Upper Respiratory Tract Toxicity of Inhaled Methylvinyl Ketone in F344 Rats and B6C3F1 Mice

Daniel L. Morgan*, Herman C. Price{dagger}, Robert W. O'Connor{dagger}, John C. Seely{ddagger}, Sandra M. Ward*, Ralph E. Wilson* and Michael C. Cunningham*

* National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709; {dagger} ManTech Environmental Technology, Inc., Research Triangle Park, North Carolina 27709; and {ddagger} PATHCO, Inc., Research Triangle Park, North Carolina 27709

Received April 24, 2000; accepted August 9, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The National Toxicology Program is conducting a chemical class study to investigate the structure-activity relationships for the toxicity of {alpha},ß-unsaturated ketones. Methylvinyl ketone (MVK) was selected for study because it is a representative straight-chain aliphatic {alpha},ß-unsaturated ketone and because of its extensive use and widespread exposure. Short-term inhalation studies of MVK were conducted to provide toxicity data for comparison with other {alpha},ß-unsaturated ketones and for use in designing chronic toxicity and carcinogenicity studies. In 2-week studies, rats and mice were exposed to 0, 0.25, 0.5, 1, 2, 4, or 8 ppm MVK 6 h/day, 5 days/week for 12 exposures. Morbidity and early deaths occurred in all male and female rats after 1 exposure and in 2 male mice after 10 exposures to 8 ppm. Rats exhibited nasal cavity toxicity and lung necrosis at 4 ppm. No toxicity was observed in animals exposed to less than 2 ppm. Based on these results a 13-week study was conducted at 0, 0.5, 1, and 2 ppm MVK. As observed in the 2-week study, the nasal cavity was the main target organ and rats were more sensitive than mice. Respiratory and olfactory epithelial necrosis were prominent by day 21 in the rat. At study termination these lesions were still evident but not as severe as noted earlier. Additionally, changes such as olfactory epithelial regeneration and metaplasia (respiratory) as well as respiratory epithelial hyperplasia and metaplasia (squamous) were clearly evident. Nasal lesions in mice were limited to a subtle squamous metaplasia of transitional and/or respiratory epithelium covering predominantly the tips of naso- and maxilloturbinates in Levels I and II. A transient, leukopenia was observed in rats exposed to 2 ppm, however, this effect was not present after 13 weeks of exposure. In mice, leukocyte counts were significantly decreased at all exposure concentrations after 13 weeks of exposure. Absolute testicular and epididymal weights and sperm counts were decreased at the high dose only. MVK can be characterized as a reactive, direct-acting gaseous irritant. MVK exposure causes the same nasal cavity lesions as the cyclic {alpha},ß-unsaturated ketone, 2-cyclohexen-1-one, although at lower exposure concentrations.

Key Words: Methylvinyl ketone; 3-buten-2-one; {alpha},ß-unsaturated ketones; inhalation; upper respiratory tract; nasal cavity; gaseous irritant.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The National Toxicology Program is conducting a chemical class study to investigate the structure-activity relationships for the toxicity of {alpha},ß-unsaturated ketones. The ketones being studied are methylvinyl ketone, and ethylvinyl ketone (representative straight chain aliphatic ketones), 2-cyclohexene-1-one (a representative cyclic ketone), and methylstyryl ketone (a representative aromatic ketone). These {alpha},ß-unsaturated ketones were selected based on demonstrated human industrial and consumer exposure and inadequate health effects testing. All these chemicals share the {alpha},ß-unsaturated ketone moiety with the more widely studied {alpha},ß-unsaturated aldehyde acrolein, a highly electrophilic and cytotoxic chemical. Short-term inhalation toxicity studies are being conducted on these structurally-related ketones using the same study design to facilitate comparisons of toxicity.

Methylvinyl ketone (MVK; 3-buten-2-one) was selected as a prototype of the straight-chain aliphatic {alpha},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., 1989Go), perfumes (Giersch and Schulte-Elte, 1990Go), plastics and resins (Papa and Sherman, 1981Go; Basavaiah et al., 1987Go), and as a pharmaceutical intermediate in the synthesis of steroids, vitamin A, and anticoagulants (Ferroni et al., 1989Go; Matsuda, 1987Go; Nakayama et al., 1985Go; Sax and Lewis, 1987Go). Consumer exposure to MVK is widespread due to its presence in cigarette smoke (0.13 mg/cigarette) (Kusama et al., 1978Go; Curvall et al., 1984Go; Florin et al., 1980Go), and its presence in vehicular exhaust (Jonsson and Berg, 1983Go; Westerholm et al., 1990Go).

