Effects of subchronic inhalation exposure of mice to a high-boiling coal liquid

FUNDAMENTALANDAPPLIEDTOXICOLOGY

9,277-286(1987)

Effects of Subchronic Inhalation Exposure of Mice to a High-Boiling Coal Liquid’ DAVID L. SPRINGER,RODNEY A. MILLER, CHERYLYN W. WRIGHT, HARVEY A. RAGAN, RAY L. BUSCHBOM,AND D. DENNIS MAHLUM Biology and Chemistry Department, Pacific Northwest Laboratory, P.O. Box 999, Richland, Washington 99352

Effects of Subchronic Inhalation Exposure of Mice to a High-Boiling Coal Liquid. SPRINGER, D. L., MILLER, R. A., WRIGHT, C. W., RAGAN, D. D. (1987). Fundam. Appl. Toxicol. 9,277-286.

H. A., BUSCHBOM,

R. L., AND MAHLUM,

Mice (CD-l) were exposed to aerosol concentrations of 0.0, 0.03,O. 14, or 0.69 mg/liter of heavy distillate (HD), a high-boiling coal liquid from the solvent-refined coal (SRC)-II process. Exposures were for 6 hr/day, 5 days/week for 13 weeks. Particle sizes ranged between 1.6 and 1.8 pm, mass median aerodynamic diameter, with a geometric standard deviation range of 1.9-2.5. Growth for high-dose males was significantly less than that of the control group. Compared to controls, weights of liver were significantly higher and those of ovaries and thymus significantly lower; these changes were significant on both absolute and relative weight bases. The number of red blood cells, volume of packed red cells, and hemoglobin concentration for animals from the high-dose group were significantly lower than those of controls. Microscopic examination of organ sections showed focal hepatic necrosis and nonspecific hepatopathy. Additionally, olfactory epithelial degeneration occurred in a dose-dependent manner. Results from this study indicated that exposure to HD caused adverse effectsat the high dose and that these changes were either less severe or absent in middledose group mice. Comparison of these results with those for rats indicated that with rats the biological effects were more severe and present at lower doses than was observed for mice. Q 1987 Society of Toxicology

Coal, an abundant natural resource in North America, is considered an alternative energy source, but its use raises health and environmental concerns. Solvent-refined coal (SRC) processes, which are methods for converting coal to liquid products that are low in sulfur and minerals, are under evaluation. Products derived from the liquefaction of coal have been tested for their ability to produce adverse biological effects in a variety of test systems with numerous endpoints. Heavy distillate (HD), a high-boiling-range material (288-454”Q is derived from the SRC-II process (Glasstone, 1982). Exposure of Salmonella typhimurium (Pelroy and Petersen, 198 1) or Chinese hamster ovary cells to HD ’ Work supported by the U.S. Department of Energy under Contract DE-AC06-76RL0 1830. 277

resulted in mutations (Frazier and Andrews, 1983). Repeated dermal application of HD to the shaved backs of mice resulted in the rapid appearance of skin tumors (Renne et al., 198 1). Papillomas developed after a single dermal application of HD, followed by repeated treatment with a phorbol ester promoter (Mahlum, 1983; Mahlum et al., 1984). When HD was administered by gavage or inhalation it also produced embryotoxicity and malformations in the offspring of pregnant rats treated on Days 12-16 of gestation (Hackett et al., 1984; Springer et al., 1982b). To expand the toxicologic database for coal liquids, we conducted a subchronic inhalation study to HD, with simultaneous exposure of rats and mice. For rats, survival through 13 weeks of exposure was greater than 90% for all groups; body weights for ex0272-0590/87 $3.00 Copyright Q 1987 by the Society of Toxicology. All rights ofreproduction in any form reserved.

