* National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina; and
Battelle Pacific Northwest, Richland, Washington
Received August 13, 1999; accepted January 10, 1999
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ABSTRACT |
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Key Words: glutaraldehyde; toxicity; carcinogenicity; nose.
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INTRODUCTION |
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Occupational exposure occurs mainly by inhalation and skin contact. Workplace concentrations ranging from less than 0.005 to 0.57 ppm have been reported (ACGIH, 1997) and routine industrial hygiene monitoring from 1977 to 1992 indicated that glutaraldehyde concentrations were generally less than 0.1 ppm in well-ventilated workplaces (ACGIH, 1997
; NICNAS, 1994
). The odor threshold for glutaraldehyde is 40 ppb (Beauchamp et al., 1992
). Glutaraldehyde is a respiratory, skin, and eye irritant (ACGIH, 1997
; Ballantyne, 1995
; NICNAS, 1994
). The minimum human irritation response level for glutaraldehyde has been reported to be 300 ppb (Ballantyne, 1995
; St. Clair et al., 1990
). Respiratory sensitization, such as in asthma and rhinitis, has been associated with glutaraldehyde in various occupational settings at concentrations as low as 32 ppb (ACGIH, 1997
; NICNAS, 1994
). Skin sensitization, contact dermatitis, and skin discoloration by glutaraldehyde have been well documented (ACGIH, 1997
; Beauchamp et al., 1992
; Jordan et al., 1972
; NICNAS, 1994
). However, Dearman and co-workers (1999) noted recently that glutaraldehyde results in a cytokine secretion profile in mice that is consistent with allergic sensitization of the respiratory tract and not the skin.
In a retrospective study on the cause of deaths among 1109 embalmers, the number of deaths due to leukemia and cancers in the brain, colon, and prostate were increased when compared to the expected number of deaths based on age-, race-, and calendar year-specific proportions of deaths for each cause among the United States male population (Walrath and Fraumeni, 1984). Deaths due to brain cancer and those that appeared to be due to leukemia were increased among 2317 men who joined the American Association of Anatomists between 1888 and 1969 (Stroup et al., 1986
). An increase in leukemia and cancer of the brain and lung was noted in pathologists (Consensus Workshop on Formaldehyde, 1984
). Embalmers, anatomists, and pathologists are often exposed to formaldehyde and glutaraldehyde.
Gene mutations in S. typhymurium strains TA100, TA102, and TA104 were found with glutaraldehyde in the absence of S9 (Dillon et al., 1998; Haworth et al., 1983
; Jung et al., 1992
). Formaldehyde also induced gene mutations in S. typhimurium strains TA100, TA102, and TA104 in the absence of S9 (Dillon et al., 1998; Haworth et al., 1983; NTP, unpublished results), at approximately the same concentrations as glutaraldehyde (100200 µg/plate),
Glutaraldehyde, formaldehyde, and acetaldehyde are low-molecular-weight, reactive aldehydes that have similar chemical properties, result in similar biological effects, and have a common metabolic pathway. The common pathway in the metabolism involves aldehyde dehydrogenases, which have been identified in most tissues (Beauchamp et al., 1992; Casanova-Schmitz et al., 1984
; Heck et al., 1990
). It is generally assumed that aldehydes initially react with amino acids to form Schiff bases with reactive amino groups (Beauchamp et al., 1992
). This has also been reported for glutaraldehyde, formaldehyde, and acetaldehyde (Beauchamp et al., 1992
; Feron et al., 1991
; Tuma and Sorrell, 1985
). The reactivity of these aldehydes is due to the electrophilic aldehyde group(s). In addition, the mutagenic potential of glutaraldehyde is strikingly similar to formaldehyde as mentioned above.
