Five strains of Salmonella typhimurium—TA98, TA100, TA1535, TA1537, and TA1538—were obtained from Dr. B. Ames of the University of California, Berkeley. The liver S9 activation system from Aroclor 1254-induced male Sprague-Dawley rats was purchased from Molecular Toxicology Inc., Annapolis, Md. The mutagenicity assays were carried out according
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to the procedures of Ames et al. (1975) and are described briefly as follows: Fractions of coprocessing products and positive controls (sodium azide, 9-aminoacridine, 2-nitrofluorene, and 2-aminoanthracene) were each dissolved in dimethyl sulfoxide to prepare appropriately diluted solutions of test chemicals. The solution (0.1 ml), inoculum (S. typhimurium), and phosphate buffer pH 7.4 (0.5 ml) were added to 2 ml histidinedeficient top agar at 45°C and mixed thoroughly. The mixture was poured onto minimal basal agar and incubated at 37°C for 48 h. After incubation, the numbers of colonies were counted by an automatic colony counter (Artek model 880). Each sample was screened at various concentrations with the five strains in the presence and absence of the S9 activation system. RESULTS Clinical Observation Animals of both sexes treated with HGOI, LCO, and CLP had skin lesions at the site of application, which consisted of dermal thickening, sclerosis and focal ulcérations. No skin lesions were seen in HGOIItreated animals, and no other signs of toxicity were observed. Growth Rate and Food Consumption Growth suppression and decreased food consumption were observed in animals of both sexes treated with LGO, HGOI, HGOII, and CLP CTable 2). Treatment with HGOI and HGOII appeared to cause a greater growth suppression in both sexes than LGO and CLP. Gross Observation Examination of visceral organs at necropsy showed that the spleens of HGOII- and CLP-treated groups displayed rough surface and white spots. Organ Weight Increased liver weights were observed in groups of both sexes treated with HGOI, HGOII, and CLP (Table 2). Biochemical Changes Decreased serum glucose was observed in animals of both sexes treated with HGOI and HGOII, and in female rats treated with CLP (Table 3). Increased serum alkaline phosphatase occurred in HGOI- and CLPtreated groups. Hepatic ER activities were increased in animals of both sexes receiving HGOI and CLP. Serum cholesterol levels were elevated in
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TABLE 2. Body Weights, Food Consumption, and Liver Weights of Rats Treated with Coal Coprocessing Products Initial weight (g)
n3
Treatment
Food intake (g/rat/d)
Liver weight
37 30" 18" 33" 18"
26 ± 3 21 ± 2 " 19 ± 1" 17 ± 4" 21 ± 1"
14.6 13.1 16.2 20 18.3
+ ± ± ± ±
2.0" 2.8" 1.6"
11 14" 13" 20" 13"
20 17 16 13 16
9.0 9.4 12.7 12.5 13.5
± ± ± ± ±
0.75 0.87 1.6" 2.4" 1.5"
Final weight (g)
(g)
Male Control HGOI HGOII CLP
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347 347 350 350 351
10 10 10 10 10
LGO
± ± ± ± ±
400 332 301 321 331
13 13 14 16 16
+ ± ± ± ±
2.8 1.8
Female Control
204 207 202 207 204
10 10 10 10 10
LGO
HGOI HGOII CLP
± ± ± ± ±
241 221 203 201 223
11 10 10 8 11
± ± ± ± ±
± ± ± ± ±
2 2" 2" 1" 2"
a
Number of animals; data represent means ± SD. "Significantly different from controls (p < .05).
