* National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709;
ManTech Environmental Technology, Inc., Research Triangle Park, North Carolina 27709; and
PATHCO, Inc., Research Triangle Park, North Carolina 27709
Received April 24, 2000; accepted August 9, 2000
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ABSTRACT |
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Key Words: Methylvinyl ketone; 3-buten-2-one; ,ß-unsaturated ketones; inhalation; upper respiratory tract; nasal cavity; gaseous irritant.
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INTRODUCTION |
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Methylvinyl ketone (MVK; 3-buten-2-one) was selected as a prototype of the straight-chain aliphatic ,ß-unsaturated ketones for which human industrial and consumer exposure has been documented, and for which health effects testing has been inadequate or lacking. Methylvinyl ketone is used commercially in the production of pesticides (Chuman et al., 1989
), perfumes (Giersch and Schulte-Elte, 1990
), plastics and resins (Papa and Sherman, 1981
; Basavaiah et al., 1987
), and as a pharmaceutical intermediate in the synthesis of steroids, vitamin A, and anticoagulants (Ferroni et al., 1989
; Matsuda, 1987
; Nakayama et al., 1985
; Sax and Lewis, 1987
). Consumer exposure to MVK is widespread due to its presence in cigarette smoke (0.13 mg/cigarette) (Kusama et al., 1978
; Curvall et al., 1984
; Florin et al., 1980
), and its presence in vehicular exhaust (Jonsson and Berg, 1983
; Westerholm et al., 1990
).
Methylvinyl ketone is highly irritating to mucous membranes, eyes (lacrymator), and the skin. Reported toxicity data for MVK include: an oral LD50 of 30 mg/kg in the rat; an oral LD50 of 33 mg/kg in the mouse; an inhalation LC50 of 7 mg/m3/4h (2.4 ppm/4h) in the rat and inhalation LC50 of 8 mg/m3/2h (2.8 ppm/2h) in the mouse (RTECS, 1991).
Methylvinyl ketone has been described as a model alkylating agent and Michael acceptor that binds to cellular protein sulfhydryl groups and glutathione (GSH). Lash and Woods (1991) studied MVK toxicity in freshly isolated rat kidney nephron cells. Methylvinyl ketone caused irreversible injury to distal tubular cells; proximal tubular cells were more resistant to MVK. Incubation of cells with MVK led to an altered cellular GSH status and pronounced inhibition of mitochondrial respiration.
Various in vitro tests of MVK genetic toxicity have produced mixed results (NTP, 1991; McMahon et al., 1979
; Florin et al., 1980
; Curvall et al., 1984
; Marnett et al., 1985
; Williams et al., 1989
) suggesting that MVK may be weakly genotoxic. There is some evidence that MVK may interact covalently with DNA. Chung et al. (1988) demonstrated the formation of two MVK-guanine adducts when deoxyguanosine was reacted with MVK in vitro. The mixed results of genetic tests and the demonstrated reaction with DNA indicate that MVK may have carcinogenic potential.
Two- and 13-week toxicity studies of MVK were conducted by inhalation in rats and mice to identify potential target organs, gender and species differences in susceptibility, and to provide exposure concentration-response data. These studies demonstrated that the primary target organ in both species was the nasal cavity, and rats were considerably more susceptible to the respiratory tract toxicity than mice.
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MATERIALS AND METHODS |
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This study was conducted under federal guidelines for the use and care of laboratory animals and was approved by the NIEHS Animal Care and Use Committee. Animals were housed in a humidity- and temperature-controlled, HEPA-filtered, mass air displacement room in facilities accredited by the American Association for Accreditation of Laboratory Animal Care. Animal rooms were maintained with a light-dark cycle of 12 h (light from 0700 to 1900 h). Sentinel animals housed in the animal facility as part of an ongoing surveillance program for parasitic, bacterial, and viral infections were pathogen-free throughout the study.
Inhalation exposure.