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, 1991Go).

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, 1991Go; McMahon et al., 1979Go; Florin et al., 1980Go; Curvall et al., 1984Go; Marnett et al., 1985Go; Williams et al., 1989Go) 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Four- to five-week-old male and female B6C3F1 mice (Charles River, Portage, MI), and male and female Fischer 344 rats (Taconic Farms, Germantown, NY) were weighed, and randomized to treatment groups (6 animals/sex/time point/exposure concentration). Animals were acclimated in holding cages for approximately two weeks before the study start and were 6 to 7 weeks old at the commencement of exposure. All animals were acclimated to the Hazleton whole body exposure chambers for three days prior to MVK exposure. Animals were fed NTP-2000 diet and given chlorinated, filtered and UV-disinfected water ad libitum. Food was removed during the exposures.

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. 78–94–4) (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, 1981Go; Boorman et al., 1990Go; Herbert and Leininger, 1999Go).

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., 1993Go). 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, 1969Go). 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).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhalation Exposures
The chamber MVK concentrations were within ± 10% of the target concentration throughout the 2- and 13-week studies. Mean chamber concentrations (± standard deviations) for the 2-week studies were 1.90 ± 0.03, 3.90 ± 0.07 and 7.90 ± 0.04 ppm MVK. Mean chamber concentrations for the 13-week studies were 0.50 ± 0.01, 1.00 ± 0.04, and 2.01 ± 0.05 ppm MVK.

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. 1Go). Relative lung weights of male rats exposed to 4 ppm for 12 days were significantly greater than control lung weights (Table 1Go). 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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FIG. 1. Body weights of male and female rats exposed to MVK 6h/day, 5 days/week for 12 exposures. Values represent means ± SE, n = 5 animals. Squares, 0 ppm; circles, 2.0 ppm; triangles, 4.0 ppm. *Significantly less than controls at respective time points (p < 0.05).

 

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TABLE 1 Lung Weights of Rats and Mice Exposed to Methylvinyl Ketone for 2 Weeks
 
Histopathology.
Histopathological effects of MVK were limited to the airways of the upper and lower respiratory tract (Table 2Go). Widespread acute necrosis of respiratory and olfactory epithelial cells was concentration-dependent in both males and females. Extensive areas of the mucosal surface were denuded or covered only by degenerated epithelium. Necrosis was accompanied by cellular and proteinaceous fluid exudate in the nasal passages. At a given concentration there was generally a decreasing severity of necrosis from anterior to posterior and central to peripheral. The epithelia of the maxillary sinuses and the nasopharyngeal duct were also necrotic. Nasal glands were not affected. At 8 ppm, necrosis was the predominant effect; at lower concentrations regenerative hyperplasia and squamous metaplasia of the respiratory epithelium and respiratory metaplasia of olfactory epithelium were also observed. At 1 ppm, exposure related lesions were limited to the anterior nasal cavity and consisted of mild squamous metaplasia of the respiratory/transitional epithelium. This change was observed in the mucosa covering the tips of the turbinates and the lateral wall of the most anterior nasal section. Metaplasia was characterized by loss of cilia and slight stratification of the epithelium with flattening of superficial cells without overt keratinization. In general, there were no gender differences in susceptibility.


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TABLE 2 Respiratory Tract Lesions in Rats and Mice Exposed to Methylvinyl Ketone for 2 Weeks
 
Acute necrosis of respiratory epithelium lining the large airways of the lower respiratory tract occurred at concentrations of 4 and 8 ppm. The necrosis was more severe in the larger airways and included inflammatory cell infiltration and luminal exudate often forming an inflammatory pseudomembrane. At 8 ppm, necrosis extended to the level of small bronchioles. A similar effect was visible in the trachea. Similar to the nasal cavity, regenerative hyperplasia and squamous metaplasia were observed in addition to necrosis at 4 ppm. The no-observed-effect level (NOEL) for lung effects in rats was 2 ppm.

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. 2Go). Relative lung weights of male and female mice were significantly increased after exposure to 8 ppm MVK for 12 days (Table 1Go). 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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FIG. 2. Body weights of male and female rats exposed to MVK 6h/day, 5 days/week for 12 exposures. Values represent means ± SE, n = 5 animals. Squares, 0 ppm; circles, 2.0 ppm; triangles, 4.0 ppm; diamonds, 8.0 ppm. *Significantly less than controls at respective time points (p < 0.05).