The biological effects of these aldehydes, as tested by various routes of exposure, suggest the involvement of a contact site mechanism. Exposure by inhalation to glutaraldehyde, formaldehyde, or acetaldehyde results in tissue damage, which is confined to the upper respiratory tract (Appelman et al., 1992; Gross et al., 1994; Heck et al., 1990
; NTP, 1993
; Woutersen et al., 1987
; Zissu et al., 1994
). Oral exposure to glutaraldehyde, formaldehyde, or acetaldehyde results in tissue damage limited to the gastric mucosa (Ballantyne, 1995
; Til et al., 1988
). Dermal exposure to acetaldehyde in humans resulted in cutaneous erythema (Wilkin and Fortner, 1985
). Dermal exposure studies with formaldehyde or glutaraldehyde have clearly shown to result in skin irritation and sensitization (Ballantyne, 1995
; IPCS, 1989; NICNAS, 1994
; Stern et al., 1989
).
Long-term inhalation studies with the structural analogues formaldehyde and acetaldehyde resulted in nasal squamous cell carcinomas (both compounds) and in nasal adenocarcinomas (acetaldehyde only) in rats (Kerns et al., 1983; Monticello et al., 1996
; Woutersen et al., 1986
).
Based on the effects in humans exposed to glutaraldehyde, the increase in nasal tumors with the structural analogues formaldehyde and acetaldehyde, the mutagenicity of glutaraldehyde in several short-term genotoxicity assays, and concerns about occupational exposure by inhalation, the National Toxicology Program (NTP, 1999) initiated 2-year inhalation studies with glutaraldehyde, in both rats and mice. Exposure concentrations were based on a previously conducted 13-week toxicity study with glutaraldehyde (Gross et al., 1994
; NTP, 1993
). This manuscript evaluates the carcinogenicity and toxicity of glutaraldehyde in 2-year, whole-body inhalation studies in male and female F344/N rats and B6C3F1 mice.
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MATERIALS AND METHODS |
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Exposure system.
Glutaraldehyde vapor was generated with a rotary evaporation system (Büchi Rotavapor, Model EL-1315, Brinkman Instruments, Westberry, NY) in a hot-water bath (112°C). A heated stream of nitrogen metered into the flask carried the glutaraldehyde and water vapors arising from the flask through the generator. The generator was maintained at a temperature sufficient to prevent condensation of the vapor as it passed through the generator to the distribution manifold where it was diluted with heated HEPA- and charcoal-filtered air. All transfer lines were heated to prevent condensation. Stainless-steel wire-bottom 14 x 11.7 x 15.2 cm (L x W x H) inhalation exposure chambers (Hazleton H-2000®; Harford Systems Division of Lab Products, Inc., Aberdeen, MD) were used with a total active mixing volume of 1.7 m3 for each chamber. The chamber airflow rate was 15 air changes per h. A small particle detector (Type CN, Gardner Associates, Schenectady, NY) was used with and without animals in the exposure chambers to ensure that glutaraldehyde vapor, and not aerosol, was produced. Chamber concentrations of glutaraldehyde as the free aldehyde were monitored by an online gas chromatography with flame ionization using a DB-5 column (15 m x 0.53-mm fused silica, 1.5 µm film, J&W Scientific) and carrier gas (nitrogen) with a flow of 30 mL/min. Each chamber was sampled approximately every 45 min. The uniformity of glutaraldehyde concentration in the exposure chamber with animals present was measured before the start of the studies and periodically during the studies. The distribution of glutaraldehyde in the chamber atmosphere varied less than 15% within a chamber. The monitor was calibrated against gravimetrically prepared standards every month.
Experimental design.
Details of the experimental design are presented in NTP technical report number 490 (1999). Four-week old male and female Fischer F344/N rats and B6C3F1 mice were obtained from Taconic Farms (Germantown, NY). Animals were quarantined for 2 to 3 weeks before the beginning of the studies. Animals were randomly distributed among groups of approximately equal weight.
Groups of 50 male and 50 female rats were exposed to glutaraldehyde by whole-body inhalation at concentrations of 0, 250, 500, or 750 ppb, 6 h plus T90 (25 min) per day, 5 days per week, for 104 weeks. Groups of 50 male and 50 female mice were exposed to glutaraldehyde by whole-body inhalation at concentrations of 0, 62.5, 125, or 250 ppb, 6 h plus T90 (25 min) per day, 5 days per week, for 104 weeks.