Methylvinyl ketone (CAS No. 78944) (purity 99%) was obtained from Aldrich Chemical Co. (Milwaukee, WI). The desired exposure chamber MVK concentrations were achieved by purging (room air, ambient temperature) the headspace of individual sealed vials each containing a measured amount of MVK. The MVK-air mixture was then mixed with conditioned air (HEPA filtered, charcoal scrubbed, temperature and humidity controlled) and delivered to the Hazleton 2000 exposure chambers at approximately 400 L/minute. Concentrations of MVK in each chamber were measured at 2.5-minute intervals using individual gas chromatographs (Photovac 10S70) containing nonpolar capillary columns (530 µmx9 meter, Cpsil 5CB, Photovac International) and precolumns (530 µmx1 meter). The columns were maintained at 30°C and medical grade breathing air (10 cc/minute) was the carrier gas. Gas chromatographs were equipped with photoionization detectors.
Animals were individually housed in the Hazleton 2000 chambers and exposed (whole body) to MVK for 6 h/day (approximately 7 AM to 1 PM), 5 days/week (weekends excluded) for either 2 or 13 weeks. Animals were exposed for two consecutive days before sacrifice. Control animals breathed conditioned air.
2-Week Studies
Rats and mice (5/sex/specie/exposure concentration) were exposed to 0.25, 0.5, 1, 2, 4, or 8 ppm MVK or conditioned air (controls) for 6 h/day, 5 days/week for 12 exposures. Animals were weighed the day just prior to initial exposure, and after 4, 7, and 12 exposures. Immediately after the last exposure, animals were euthanized by CO2 asphyxiation and necropsied. Prior to fixation or formalin infusion the right kidney, liver, and lungs were weighed. Lungs, nasal cavity, kidney, liver, spleen, brain, stomach, heart, thymus, and adrenal glands were fixed in 10% formalin, embedded in paraffin, sectioned at 5 µ, and stained with hematoxylin and eosin. Slides were evaluated by light microscopy.
13-Week Study
Rats and mice (10/sex/specie/exposure concentration) were exposed to 0.5, 1, or 2 ppm MVK, or conditioned air (controls) for 6 h/day, 5 days/week for 13 weeks. Body weights were recorded on day 1 just prior to exposure and weekly thereafter. On the morning after the last exposure animals were anesthetized (70:30 mixture of CO2:O2), blood was collected for clinical pathology, and then animals were euthanized by CO2 asphyxiation. Tissue weights were obtained for liver, thymus, right kidney, right testicle, heart, and lungs.
Clinical pathology.
An additional 10 rats/sex/exposure concentration were included for clinical chemistry and hematology. Immediately after exposure for 4 and 21 days, rats were anesthetized (CO2:O2) and blood was collected from the retro-orbital plexus. Rats dedicated to clinical pathology were euthanized after the 21-day blood collection. Blood was collected from the remaining rats and mice (retro-orbital plexus) on the morning after the last exposure (13 weeks). Blood from rats was analyzed for clinical chemistry and hematology, and blood from mice was used for hematology only.
Blood samples were centrifuged in serum collection vials at 100xg for 10 min. Serum samples were analyzed for creatinine, urea nitrogen (UN), alanine aminotransferase (ALT), alkaline phosphatase, creatine kinase (CK), sorbitol dehydrogenase (SDH), albumin, total protein, urea nitrogen, creatinine, and total bile acids using an automated analyzer (Monarch System 2000, Instrumentation Laboratory, Lexington, MA) and commercially available reagents.
An additional blood sample was collected for hematology. Blood was collected using heparin-rinsed micropipettes and transferred to 2.5 ml EDTA tubes. Complete blood counts, white blood cell counts and differentials were performed using a Technicon H*1 hematology analyzer (Miles, Inc., Tarrytown, NY). Reticulocyte counts were performed manually on blood smears stained with New Methylene Blue (EK Industries, Joliet IL). On the morning after the last exposure, remaining rats and mice were anesthetized (CO2:O2), and blood collected for clinical pathology. Blood from rats was analyzed for clinical chemistry and hematology, and blood from mice was used for hematology only.
Histopathology.