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posed animals were decreased in a dose-dependent manner (Springer et al., 1986). Significant increases in liver weights and decreases in thymus and ovary weights were observed for treated animals relative to controls. There were also significant treatmentrelated decreases in erythrocytes, hemoglobin, volume of packed red blood cells, lymphocytes, eosinophils, and total white blood cells. After 5 weeks of exposure, serum cholesterol concentrations increased in a dosedependent manner for both sexes and serum triglyceride amounts decreased for males but not for females. After 13 weeks of exposure, high-dose animals had significant increases in cholesterol (males only), triglycerides, blood urea nitrogen, and serum glutamic-pyruvic transaminase (SGPT; males) and significant decreases in albumin, SGPT (females), and lactate dehydrogenase (LDH). Examination of bone marrow preparations from exposed animals demonstrated consistent decreases in the degree of cellularity, suggesting that this organ is a target for HD. Microscopic evaluation of organ sections indicated exposure-related changes for nasal mucosa, pulmonary macrophages, thymus, liver, kidney, bone marrow, ovaries, and cecum. Results for rats indicated dose-dependent increases in the severity of the lesions observed, with few effects in the low-exposure group that were attributable to the exposure. This paper describes the results for survival, growth, organ weights, hematology, and histopathology for mice after 13 weeks of exposure to HD.

METHODS Material. The SRC-II material used in this study, HD (boiling range, 288-454”C), was obtained from the Ft. Lewis, Washington pilot plant, which was operated by the Pittsburg & Midway Coal Mining Company. The coal liquid sample, collected on April 3-4, 1980, while processing Powhatan No. 6 coal, was stored at 4°C under nitrogen in Teflon-lined drums. Characterization of major components of HD has been reported by Wright et al. ( 1984).

The chemical class composition of the HD, determined by separation on neutral alumina according to the method of Later et al. ( 198 1,1985) and given as a weight percentage, was 20% aliphatic hydrocarbons (AH), 53% polycyclic aromatic hydrocarbons (PAH), 19% nitrogencontaining polycyclic aromatic compounds (NPAC), and 10% hydroxylated PAH (HPAH). The major components of the AH fraction were straight-chain AH containing 16 to 29 carbon atoms. Components of the HPAH fraction were mono-hydroxy-substituted parent and alkylated PAH containing two to four aromatic rings. Components ofthe PAH and NPAC fractions were quantified in their respective fractions by high-resolution gas chromatography according to the method described by Wilson et al. (1984) using 2-chloroanthracene as an internal standard, with external standard instrument calibration. Concentrations (mg/g) of selected components in the HD from these two fractions are listed in Table 1 as the average of two replicates, each analyzed at concentrations of 2.50,5.00, and 10.0 mg/ml. The following gas chromatographic conditions were used: a Hewlett-Packard 5880A gas chromatograph equipped with 25 m X 0.25 mm i.d. fused silica capillary column coated with 0.25-pm film thickness DB-5 (J&W Scientific, Inc.. Folsom, CA); helium carrier gas at 50 cm/sac linear velocity, splitless injection, flame ionization detection, and an oven temperature program rate of 3’C/min from 50 to 300°C after 2 min isothermal operation at the initial temperature. Samples of HD were evaluated for changes in chemical composition and biological activity to determine whether the material was stable during storage. Data from these samples indicated that both the composition and the mutagenic activity remained constant for the period between sample collection and exposure of mice for this study (Wright and Weimer, 1984). Exposure system. The inhalation exposure system employed in the current study was described previously (Moss et al., 1982); it was modified for whole-body animal exposure to coal liquids (Springer et al.. 1982a). Air flow through the chambers was from top to bottom at a rate of 283 liters/min. The HD aerosol, produced by two Solo-sphere nebulizers (McGaw Respiratory Therapy, Irvine, CA), was tran,sferred to a 7.62-cm stainless-steel manifold, where appropriate amounts were withdrawn and diluted with HEPA-filtered room air to the desired concentration in three Battelle exposure chambers (Lab Products, Inc., Maywood, NJ). The fourth chamber, which housed control animals, was supplied with HEPAfiltered room air. The concentration of HD was determined by impinging samples ofaerosol onto Metricel filters (0.45 rrn; Gelman Science, Ann Arbor, MI), eluting the samples into chloroform, and quantitating by ultraviolet (uv) absorbance at 254 nm. Triplicate aerosol samples, collected at three equal time intervals during each 6-hr exposure period, were averaged to give a daily concentration.