The highest exposure concentration in the 2-year study with rats (750 ppb) was chosen based on decreased body weights and significant histopathological lesions in the anterior part of the nose at 1000 ppb in a 13-week toxicity study, which were expected to become life threatening in a 2-year bioassay. The middle exposure concentration selected, 500 ppb, was based on the slight increase in the rate of cell replication and mild lesions in the anterior part of the nose. The lowest exposure concentration selected, 250 ppb, was based on the absence of squamous exfoliation. In the 2-year study with mice, the highest exposure concentration of 250 ppb was based on the decrease in body weight, deaths, and absence of significant nasal lesions as observed at 500 and 1000 ppb in the 13-week toxicity studies. The 125 and 62.5 ppb concentrations were based on the no-effect concentration for squamous exfoliation.
Rats and mice were housed individually. Water was available ad libitum during non-exposure periods. Feed (NIH-07, open formula pellet diet, Zeigler Brothers, Inc., Gardners, PA) was available during non-exposure periods. Chambers and cages were rotated weekly. Chambers were maintained at an average daily temperature of 24 ± 2°C. The daily average relative humidity was 55 ± 15%.
All animals were observed twice daily. Body weights were recorded initially, and body weights and clinical observations were recorded every 4 weeks from week 5 through week 89, and every 2 weeks from week 92 (rats) or 93 (mice) until the end of the studies. A complete necropsy and microscopic examination were performed on all rats and mice.
At necropsy, all organs and tissues were examined for grossly visible lesions, and all major tissues were fixed and preserved in 10% neutral-buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 5 to 6 µm, and stained with hematoxylin and eosin for microscopic examination. A complete histopathologic examination was performed on all rats and mice. Tissues and organs examined microscopically included adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder (mice), heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lung, lymph nodes (mandibular, mesenteric, bronchial, mediastinal), mammary gland, nose, ovary, pancreas, pancreatic islets, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus. Because of the length and complexity of the nasal cavity, multiple sections are necessary to adequately assess the various morphological and functional regi ons. Three sections of the nasal cavity are routinely taken for microscopic examination in NTP carcinogenicity studies and were taken in this study. In addition, because glutaraldehyde was expected to affect the anterior nasal region, a fourth section was taken in this study (Fig. 1). This section (Level I) was taken from the most anterior region of the nasal cavity just behind the external nares. Histologically, it included the nasal squamous epithelium that is continuous rostrally with the external opening of the nares and caudally with the respiratory epithelium. The other 3 sections were taken from the usual locations. Level II was taken just posterior to the upper incisor teeth and includes the naso and maxilloturbinates; this generally represents the area where there is transition from squamous to respiratory epithelium with both types of epithelium present in section. Level III is taken further caudally, between the incisive papilla and rostral to the first palatal ridge. It includes the most caudal regions of the naso and maxilloturbinates that are covered predominately by respiratory epithelium. Some squamous and olfactory epithelium is usually observed in this section as well. The most caudal section (level IV) was taken at the level of the second upper molar and includes the tortuous ethmoid turbinates that are covered by olfactory epithelium.
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RESULTS |
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Nose.
Nonneoplastic lesions were most common and severe in the squamous epithelium in Level I, less common and severe in the second section, infrequent in the third section, and rarely present in the fourth section.
The changes observed in exposed male and female rats included increased incidences of hyperplasia and inflammation of the squamous epithelium; hyperplasia, goblet cell hyperplasia, inflammation, and squamous metaplasia of the respiratory epithelium; and hyaline degeneration (often referred to as hyaline droplets) of the olfactory epithelium (Table 1). Inflammation was a minimal to marked change consisting of multifocal to locally extensive infiltrates of neutrophils, lymphocytes, plasma cells, and sometimes macrophages within the lamina propria and, in severe cases, within the epithelium itself. In more severe cases of inflammation, sizable aggregates of neutrophils were present in the nasal passage.
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Slightly increased incidences of hyaline degeneration of the olfactory epithelium were observed in exposed groups of males and females. The microscopic appearance of this lesion was characteristic of that seen with the spontaneously occurring hyaline degeneration of the olfactory epithelium in F344/N rats; the lesion consisted of an accumulation of homogeneous eosinophilic droplets within the cytoplasm of epithelial cells. The change was of minimal to mild severity and was observed in the olfactory epithelium lining the dorsal meatus of Level III. No neoplasms were observed in the nasal cavity.