After blood collection, animals were euthanized by CO2 asphyxiation and a complete necropsy conducted. Tissues were collected, fixed in 10% formalin, embedded in paraffin, sectioned at 5 µ, and stained with hematoxylin and eosin. Tissues saved for histological evaluation at the 90-day sacrifice include: adrenal glands, brain (3 sections including frontal, cortex and basal ganglia, parietal, cortex and thalamus, and cerebellum and pons), clitoral glands, esophagus, femur, including diaphysis with marrow cavity and epiphysis, gallbladder (mouse), heart and aorta, intestine, large (cecum, colon, rectum), intestine, small (duodenum, jejunum, ileum), kidneys, liver (2 sections including left lateral lobe and median lobe), lungs and mainstem bronchi, lymph nodes (mandibular and mesenteric), mammary gland with adjacent skin, nasal cavity and nasal turbinates (3 sections), ovaries, pancreas, parathyroid glands, pituitary gland, preputial glands, prostate, salivary glands, seminal vesicle, spleen, stomach (forestomach and glandular), testes with epididymis, thymus, thyroid glands, trachea, urinary bladder, and uterus.
In addition to the tissues collected at the 13-week necropsy, nasal cavity was collected for histopathological evaluation from the clinical pathology rats euthanized on day 21. Three standard nasal sections were prepared: Level I represented a section immediately posterior to the upper incisor teeth; Level II represented a section in between the incisive papilla and first palatal ridge, and Level III represented a section posterior to the first upper molar teeth (Young, 1981; Boorman et al., 1990
; Herbert and Leininger, 1999
).
Sperm motility and vaginal cytology.
At necropsy, the left testis and epididymis from rats and mice were collected and weighed. Sperm motility and sperm density counts were conducted. The left testis was collected and frozen for later spermatid counts. Vaginal smears were prepared for female rats and mice for the last 12 consecutive days of exposure. The vaginal cytology slides were evaluated and the estrous cycle stage (proestrus, estrus, metestrus, or diestrus) was determined for each day (Cooper et al., 1993). The cycle length, number of cycles, number of cycling females, and number of females with a regular cycle were determined.
Statistics.
For weight data and clinical pathology data, statistically significant differences (p<0.05) between treatment groups were determined by one-way ANOVA and Dunnett's multiple comparison tests (Sokal and Rohlf, 1969). Sperm motility and vaginal cytology data were evaluated using the non-parametric multiple comparisons procedures of Dunn (1964) or Shirley (1977) as modified by Williams (1986).
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RESULTS |
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2-Week Study, Rats
Mortality.
After one exposure to 8 ppm MVK, all male and female rats were either found dead or were euthanized in moribund condition. The cause of morbidity and mortality was attributed to airway necrosis.
Body and organ weights.
Body weights of male and female rats exposed to 2 or 4 ppm were significantly less than those of controls (Fig. 1). Relative lung weights of male rats exposed to 4 ppm for 12 days were significantly greater than control lung weights (Table 1
). Absolute lung weights of female rats exposed to 4 ppm were significantly less than controls; however, the biological significance of this effect is not clear. Exposure to 0.5 or 1.0 ppm MVK for 2 weeks had no significant effects on body or organ weights of rats (data not shown).
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2-Week Study, Mice
Mortality.
Two of five male mice were found dead after ten exposures to 8 ppm. The cause of mortality was attributed to airway necrosis. No mortality or morbidity was observed at exposure concentrations below 8 ppm.
Body and organ weights.
Body weights of mice exposed to 4 or 8 ppm were significantly less than controls after 4, 7, and 12 exposures (Fig. 2). Relative lung weights of male and female mice were significantly increased after exposure to 8 ppm MVK for 12 days (Table 1
). Exposure to MVK concentrations below 2 ppm had no significant effects on body or organ weights of mice (data not shown).
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Acute necrosis of the respiratory epithelium lining the large airways of the lower respiratory tract was observed in mice exposed to 8 ppm MVK. In contrast to rats, necrosis was limited to this group of mice, and metaplastic changes were not observed. The NOEL for lung effects in mice was 4 ppm.
13-Week Study, Rats
Body and organ weights.
Body weights of male and female rats exposed to 2 ppm MVK were significantly less than controls after one week of exposure and remained at approximately 20% (males) and 1015% (females) less than controls thereafter (Fig. 3). Relative lung and kidney weights were significantly increased in female rats exposed to 2 ppm (Table 3
).