SUBCHRONIC

INHALATION

TABLE 1 CONCENTRATION OF SELECTED COMPOUNDS OF SOLVENT-REFINED COAL HEAVV DISTILLATE Compound’ Naphthalene 2-Methylnaphthalene Fluorene 2-Methylfluorene Phenanthrene Anthracene 3-Methylphenanthrene 2-Methylphenanthrene 9- or 4-Methylphenanthrene 1-Methylphenanthrene Fluoranthene Pyrene Benzo[a]fluorene Benzo[b]fluorene, 2- or 4-methylpyrene 1-Methylpyrene Benz[a]anthracene Chrysene 6- or 4-Methylchrysene Benzo[j or blfluoranthene Benzo[k]fluoranthene Benzo[e]pyrene Benzo[a]pyrene Indeno[ 1,2,3-cdlpyrene Benzo[ghi]perylene Benzo[h]quinoline Acridine Benzo[flquinoline, phenanthridine Carbazole 2- or 3-Methylbenzo[flquinoline 1-Methylcarbazole 3-Methylcarbazole 2-Methylcarbazole 4-Methylcarbazole I- or 4-Azapyrene Benzo[a]carbazole Benzo[b]carbazole Benzo[c]carbazole Dibenzo[a, g or c, g]carbazole

Concentration @p/g) b 0.05 0.14 1.24 3.95 52.2 I .67 14.4 16.7 1.51 3.70 5.73 31.2 4.76 17.7 1.83 1.64 2.83 1.23 0.97 0.57 1.01 0.65 0.27 0.64 1.68 0.97 3.31 15.3 1.15 2.20 5.80 2.99 1.41 2.36 1.42 0.72 1.21 0.08

a Identified by retention time and mass spectral information. b Average of two replicates, three determinations each. Mean concentrations over the 13 weeks ofexposure were 0.00, 0.029 + 0.003, 0.14 ? 0.01, and 0.69 + 0.03 mg/ liter of air for the control, and low-, middle-. and highexposure groups, respectively. Aerosol samples for particle size analysis were collected either once or twice weekly from each chamber

COAL LIQUID

MICE

279

using a Mercer cascade impactor. The particle size distributions were estimated (mass median aerodynamic diameter; MMAD), using the computer program NEWCAS (Pacific Northwest Laboratory, 1977). Mean MMAD (X f SE) for the low-, middle-, and high-exposure chambers over the 13 weeks were 1.7 f 0.04, 1.6 f 0.04, and 1.8 + 0.05 pm, respectively; geometric Standard deviations (GSD) for the size distributions for samples taken from all chambers ranged from 1.9 to 2.5. At the end of each exposure period, chamber air 50~s were increased to 340 liters/min to decrease the time required to clear aerosol from the chambers. Ammonia levels were measured several times during the 13-week exposure using low-range ammonia detection tubes (Gastec Corp., Yokohama, Japan; available from Safety and Supply Co., Seattle, WA). It was necessary to place deotized cage board (Shepherd Specialty Papers, Kalamazoo, MI) in the catch pans under each cage unit during nonexposure periods to maintain ammonia concentrations at less than 5 ppm. Animals and experimental design. Sixty male and 60 female CD-I mice (Charles River, Kingston, NY) were quarantined for 3 weeks prior to exposure. Mice were individually identified and randomly assigned by weight to one of the three treatment groups or the control group (1 S/sex/group). Beginning at 9 weeks of age, mice were exposed to the HD aerosol for 6 hr daily, 5 days/week for 13 weeks. Throughout the 13-week exposure period, the mice were observed twice daily and weighed weekly. Animals were continuously housed in the chambers and had free access to water at all times; food (Wayne Lab Blox; Chicago, IL; 24.5% protein) was available ad libitum during nonexposure hours. Room fluorescent lighting was on a 12-hr electronically controlled cycle. Temperatures within the chambers were continuously monitored and averaged 23 f 1°C (X? SD) during the study. Relative humidities, recorded three times daily, averaged 68 f 7%. After 13 weeks of exposure, blood was collected into tubes containing potassium EDTA by retroorbital bleeding of ether-anesthetized animals. A Coulter Model S counter was used to determine erythrocyte (RBC) and leukocyte (WBC) counts, hemoglobin (Hgb) concentration, volume of packed red cells (VPRC), and the red cell indices (MCV, MCH, and MCHC). Blood smears were made and stained with Wright-Giemsa stain prior to classifying a minimum of 200 leukocytes. A second blood smear was made from each animal and stained with new methylene blue for reticulocyte counts. Serologic tests for pneumonia virus of mice, Reo 3, GDVII, polyoma virus, minute virus of mice, Ectromelia, Sendai virus, mouse adenovirus, lymphocytic choriomeningitis virus, mouse hepatitis virus, and Mycoplasma pulmonis were performed on samples from 15 mice selected randomly from all chambers. Serological assays were performed by Microbiological Associates (Bethesda, MD). Blood samples were negative for evidence of these pathogens.