TABLE 3. Biochemical Changes of Rats Administered Dermally Coal Coprocessing Products
Treatment
Glucose (mg/dl)
Alkaline phosphatase (mlU/ml)
156 157 122 119 145
144 162 295 123 212
Cholesterol (mg/dl)
Hepatic ethoxyresorufin deethylase activity (nmol/mg protein/h)
51 45 54 110 61
+ ± + ± ±
15 17 8 25" 20
0.58 0.64 0.90 0.45 1.0
± + ± ± ±
0.17 0.17 0.29" 0.06 0.16"
116 86 100 198 116
± ± ± ± ±
24 14 6 27" 33
0.47 0.52 0.83 0.32 0.76
± ± + ± ±
0.08 0.07 0.22" 0.08 0.18"
Male Control LGO
HGOI HCOII CLP
10 10 10 10 10
+ ± ± ± ±
20 47 15" 21" 14
± + ± ± ±
36 42 (9) 56" 32 51"
Female Control LGO
HCOI HCOII CLP a
10 10 10 10 10
210 183 151 128 176
± + + ± ±
21 39 (9) 21" 22" 28"
154 138 276 218 179
+ ± ± ± ±
48 39 92" 88 56
Unless otherwise given in parentheses; data denote means ± SD of 10 animals. "Significantly different from controls (p < .05).
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HGOII groups. No apparent differences in biochemical changes between males and females were observed. Hepatic aniline hydroxylase and aminopyrine A/-demethylase activities were not affected by treatment.
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Hematological Changes Hematological data are presented in Table 4. Treatment with LGO, HGOI, and CLP resulted in decreased erythrocyte counts and hemoglobin and hematocrit values in both sexes. Increased lymphocytes and neutrophils were also observed in these groups. HGOII had no effects on erythrocyte and leukocyte counts. Histopathology Treatment with coprocessing products caused mild changes in liver, thyroid, thymus, bone marrow, kidney, and skin of animals of both sexes, and the changes were similar to those produced by CLP. Skin was a major site of action. Crust formation, epidermal inflammation, and parakeratosis were observed. Superficial dermis changes consisted of focal edema and occasionally of sclerosis. Changes in the liver were proliferative in nature and consisted of cytoplasmic vacuolation, nuclear vesiculation, and increased cytoplasmic density. There were degenerative changes in the thyroid characterized by angular collapse of follicles, increased epithelial height, and focal areas of papillary proliferation. The thymus was a target organ with cortical atrophy of varying degree. Changes in the kidney were characterized by the presence in the proxiTABLE 4. Hematological Changes of Rats Treated with Coal Coprocessing Products Hemoglobin (g/dl)
Treatment
Hematocrit (%)
Erythrocytes (x 106/Ml)
Neutrophils
Lymphocytes
3 (x 10 /itl)
(x
IO 3 /MD
Male Control LGO
HGOI HGOII CLP
10 9 10 10 10
15.0 13.7 12.5 14.4 12.2
± ± ± ± ±
1.4 1.2b 0.9b 1.0 0.6b
41 37 34 39 32
± ± ± ± ±
4.5 3.4b 2.3b 2.9 1.4b
10 10 10 10 10
14.2 13.0 11.1 14.3 11.4
± ± ± ± ±
2.5 3.1b 7.8b 1.6 0.6b
40 35 31 40 32
± ± ± ± ±
1.1 1.2b 2.1 b 4.1 1.8b
7.6 6.8 6.2 7.4 6.1
± ± ± ± ±
0.85 0.72b 0.48b 0.57 0.31b
1.1 7.2 15 2.9 12
± ± ± ± ±
0.6 3.2b 4.4b 4.1 4.1 b
6.2 9.9 13 6.5 11
± ± ± ± ±
1.5 2.5b 4.9b 2.1 5.0b
± ± ± ± ±
0.26 0.21b 0.51b 0.90 0.366
0.8 2.5 12 0.9 4.2
± ± ± ± ±
0.5 1.4b 4.6b 0.5 3.3b
3.7 5.5 8.7 6.2 5.4
± ± ± ± ±
0.9 1.4 2.4b 2.6b 2.4
Female Control LGO
HGOI HGOII CLP
"Number of animals. Significantly different from controls (p < .05).