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At day 21, treatment-related lesions were evident in the nose of the 1 and 2 ppm males and females. In rats exposed to 2 ppm, patchy areas of respiratory epithelial degeneration and necrosis were observed in both Levels I and II of the nasal cavity (Fig. 4). Non-keratinizing, squamous metaplasia of the respiratory epithelium was evident in the 1 and 2 ppm rats at this time point as well. In addition, minimal chronic inflammation and serous exudation were also present. Olfactory epithelial necrosis was generally limited to the dorsal meatus of Level II and occasionally Level III of rats exposed to 2 ppm MVK (Fig. 5
). Olfactory epithelial necrosis was not evident in the 1 ppm animals. Nasal lesions were not present at day 21 in rats exposed to 0.5 ppm.
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13-Week Study, Mice
Body and organ weights.
Body weights of MVK-exposed male and female mice were not significantly different from controls throughout the study (data not shown). However, relative liver weights of male mice were significantly increased at all exposure concentrations (Table 7); absolute liver weight and heart weights were increased at 2 ppm. In female mice relative kidney and lung weights were increased at 2 ppm MVK; relative liver weights were significantly increased at 1 and 2 ppm.
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Histopathology.
Liver, heart, ovary, uterus, testis, epididymis spleen, bone, kidneys, lung, larynx and nasal cavity were examined from the control and high-dose mice. Treatment-related lesions were identified only in the nose and therefore this tissue was examined from all dose groups. Treatment-related lesions in the nose were only observed in the 2 ppm group. These lesions include squamous metaplasia of the transitional and/or respiratory epithelium on the tips of the naso- and maxilloturbinates in the Level I to II sections (Fig. 8). Squamous metaplasia was characterized by an increased thickness due to replacement of normal ciliated epithelium by increased numbers of oval to slightly flattened cells that lacked cilia. Lesions were minimal in severity and were present in 8/10 male and 7/10 female mice. Treatment-related lesions were not identified in the larynx or lungs of exposed mice.
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DISCUSSION |
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Initial 2-week studies were conducted to identify relevant exposure ranges and potential species differences in acute toxicity. Methylvinyl ketone was considerably more toxic for rats than mice. In rats, MVK exhibited a relatively steep dose-response curve with 100% mortality at 8 ppm and no mortality at 4 ppm. All rats were found dead or moribund after one exposure to 8 ppm, while only two male mice died after ten exposures to 8 ppm. The cause of morbidity and mortality in both species was attributed to airway necrosis.
The LC50 of MVK was previously reported as 2.4 ppm/4 h exposure in rats and approximately 2.8 ppm/2 h exposure in mice (RTECS, 1991). These reported LC50 values indicate that mice were more susceptible than rats to the lethal effects of MVK, and that MVK was considerably more toxic for both species than in the current study. In our study, animals survived exposure to higher MVK concentrations for a longer exposure period (6 h/day for 12 exposures). Based upon our mortality data, the LC50 for F344 rats was estimated to be between 4 and 8 ppm/6 h exposure, and considerably greater than 8 ppm/6 h exposure for B6C3F1 mice. The discrepancies between these two studies are likely a result of differences in inhalation exposure technology, animal strains, moribund sacrifice criteria and other differences in experimental design.
Histopathological evaluation of tissues from surviving animals also indicates that toxicity was greater in rats than mice. In rats, respiratory lesions occurred at lower exposure concentrations than in mice after two weeks exposure to non-lethal concentrations. The severity and pattern of nasal epithelial necrosis was similar in rats and mice; however, in mice the olfactory epithelium was spared and metaplastic changes were not apparent. This species difference in susceptibility was more apparent after a longer exposure period. In the 13-week study, exposure to the highest concentration (2 ppm) caused nasal lesions in rats but had no effect on the nasal cavity of mice.
The mechanism(s) for the species differences in susceptibility to MVK is (are) not clear. Differences in breathing patterns, as well as anatomical differences and airflow patterns, may contribute to species differences in the tissue dose of MVK in the upper respiratory tract. Methylvinyl ketone is a reactive alkylating agent and Michael acceptor that readily binds to sulfhydryl groups of proteins and glutathione (Zollner 1973; Lash and Woods, 1991
). Differences in susceptibility may be due to species differences in protective mechanisms such as the availability of protein sulfhydryls, glutathione, and protective enzymes in the upper and lower respiratory tract.