280

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All mice were necropsied; body weights and weights for adrenals, brain, gonads, heart, kidneys, liver, spleen, and thymus were recorded. Organs were preserved in 10% neutral buffered Formalin. Organs from 10 randomly selected mice of each sex from each of the treatment and control groups were examined histologically. Organs evaluated included adrenal glands. aorta, bone marrow, brain, cecum, colon, duodenum, esophagus, heart, kidneys, larynx, liver, lungs, mammary glands, mandibular lymph nodes, mesenteric lymph nodes, nasal cavity, ovaries, pancreas, parathyroid gland, pituitary gland, prostate, salivary gland, skin, spleen, stomach, testis, thyroid gland, trachea, tracheobronchial lymph nodes, thymus, uterus, and urinary bladder; grossly abnormal tissues were also examined. Nasal sections were taken immediately posterior to the incisor teeth (anterior). midway between the incisor teeth and the first molar (middle), and through the second molar (posterior). Tissue sections were embedded in paraffin, microtomed at 4-6 pm, and stained with hematoxylin and eosin. All organ sections from controls and high-dose animals were examined by light microscopy; target organs identified in the high-dose group were evaluated in the next lower dose group until either no effect was observed or the lowest-dose-group organs were examined. Statistics. Body-weight growth curves were analyzed by a randomization test (Lindgren, 1963); this test is a nonparametric statistical test based on the absolute area between growth curves. Analysis of variance (Steel and Torrie, 1960) was used to analyze tissue weight, erythrocyte, and WBC measurements. Treatment means were compared with control means by Dunnett’s test (Dunnett, 1955). The level of significance for each statistical comparison was 0.05.

RESULTS Mortality was limited to one high-dose male that died during the first week of exposure and one low-dose female that died during the 11th exposure week; both deaths were unrelated to the exposure. Growth for highdose male mice was significantly less than that of the control group; this effect developed after 1 week and persisted throughout the exposure (Fig. 1). Growth for females followed similar trends, although differences between controls and high-dose females were not statistically significant. Liver weights were increased for males from the middle- and high-dose groups and for females from the high-dose group (Table 2). Thymus weights were decreased for fe-

ET AL.