7.0 6.3 5.5 7.7 6.0
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TABLE 5. Mutagenicity of HCOI Salmonella typhimuríum revertants/plate
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A
Control, 0 10 50 100 250 500
17 20 21 19 18 9
B
± ± ± ± ± ±
2 1 3 1 2 1
19 25 51 90 28 12
TA1538
TA100
TA98 HGOI (/ig/plate)
B
A
± ± ± ± ± ±
3 3 13a 4a 10 2
144 162 202 203 32 30
± ± ± ± ± ±
22 28 36 20a 12 19
142 177 375 541 151
± ± ± ± ±
26 25 61 a 78a 13
40 ± 8
A
B
13 + 5 11 + 3 8 ± 1 10 ± 3 13 ± 2 8 ± 1
12 15 44 80 122 37
± ± ± ± ± ±
6 4 3a 14a 4a 6a
Note. Data represent mean ± SD obtained from three determinations; A, without metabolic activation; B, with S9 metabolic activation. Mutagenicity data of positive controls are given in Table 6. a Significantly increased over the control (p < .05).
mal tubules of hyaline droplets, which occurred more frequently in the treated groups than the control. Morphological alterations in the spleen were characterized by follicular atrophy with reduced blood in splenic interstitium and increased levels of extramedullary hematopoiesis. Hyperplastic changes were observed in the bone marrow with increased proportions of granulocytes and dyserythropoiesis. In general, histological changes were mild in nature and were more severe in males than in females. Mutagenicity Of the four coprocessing fractions tested, naphtha and LGO were devoid of any mutagenic activity with or without metabolic activation. HGOI was weakly mutagenic in 5. typhimurium TA98, TA100, and TA1538, only after S9 metabolic activation (Table 5). In contrast, HGOI I was mutagenic in all five strains of S. typhimurium with and without metabolic activation. In the presence of rat liver S9 fraction, the mutagenic activity of HGOII was greatly enhanced (Table 6). Compared to the known mutagen 2-aminoanthracene and others, both HGOI and HGOII were still considered to be weak mutagens. As shown in Table 6, HGOII has a higher degree of aromaticity than HGOI, and it is more mutagenic than the latter. Thus these two parameters of the coal liquid appear to be related. Chemicals such as those distillable in the low-boiling LGO fraction have no mutagenicity. DISCUSSION Topical application of coprocessing products resulted in growth suppression, increased liver weights, increased hepatic microsomal activity,
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TABLE 6. Mutagenicity of HGOII Salmonella typhimurium revertants/plate TA98 Test material (ftg/plate)
w
Control HGOII 100 500 1000 2500 5000 2-Aminoanthracene 2 Sodium azide 10 9-Aminoacridine 100 2-Nitrofluorene 50
A
TA100
B 19 ± 2 17 24 26 48 12
± ± ± ± ±
1 3' 7 7' 7
18 ± 2
A 19 ± 3
155 396 488 644 425
± ± ± ± ±
10' 47a 60' 89a 86a
1417 ± 613'
403 457 558 279 72
A
B
144 ± 22
± ± ± ± ±
17a 60a 73' 17' 17
145 ± 10
146 ± 26 570 1109 1418 985 120
± ± ± ± ±
90' 82a 83 a 100a 14
1953 ± 131' •
1203 ± 137'
TA1537
TA1535
18 ± 1 94 138 98 83 13
A
B
± ± ± ± ±
7a 12' 10' 8' 2'
15 ± 3
17 ± 5 131 355 612 390 55
± ± ± ± ±
11' 67a 82 a 29 a 11'
314 ± 102'
± ± ± ± ±
1 6 1 3 1
9 ± 3
A 7 ± 2
239 508 644 762 14
± ± ± ± ±
30a 39' 84' 58' 3'
1243 ± 154a
B
13 ± 5 19 15 21 20 19
± ± ± ± ±
1 2 6 2 2
12 ± 6 540 740 865 1090 428
± ± ± ± ±
89a 56' 110' 57' 59a
16 ± 4
1163 ± 62a
—
1065 ± 150a
774 ± 119' 1515 ± 253 a
— 1091 ± 129;
B
7 ± 3 8 12 7 5 2
TA1538
—
—
—
—
—
—
Note. Data denote means ± SD obtained from three determinations; A, without metabolic activation; B, with metabolic activation system. 'Significantly increased over the control (p < .05).