These results indicate that MVK is a direct acting irritant, i.e., does not require metabolism for toxicity. Indeed, the chemical structure of MVK suggests that metabolism would likely be a detoxification reaction producing less reactive metabolites such as alcohols and mercapturic acids. The toxicity of inhaled MVK vapors was primarily restricted to the respiratory tract as would be predicted for a reactive, direct-acting, gaseous irritant. The non-specific distribution of lesions in both respiratory and olfactory epithelium and the distinct anterior-posterior gradient in severity of damage are typical of direct acting nasal toxicants (Gaskell, 1990). Similar upper respiratory tract lesions have been described for direct-acting gaseous irritants such as chlorine (Jiang et al., 1983
), formaldehyde (Monteiro-Riviere and Popp, 1986
), ammonia (Broderson et al., 1976
), acetaldehyde (Kruysse et al., 1975
), acrolein (Feron et al., 1978
) and cigarette smoke (Vidic et al., 1974
).
At 13 weeks respiratory and olfactory epithelial necrosis were still evident in the high-dose rats, but were less severe than that noted at 21 days. Respiratory epithelial metaplasia, hyperplasia and squamous metaplasia, as well as olfactory epithelial regeneration and mineralization were present and were typical of changes that result from epithelial necrosis or associated with longer exposure periods. Adaptive squamous metaplasia of the respiratory epithelium is a common response in rodent nasal passages following chronic exposure to cytotoxic irritants. This metaplastic change is characterized by the replacement of the more susceptible respiratory epithelium by squamous epithelium which is more resistant to injury by inhaled toxicants (Monticello et al., 1990). Similarly, repair of olfactory epithelium can result in squamous or respiratory metaplasia, or in complete recover of olfactory epithelium (Hardisty et al., 2000
).
Clinical chemistry parameters and microscopic evaluation of extrapulmonary tissues revealed no significant evidence of systemic toxicity in either species. Significant reductions in testis weight and sperm numbers were observed in the high concentration male rats; however, these effects were not concentration related, and there was no histopathological evidence of toxicity. If the testis is a target organ for MVK in rats these effects may become more prominent after chronic exposure. A transient, mild leukopenia was observed in rats exposed to 2 ppm for 4 and 21 days, however, this effect was not present after 13 weeks of exposure. Leukopenia was more prominent in mice; after 13 weeks of exposure leukocyte counts were significantly decreased at all exposure concentrations. Leukopenia and slight increases in organ weights in mice may be indicative of mild systemic toxicity of MVK caused by absorption of MVK from the respiratory tract, and/or ingestion during grooming. Alternatively, these effects may be secondary to the inflammation in the nasal cavity.
As a part of the chemical class study of ,ß-unsaturated ketones, MVK was studied using the same short-term inhalation study designs as those used for 2-cyclohexen-1-one (CHX), a cyclic
,ß-unsaturated ketone (Cunningham et al., manuscript submitted). The short-term toxicity of MVK was similar to that of CHX although MVK was considerably more potent. In general, both ketones were more toxic for rats than for mice. The nasal cavity was the primary target organ for both ketones, and the pattern of hyperplasia and squamous metaplasia of the respiratory epithelium was similar for MVK and CHX. The greater nasal toxicity of MVK may be attributed to its greater reactivity. The ring-stabilized ketone of CHX may be less likely to undergo Michael addition reactions than the ketone functionality of MVK. It is also possible that steric hindrance from the cyclohexene ring may also limit reactivity of CHX with cellular components.
These short-term inhalation studies were conducted to characterize the toxicity of MVK in F344 rats and B6C3F1 mice. Based upon the study results, inhaled MVK can be characterized as a direct-acting upper respiratory tract irritant. The primary target organ in both species was the nasal cavity, and rats were considerably more susceptible to respiratory tract toxicity than mice. Qualitatively, the toxicity of MVK is similar to that observed for CHX, a structurally-related ,ß-unsaturated ketone; however, MVK is a more potent toxicant. In designing a chronic MVK inhalation study the respiratory tract of rats and mice should be considered a potential target organ. Other potential target organs in a chronic study include testes in the male rat, and possibly liver and kidney in mice.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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Boorman, G. A., Morgan, K. T., and Uriah, L. C. (1990). Nose, Larynx and Trachea. In, Pathology of the Fischer Rat: Reference and Atlas, (G. A. Boorman, S. L. Eustis, M. R. Elwell, G. A. Montgomery, and W. F. Mackenzie, Eds.). pp. 315337. Academic Press, San Diego, CA.