FIG.1. Body weights for male and female mice during the 13-week inhalation exposure to solvent-refined coalII heavy distillate.

males from the middle- and high-dose groups and for males from the high-exposure group. Ovary weights were decreased for high-dose females. Changes in these organ weights were significant both on an absolute basis and on a relative weight basis. After 13 weeks of exposure to HD, RBC, Hgb, and VPRC values were decreased for high-dose animals relative to controls (Table 3). This effect was observed for both males and females. Other hematologic parameters were unaffected by exposure. Histologically, a subtle treatment-related change was observed in liver sections of highdose male and female mice (Table 4). This hepatopathy was characterized by a slight increase in cytoplasmic basophilia, slightly more variability than normal in hepatocellular size, the presence of hepatomegalocytes, increased variability in nuclear size, and minimal loss of cording and lobular pattern (Fig. 2). Based on these kinds of changes, middle-

SUBCHRONIC

INHALATION

COAL

TABLE

LIQUID

281

MICE

2

BODY WEIGHT AND ORGAN TO BODY WEIGHT RATIOS FOR MICE EXPOSED BY INHALATION FOR 13 WEEKS TO SOLVENT-RELINED COAL-II HEAVV DISTILL.ATE~ Exposure Sex Male

Female

Observation

Control

N Body weight b Adrenals’ Brain Gonads Heart Kidney Liver Spleen Thymus

39.2 0.023 1.321 0.664 0.556 1.764 5.550 0.266 0.102

N Body weight’ Adrenals Brain Gonads Heart Kidney Liver Spleen Thymus

15 28.8 f 0.579 0.049 f 0.002 1.716+0.041 0.086 + 0.008 0.559 + 0.025 1.473 f 0.04 1 5.641 c 0.205 0.373 + 0.016 0.159 -co.012

“.Tt SE, ’ Grams. ’ Organ to body weight * Significantly different

ratio, from

(%). control

15 k + + f f f + _t iz

Low

group

0.658 0.003 0.025 0.034 0.032 0.045 0.160 0.015 0.01 I

15 39.3 f 0.021 + 1.314 + 0.647 f 0.550 * 1.673 f 5.775 + 0.300 * 0.10 1 *

29.0 0.044 1.755 0.085 0.584 1.506 5.759 0.352 0.139

group Middle

0.950 0.002 0.028 0.02 1 0.02 1 0.044 0.102 0.013 0.009

14 + 0.760 f 0.003 + 0.048 f 0.006 + 0.044 f 0.065 + 0.110 + 0.019 *0.010

39.1 0.023 1.276 0.718 0.519 1.745 6.107 0.269 0.101

15 f 0.053 f 0.002 f 0.033 f 0.012 f 0.018 + 0.056 + 0.215* i 0.0 17 f 0.008

15 29.2 f 0.358 0.046 i 0.005 1.690 f 0.032 0.074 + 0.007 0.562 + 0.022 1.373 kO.071 6.009 kO.152 0.378 + 0.021 0.109+0.010*

High

37.0 0.027 1.376 0.675 0.545 1.812 8.175 0.264 0.072

14 + 0.507 f 0.002 + 0.03 1 f 0.018 f 0.018 f 0.056 + 0.186* t 0.0 15 f 0.006*

15 27.4 f 0.707 0.046 f 0.003 1.743 f 0.055 0.06 1 + 0.004* 0.594 f 0.02 1 1.514~0.031 7.730 f 0.212* 0.332 + 0.022 0.101 + 0.007*

mean (p d 0.05).

and low-dose mouse livers from either sex factory epithelial degeneration was charactercould not be distinguished from those of con- ized by increased basophilia of the olfactory trols. Minimal scattered focal hepatic necro- epithelium, scattered pyknotic and karyorsis was also observed in four male and three rhectic nuclei, disorganization and loss of female high-dose mice and in one middlesensory neurons, and decreased sustentacular dose male and two low-dose males; this lesion cell cytoplasm. A few mice had small foci of was not observed in control animals of either basal cell hyperplasia and occasional hypersex. Transmission electron microscopic ex- trophic ducts of Bowman’s glands. Fewer leamination of selected liver samples indicated sions were observed in middle-dose mice of that there was proliferation of the smooth en- both sexes and they were present mainly in doplasmic reticulum and, possibly, an in- the middle nasal sections. Nasal sections of crease in secondary lysosomes in high-dose low-dose mice could not be distinguished animals relative to controls. from controls. Although olfactory epithelial Minimal olfactory epithelial degeneration degeneration was diagnosed in one control was observed along the dorsal meatus of the male and two control females, it was more fomiddle and posterior nasal sections of the cal and associated with areas of prominent high-dose male and female mice (Fig. 3). Ol- eosinophilic globules. No lesions were ob-