 
Histopathology.
Nasal epithelial necrosis of a similar severity and pattern to that seen in the rats was also observed in mice (Table 2Go). In contrast to rats, necrosis of the nasal vestibule was present; olfactory epithelium was spared at lower concentrations, and metaplastic changes were not apparent at lower concentrations. At 1 ppm MVK, exposure related findings in mice were less severe and widespread than observed in rats.

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 10–15% (females) less than controls thereafter (Fig. 3Go). Relative lung and kidney weights were significantly increased in female rats exposed to 2 ppm (Table 3Go).



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FIG. 3. Body weights of male and female rats exposed to MVK 6h/day, 5 days/week for 13 weeks. Values represent means ± SE, n = 10 animals. *Significantly less than controls at respective time points (p < 0.05).

 

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TABLE 3 Selected Organ Weights of Rats Exposed to Methylvinyl Ketone for 13 Weeks
 
Clinical pathology.
There were no biologically significant changes in any of the serum chemistry parameters measured in rats after 4, 21 or 90 days of exposure (data not shown). Total leukocyte counts were significantly decreased in male rats exposed to 2 ppm MVK for 4 or 21 days (Table 4Go). Decreased leukocyte counts were attributed to significant decreases in lymphocyte counts at this exposure concentration.


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TABLE 4 Decreased Leukocytes in Male Rats and Mice Exposed to Methylvinyl Ketone for 13 Weeks
 
Histopathology.
Lung, larynx, nasal cavity, liver, heart, ovary, uterus, testis, epididymis, spleen, bone, and kidneys were examined from the control and high-concentration rats. Treatment-related lesions were identified only in the nose and therefore this tissue was examined from all concentration groups. In addition, the nasal cavities from 5 rats/sex/concentration were examined from clinical pathology animals after 21 days exposure.

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. 4Go). 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. 5Go). 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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FIG. 4. (A) Nasal septum, Level I, control rat at day 21. Normal appearing respiratory mucosa lines the nasal septum. The ciliated respiratory epithelium overlies the lamina propria, which contains nasal glands and blood vessels. Bar = 30 µ.(B) Nasal septum, Level I, of a rat exposed to 2 ppm MVK for 21 days. Note the focal area of respiratory epithelial degeneration and necrosis accompanied by mononuclear cell inflammation within the lamina propria. Bar = 30 µ.

 


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FIG. 5. (A) Dorsal meatus, Level II, control rat. Normal appearing olfactory epithelium. Bar = 200 µ.(B) Dorsal meatus, Level II, of a rat exposed to 2 ppm MVK for 21 days. Extensive necrosis and desquamation of the olfactory epithelium. Bar = 200 µ. (C) Dorsal meatus, Level II, control rat. Olfactory epithelium overlying the lamina propria which contains bundles of nerve fibers. Bar = 40 µ. (D) Dorsal meatus, Level II, of a rat exposed to 2 ppm MVK for 21 days. Inflammation, edema, and loss of nerve fibers are evident within the lamina propria. Bar = 40 µ.

 
At 13 weeks treatment-related lesions were again identified in the nose (Table 5Go). The distribution of these lesions was similar to that at day 21; however, the lesions were less severe at 13 weeks. Respiratory epithelial hyperplasia was present in all exposed groups, and the incidence was dose-related. Areas of respiratory epithelial hyperplasia along the nasal septum (Level I) were characterized by an increased number of epithelial cells which contributed to the overall loss of the orderly appearance of the respiratory epithelium (Fig. 6Go). Many cells appeared hypertrophied and the presence of ciliated cells was reduced. Squamous metaplasia was also noted primarily in the respiratory epithelium covering the nasal septum and the naso- and maxilloturbinates (Fig. 7Go). Squamous metaplasia at this time point was predominantly non-keratinizing. Treatment-related lesions were not present in the larynx or lungs of exposed rats.


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TABLE 5 Nasal Cavity Lesions in Rats Exposed to Methylvinyl Ketone for 13 Weeks
 


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FIG. 6. (A) Nasal septum, Level I, of a rat exposed to 2 ppm MVK for 90 days. The respiratory epithelium has become hyperplastic. Bar = 25 µ. (B) Nasal septum, Level I, of a rat exposed to 2 ppm MVK for 90 days. Squamous metaplasia of the respiratory epithelium. Bar = 25 µ.