Broderson, J. R., Lindsey, J. R., and Crawford, J. E. (1976). The role of environmental ammonia in respiratory mycoplasmosis of rats. Am. J. Pathol. 85, 115130.[Abstract]
Chuman, T., Guss, P. L., Doolittle, R. E., McLaughlin, J. R., and Tumlenson, J. H., III (1989). Isolation and preparation of 6,12-dimethylpentadecan-2-one as attractant for the banded cucumber beetle. Patent US 207591-A0 issued to the US Dept of Agriculture. [Abstract: CA102:119238].
Chung, F. L., Roy, K. R., and Hecht, S. S. (1988). A study of reactions of ,ß-unsaturated carbonyl compounds with deoxyguanosine. J. Org. Chem. 53, 1417.[ISI]
Cooper, R. L., Goldman, J. M., and Vandenbergh, J. G. (1993). Monitoring of the estrous cycle in the laboratory rat by vaginal lavage. In Female Reproductive Toxicity (R. E. Chapin and J. J. Heindel, Eds. ), pp. 4556, Academic Press, New York.
Cunningham, M. L., Price, H. V., O'Connor, R. W., Moorman, M. P., Mahler, J. F., Nold, J. B., and Morgan, D. L. (2000). Inhalation toxicity studies of the ,ß-unsaturated ketones: 2-cyclohexen-1-one. Inhal. Tox. (in press).
Curvall, M., Enzell, C. R., Jansson, T., Petterson, B., and Thelestam, M. (1984). Evaluation of the biological activity of cigarette smoke condensate fractions using six in vitro short-term tests. J. Toxicol. Env. Health 14, 163180.[ISI][Medline]
Dunn, O. J. (1964). Multiple comparisons using rank sums. Technometrics 6, 241272.[ISI]
Feron, V. J., Kruysse, A., Til, H. P., and Immel, H. R. (1978). Repeated exposure to acrolein vapor: subacute studies in hamsters, rats, and rabbits. Toxicology 9, 4757.[ISI][Medline]
Ferroni, R., Milani, L., Simoni, D., Orlandini, P., Guarneri, M., Franze, D., and Bardi, A. (1989). 4-(1H-Pyrazol-1YL)-2-butylamine derivatives as inhibitors of blood platelet aggregation. Il Farmaco, 44, 495502.[Medline]
Florin, I., Rutber, L., Curvall, M., and Enzell, C.R. (1980). Screening of tobacco smoke constituents for mutagenicity using the Ames test. Toxicology 15, 219232.[ISI][Medline]
Gaskell, B. A. (1990). Nonneoplastic changes in the olfactory epithelium Experimental studies. Environ. Health Perspect. 85, 275189.[ISI][Medline]
Giersch, W. and Schulte-Elte, K. H. (1990). Preparation of tricyclic spiroketones as perfumes. Patent EP 382934-A2. [Abstract: CA 114:207551].
Hardisty, J. F., Garman, R. H., Harkema, J. R., Lomax, L. G. and Morgan, K. T. (2000). Histopathology of nasal olfactory mucosa from selected inhalation toxicity studies conducted with volatile chemicals. Tox. Pathol. 27, 618627.[ISI]
Herbert, R. A. and Leininger, J. R. (1999). Nose, Larynx and Trachea. In, Pathology of the Mouse: Reference and Atlas, (R. R. Maronpot, Ed. ), pp. 259292, Cache River Press, Vienna, IL.
Jiang, X. Z., Buckley, L. A., and Morgan, K. T. (1983). Pathology of toxic responses to the RD50 concentration of chlorine gas in the nasal passages of rats and mice. Toxicol. Appl. Pharmacol. 71, 225236.[ISI][Medline]
Jonsson, A. and Berg, S. (1983). Determination of low molecular weight oxygenated hydrocarbons in ambient air by cryogradient sampling and two-dimensional gas chromatography. Chromatog. 279, 307322.
Kruysse, A., Feron, V. J., and Til, H. P. (1975). Repeated exposure to acetaldehyde vapor. Studies in Syrian golden hamsters. Arch. Environ. Health 31, 449452.