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ET AL.

served in the respiratory epithelium of HDexposed mice. Olfactory epithelial lesions were not originally observed in rats exposed to the same concentrations of HD (Springer et al., 1986). Review of the rat nasal sections showed that, in addition to the respiratory epithelial lesions previously diagnosed, exposed rats had subtle olfactory epithelial changes similar to the mice. No olfactory lesions were seen in control rats. Most high-dose rats of both sexes had olfactory epithelial lesions in the middle and posterior nasal sections. Most middledose rats of both sexes had similar changes primarily in the middle nasal sections. Half of the low-dose male rats had lesions restricted to the middle nasal sections whereas few low-dose female rats had lesions in this region. Examination of ovarian sections from high-dose animals showed an apparent decrease in the amount of luteal tissue in three animals; other ovarian structures appeared normal. Ovaries from middle- and low-dose mice were indistinguishable from those for controls. DISCUSSION

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Comparison of the effects observed in mice following inhalation exposure to HD with those for similarly exposed rats demonstrated both differences and similarities in the responses of the two species (Springer et al., 1986). Generally, similar effects were less pronounced in mice than in rats. Although mortality during exposure was less than 10% for both species, body weight gain was more severely depressed for rats than for mice. Even though at the start of the exposure the ages of mice and rats were similar (i.e., 9 and 12 weeks, respectively) the observation that the body weight effects appeared more severe for rats was probably due to rapid growth of the rats, whereas growth for mice was nearly complete. Significant changes in mice were observed in weights of liver, ovaries, and thy-

SUBCHRONIC

INHALATION

COAL LIQUID

283

MICE

TABLE 4 LES~ONS(NUMBEROFLESIONS/NUMBEREXAMINED)FORMICEEXPOSEDBYINHALATION TOSOLVENT-REFTNEDCOAL-IIHEAWDISTILLATE High

Control

Liver Hepatopathy Necrosis Nose Olfactory epithelial atrophy Ovary Decreased luteal tissue

Male

Female

2110 o/10

o/10 o/10

l/IO

2110

Male

Middle

Low

Female

Male

Female

Male

Female

8/10 4110

8/10 3110

l/10 l/IO

o/10 o/10

o/10 2110

NE” NE

lO/lO

9110

3110

4/10

o/10

l/10

019

319

o/10

NE

a Not examined.

mus, with effects present primarily in animals from the high-dose group. These changes were also observed for rats; however, most effects were present in both the middle- and high-dose groups. For mice from the highdose group, RBC parameters were below those of controls, but WBC counts were not significantly affected. In contrast, lower RBC values were observed for rats from both the middle- and high-dose groups, and total WBC were significantly lower primarily due to fewer lymphocytes. Microscopic lesions in mice were limited to liver, olfactory epithelium, and ovaries, whereas in rats the lesions were present in these and several other organs. Even though changes were observed in RBC parameters for mice, hypocellularity of bone marrow, as seen in the rat, was not observed. Since effects on RBC values were less pronounced in mice, bone marrow changes may have been less severe and thus not detected by microscopic examination; this interpretation is consistent with the reported insensitivity of light microscopic evaluation of paraffin sections of bone marrow (Collan, 1982). Other effects observed in rats but absent in mice included (1) pelvic epithelial hyperplasia of the kidney, (2)

atrophy of the thymus, (3) squamous metaplasia of the respiratory epithelium and suppurative inflammation of anterior nasal sections, (4) histiocytosis of the lung, (5) decreased numbers of megakaryocytes in the spleen, and (6) epithelial hyperplasia, ulceration, and chronic active inflammation of the cecum. Increased liver weights in male and female high-dose mice correlated with hepatocytomegaly and proliferation of smooth endoplasmic reticulum. The functional significance of the hepatocellular lesions in mice is probably minimal. The hepatocellular necrosis was scattered and focal and involved only small portions of liver parenchyma. The hepatocytomegaly seen in the mice was much less obvious than that seen in the rats, where there was some clinical pathologic evidence of hepatocellular dysfunction. Lower ovarian weights may be correlated with a decrease in luteal tissue, which may be the result of suppressed ovarian cycling. Thymic weight decreases were not severe enough to permit identification of changes by light microscopy. Degeneration of the olfactory epithelium, varying from loss of isolated sensory cells to