 


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FIG. 7. (A) Nasoturbinate, Level I, control rat. Normal appearing transitional epithelium covering the tip of the turbinate. Bar = 40 µ. (B) Nasoturbinate, Level I, of a rat exposed to 2 ppm MVK for 90 days. Hyperplasia and squamous metaplasia of the epithelium. Bar = 40 µ.

 
Sperm motility and vaginal cytology evaluation.
In male rats exposed to 2 ppm MVK, left epididymis, and the left testis absolute weights were 10% and 5% less than those of controls (Table 6Go). However, the relative weight of left epididymus weight was not significantly different from control, and the relative weight of the left testis was significantly greater than control. Changes in absolute and relative epididymus and testis weights were not concentration related. The total number of sperm per cauda was 22% less than controls at 2 ppm. However, the number of sperm/mg cauda, sperm motility, number of spermatids/mg testis, and total spermatids/testis were not significantly different between groups.


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TABLE 6 Reproductive Toxicity in Male Rats Exposed to Methylvinyl Ketone for 13 Weeks
 
Evaluation of vaginal smears revealed no differences between treated and control rats in the amount of time spent in different estrous stages, cycle length, number of cycles, number of cycling females, or the number of females with regular cycles (data not shown).

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 7Go); 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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TABLE 7 Selected Organ Weights of Mice Exposed to Methylvinyl Ketone for 13 Weeks
 
Clinical pathology.
There were no biologically significant changes in any of the serum chemistry parameters measured in mice after exposure for 13 weeks (data not shown). In male mice, total leukocyte counts were significantly decreased at all MVK exposure concentrations after 13 weeks of exposure (Table 4Go). Decreased leukocyte numbers in male mice were attributed to significant decreases in lymphocyte and neutrophil counts at all exposure concentrations.

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. 8Go). 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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FIG. 8. (A) Maxilloturbinate, Level I, control mouse. Normal appearing transitional epithelium. Cells are generally cuboidal in shape and sparsely ciliated. Bar = 20 µ.(B) Maxilloturbinate, Level I, of a mouse exposed to 2 ppm MVK for 90 days. Note squamous metaplasia characterized by several layers of flattened cells and reduction in cilia. Bar = 20 µ.

 
Sperm motility and vaginal cytology evaluation.
The total number of sperm per cauda, the number of sperm/mg cauda, sperm motility, number of spermatids/mg testis, and total spermatids/testis in treated male mice were not significantly different from those of controls (data not shown). Evaluation of vaginal smears revealed no differences between treated and controls in the amount of time spent in different estrous stages, cycle length, number of cycles, number of cycling female mice, or the number of females with regular cycles.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Short-term inhalation studies of MVK were conducted as part of a chemical class study of the {alpha},ß-unsaturated ketones. These studies were designed to characterize and compare the toxicity of MVK, a prototypical straight-chain {alpha},ß-unsaturated ketone, with that of other {alpha},ß-unsaturated ketones. In addition, these data will be used to design chronic toxicity and carcinogenicity studies of MVK.

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, 1991Go). 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 1973Go; Lash and Woods, 1991Go). 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, 1990Go). Similar upper respiratory tract lesions have been described for direct-acting gaseous irritants such as chlorine (Jiang et al., 1983Go), formaldehyde (Monteiro-Riviere and Popp, 1986Go), ammonia (Broderson et al., 1976Go), acetaldehyde (Kruysse et al., 1975Go), acrolein (Feron et al., 1978Go) and cigarette smoke (Vidic et al., 1974Go).

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., 1990Go). Similarly, repair of olfactory epithelium can result in squamous or respiratory metaplasia, or in complete recover of olfactory epithelium (Hardisty et al., 2000Go).

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 {alpha},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 {alpha},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 {alpha},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.


    ACKNOWLEDGMENTS
 
Inhalation exposures were conducted at the NIEHS inhalation facility under contract to METI, RTP, NC. SMVCE evaluations were conducted by R.O.W. Sciences, Inc., Gaithersburg, MD. The authors acknowledge the technical assistance of C. Best, R. Bousquet, R. Chapin, C. Colegrove, D. Crawford, M. Diehl, N. Gage, M. Goods, J. Mahler, M. Moorman, S. Philpot, P. Rydell, A. Savitz, Y. Wang, G. Wolfe, and E. Zeiger.


    NOTES
 
To whom correspondence should be addressed at Respiratory Toxicology, Mail Stop IF-00, NIEHS, P.O. Box 12233, Research Triangle Park, NC 27709. Fax: (919) 541-0356. E-mail: morgand{at}niehs.nih.gov.


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