Kusama, M., Sakuma, H., and Sugawara, S. (1978). Low boiling compounds in cellulose cigarette smoke. Agric. Biol. Chem. 42, 479481.[ISI]
Lash, L. H. and Woods, E. B. (1991). Cytotoxicity of alkylating agents in isolated rat kidney proximal tubular and distal tubular cells. Arch. Biochem. Biophys. 286, 4656.[ISI][Medline]
Marnett, L. J., Hurd, H. K., Hollstein, M. C., Levin, D. E., Esterbauer, H., and Ames, B. N. (1985). Naturally occurring carbonyl compounds are mutagens in Salmonella tester strain TA104. Mutat. Res. 148, 2534.[ISI][Medline]
Matsuda, I. (1987). Michael-type addition of O-ethyl-C-O-bis(trimethylsilyl)ketene acetal and its application to the synthesis of -xylidene-
-lactones. J. Organomet. Chem. 321, 307316.[ISI]
McMahon, R. E., Cline, J. C., and Thompson, C. Z. (1979). Assay of 855 test chemicals in ten tester strains using a new modification of the Ames test for bacterial mutagens. Canc. Res. 39, 682693.[Abstract]
Monteiro-Riviere, N. A. and Popp, J. A. (1986). Ultrastructural evaluation of acute nasal toxicity in the rat respiratory epithelium in response to formaldehyde gas. Fundam. Appl. Tox. 6, 251262.[ISI]
Monticello, T. M., Morgan, K. T., and Uraih, L. (1990). Nonneoplastic nasal lesions in rats and mice. Environ. Health Perspect. 85, 249274.[ISI][Medline]
Nakayama, M., Tanimore, S., Hashio, M., and Mitani, Y., (1985). Stereoselective synthesis of C/D/E/rings in steroids containing E (lactone) ring. Synthesis of (+)-1-(1'-hydroxyethyl)-7,7a-dihydro-5(6H)-indanone-7a-carboxylic acid-7a,1'-lactone. Chem. Lett. 5, 613614.
NTP (1991). National Toxicology Program: Results and status information on all NTP chemicals, NTP Chemtrack System, 4/3/91.
Papa, A. J., and Sherman, P. D., Jr. (1981). Ketones. In Kirk-Othmer Encyclopedia of Chemical Technology, 3rd Ed., (H. F. Mark, D. F. Othmer, C. G. Overberger, G. T. Seaborg, , and M. Grayson, eds.) Vol. 13, pp. 894941. New York, John Wiley and Sons.
RTECS (1991). Registry of Toxic Effects of Chemical Substances Database, National Library of Medicine, Bethesda, MD, October, 1991.
Sax, N. I., and Lewis, R. J., Eds. (1987). Hawley's Condensed Chemical Dictionary, 11th Ed. Van Nostrad Reinholt Co., New York.
Shirley, E. (1977). A non-parametric equivalent of William's test for contrasting increasing dose levels of a treatment. Biometrics 33, 386389.[ISI][Medline]
Sokal, R. R., and Rohlf, F. J. (1969). Biometry. The Principles and Practice of Statistics in Biological Research. W. H. Freeman, San Francisco.
Vidic, B., Rana, M. W., and Bhagat, B. D. (1974). Reversible damage of rat upper respiratory tract caused by cigarette smoke. Arch. Otolaryngol. 99, 110113.[ISI]
Westerholm, R., Almen, J., Hang, L., Runnug, U., and Rosen, A. (1990). Chemical analysis and biological testing of exhaust emissions from two catalyst equipped light duty vehicles operated at constant cruising speeds 70 and 90 km/h, and during acceleration conditions from idling up to 70 and 90 km/h. Sci. Total Environ. 93, 191198.[ISI]
Williams, D. (1986). A note on Shirley's non-parametric test for comparing several dose levels with a zero-dose control. Biometrics 42, 183186.[ISI][Medline]
Williams, G. M., Mori, H., and McQueen, C. A. (1989). Structure-activity relationships in rat hepatocyte DNA-repair test for 300 chemicals. Mutat. Res. 221, 263286.[ISI][Medline]
Young, J. T. (1981). Histopathologic examination of the rat nasal cavity. Fundam. Appl. Toxicol. 1, 309312.[Medline]
Zollner, H. (1973). Inhibition of some mitochondrial functions by acrolein and methylvinyl ketone. Biochem. Pharmacol. 22, 11711178.[ISI][Medline]