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ET AL.

FIG. 2. Photomicrographs of liver sections from CD- 1 mice demonstrating an increase in variability of hepatocyte and nuclear size and hepatomegalocytes. (A) Control and (B) a section from a mouse exposed by inhalation to 0.69 mg/liter of HD for 13 weeks (H&E stain; original magnification, x50).

more expansive loss of sensory cells, sustentacular cells and underlying nerves, is not uncommon in inhalation exposure of mice to irritants (Buckley et al., 1984). Nasal olfactory epithelial lesions seen in the HD-exposed mice are comparable in locations and descriptions to those reported for other recent inhalation toxicity studies (Jiang et al., 1986). The presence of olfactory epithelial lesions in the middle and posterior nasal sections in the high-dose mice and similar lesions more restricted to the middle nasal section in the middle dose is consistent with the anteriorposterior severity gradient commonly seen with inhaled irritants. When olfactory epithelium is affected, the anterior-dorsal extension of the olfactory epithelium is the most commonly involved site (Jiang et al., 1986; Buckley et al., 1984). The HD-exposed rats had nasal olfactory lesions similar to the

mice, but lesions extended to lower doses. Additionally, the rats had squamous metaplasia and inflammation of the respiratory epithelium. Thus, rats had lesions more compatible with conventional irritants than did mice. It has been shown that mice were more capable of reducing minute ventilation than were rats after repeated exposure of formaldehyde, thereby reducing the amount of formaldehyde available for deposition in the nasal cavity (Chang et al., 1983). Since the respiratory rate and tidal volume were not measured in the present study, it was not possible to quantify the amount of HD inhaled in rats versus mice. Therefore, whether the respiratory tract differences (or the other differences for that matter) between HD-exposed rats and mice were due to rats receiving a higher “dose” of HD than the mice or if they repre-

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FIG. 3. Photomicrographs of nasal olfactory epithelium from the middle nasal section along the dorsal meatus of CD- 1 mice demonstrate disorganized olfactory epithelium, pyknotic nuclei, ,overall thinning of the olfactory epithelium, and decreased sustentacular cell cytoplasm. (A) Control and (B) a section from a mouse exposed by inhalation to 0.69 mg/liter of HD for 13 weeks. (H&E stain; original magnification, X50).

sent more species-specific effects is unknown. It is possible that HD aerosols affect the olfactory epithelium at lower doses but at higher doses they act more like a conventional irritant and affect both types of epithelia. Even though the aerosol concentration used for the high-exposure group of this study is high relative to anticipated human exposures, the fact that certain effects were observed in the middle- and high-exposure groups indicates that these doses may be considered in setting exposure limits for humans. The lowest dose, 0.03 mg/liter, is three times the threshold limit value (TLV) for exposure to nuisance dust and a factor of 150 times greater than the TLV for coal tar pitch volatiles (American Conference of Governmental Industrial Hygienists, 1986). Conformance to limits similar to those for coal tar pitch vola-

tiles should result in human doses below those which we have found to cause effects in animals.

ACKNOWLEDGMENTS The authors particularly thank R. R. Adee, G. A. Apley, J. A. Brower, K. H. Debban, C. J. Gerdes, P. S. Lytz, K. M. McCarty, M. C. Perkins, M. A. Pope, and D. C. Snyder for excellent technical assistance. We also thank L. D. Winter and D. L. Felton for assistance in preparation of the manuscript.

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