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anemia, and mild histological changes in liver, thyroid, kidney, spleen, bone marrow, and thymus. Some of these effects were also observed in rats treated with CLP. CLP was used as a positive control and its toxic effects were well characterized in our previous studies (Chu et al., 1988a, 1988b, 1989). Among the three coprocessing products tested, HGOI appeared to have more toxicological properties in common with CLP than the other two fractions since both HGOI and CLP caused elevations of liver ER and SAP activities. The increased ER activity was consistent with the histological changes in the liver such as proliferative changes in this organ. An examination of physicochemical characteristics of the fractions revealed the similarity in boiling ranges for CLP (154-378°C) and HGOI (243-409°C). Thus, it would appear that the hydrocarbon components in the coal liquids distillable at these boiling ranges were responsible for the changes observed. Rat kidney is known to have high levels of SAP (Clampitt and Hart, 1978); increased serum levels may reflect the histopathological changes observed in the kidneys of rats treated with these liquid fuels. Elevated serum cholesterol levels were observed in HGOII-treated groups and might be related to the effects of the test material on fatty acid metabolism. Decreased serum glucose was noted in the HGOI and HGOII groups, and this effect may be a result of decreased food intake and poor health status rather than specific organ damages. Hematological changes were perhaps the most prominent toxic effects that occurred following administration of these liquid fuels. Studies from our laboratory in rats treated with HGOII for 3 mo showed increased serum iron and total iron-binding capacity, suggesting that the observed anemia may be a result of defective heme synthesis. In general, some biochemical and/or hematological changes observed here may represent an adjustment in the physiological state of the animals suffering decreased food intake and weight loss. HGOI and HGOII were also found to be more toxic than LGO in the mutagenicity assays. The fact that HGOI and HGOII were more active after metabolic activation also suggests the presence in these fractions of mutagenic PAHs components that require metabolic activation into biologically active intermediates. Compared to known mutagens such as 9aminoanthracene and other positive controls, the coprocessing products are still considered to be weak mutagens. In conclusion, treatment with coal coprocessing products resulted in toxicological changes including growth suppression, increased liver weight, increased hepatic ER activity, hematological aberrations, and histological changes in liver, thyroid, kidney, bone marrow, and thymus. These effects are considered to be in the same order of magnitude to those observed for CLP. Based on the data presented above, HGOI and HGOII appear to be more toxic than LGO, and are selected for longerterm studies.
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REFERENCES Ames, B. M., McCann, J., and Yamasaki, E. 1975. Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsomes mutagenicity test. Mutat. Res. 31:347-364. Burke, M. D., and Mayer, R. T. 1974. Ethoxyresorufin: Direct fluorimetric assay of a microsomal Odealkylation which is preferentially inducible by 3-methylcholanthrene. Drug. Metab. Dispos. 2:583-588. Chu, I., Villeneuve, D. C , Côté, M., Valli, V. E., and Otson, R. 1988a. The dermal toxicity of a medium-boiling (154-378°C) coal liquefaction product in the rat (Part I). J. Toxicol. Environ. Health 23:193-206. Chu, I., Villeneuve, D. C , Côté, M., Secours, V., Otson, R., and Valli, V. E. 1989b. Dermal toxicity of a high-boiling (bp 250-450°C) coal liquefaction product in the rat (Part II). J. Toxicol. Environ. Health 25:509-525. Chu, I., Rinehart, W., Hoffman, G., Villeneuve, D. C , Otson, R., and Valli, V. E. 1989. Subacute inhalation toxicity of a medium-boiling coal liquefaction product (154-378°C) in the rat (Part III). J. Toxicol. Environ. Health 28:195-204.
Clampitt, R. B., and Hart, R. J. 1978. The tissue activities of some diagnostic enzymes in ten mammalian species. J. Comp. Pathol. 88:607-621. Cochin, J., and Axelrod, J. 1959. Biochemical and pharmacological changes in the rat following chronic administration of morphine, nalorphine and normorphine. J. Pharmacol. Exp. Ther. 125:105-110. Fouts, J. R. 1963. Factors influencing the metabolism of drugs in liver microsomes. N. Y. Acad. Sci. 104:875-880. Nie, N. H., Hull, C. H., Jenkins, J. G., Steinbrenner, K., and Bent, O. H. (1977). Statistical Programs for the Social Sciences. Chicago: SPSS, Inc. Received August 28, 1990 Accepted February 15, 1991