Mammary gland.
The incidences and multiplicity of fibroadenoma occurred with a negative trend in females (chamber control, 24/50; 250 ppb, 23/50; 500 ppb, 18/50; 750 ppb, 10/50). The incidence of fibroadenoma or carcinoma (combined) in 750 ppb female rats was significantly decreased (26/50, 27/50, 21/50, 11/50).
Pituitary gland (pars distalis).
The incidence of adenoma was significantly decreased in 500 ppb females (37/50, 37/50, 27/50, 24/49) and occurred with a negative trend.
Mice
Survival, Body Weights, and Clinical Findings
Survival of exposed mice was similar to that of the chamber controls (Fig. 8). There were no exposure-related effects on mean body weights of males (Fig. 9
). Mean body weights of females exposed to 250 ppb were generally less than those of the chamber controls throughout the study. No clinical findings were attributed to glutaraldehyde exposure.
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Nose.
There were increased incidences of several nonneoplastic lesions within the various sections of the nose. In general, the lesions observed in the noses of mice were qualitatively similar to those that occurred in rats. Female mice were more severely affected than were male mice. Incidences of squamous metaplasia of the respiratory epithelium were increased in 250-ppb males and females and 125-ppb females (Table 2). Incidences of hyaline degeneration of the respiratory epithelium were increased in all exposed groups of females. Incidence of inflammation of the nose was marginally increased in 250-ppb females. Turbinate necrosis was observed in 2 125-ppb males and in all exposed groups of females. While the increased incidence was not statistically significant, this is not a common spontaneous lesion.
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Turbinate necrosis usually consisted of a small focus of necrosis extending the full thickness of the epithelium into the underlying lamina propria and sometimes affecting the turbinate bone.
The microscopic appearance of the hyaline degeneration of the respiratory epithelium was typical of that seen with the spontaneously occurring hyaline degeneration of the olfactory and respiratory epithelium in B6C3F1 mice, and consisted of accumulation of homogeneous eosinophilic material within the cytoplasm of epithelial cells. No neoplasms were observed in the nasal cavity.
Liver.
Incidences of hepatocellular adenoma were decreased in 62.5- and 250-ppb male mice and 250-ppb female mice (males: 19/49, 10/50, 20/50, 11/49; females: 11/50, 11/48, 7/50, 3/50).
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DISCUSSION |
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The exposure concentration-dependent, nonneoplastic lesions found in the nose in the current studies were similar to those found in the 13-week studies (NTP, 1993) and in a 78-week inhalation study in B6C3F1 mice with glutaraldehyde (Zissu et al., 1998
). These lesions included a spectrum of inflammatory, degenerative, and proliferative lesions that were more severe in the anterior than in the posterior portions of the noses of rats and mice.
In the squamous epithelium of the nasal cavity, minimal to marked hyperplasia and inflammation in rats and minimal to mild inflammation in mice were observed in the 2-year studies, especially at the highest exposure concentrations (750-ppb for rats and 250-ppb for mice). In severe cases in rats, there was accumulated keratin that partially filled the lumen of the nasal passages (squamous exfoliation). It is possible that the increased keratin represents increased production (hyperkeratosis), but it is more likely that the keratin became fixed by the glutaraldehyde and accumulated within the lumen rather than being sloughed and cleared from the nasal passage. It appeared that accumulated keratin produced significant obstruction of the nasal passage in some animals, resulting in mouth breathing. In general, the nasal lesions were more severe in exposed female rats than in males, and that may explain the increased mortality among females. Squamous exfoliation was observed at 250-ppb and greater in the 13-week rat and mouse studies, and it was thought to be responsible for the breathing difficulties and subsequent removal of mice from the 13-week studies (NTP, 1993).
In the 2-year rat study, hyperplasia and inflammation, as well as squamous metaplasia and goblet cell hyperplasia, were observed in the respiratory epithelium. These lesions were slightly less severe than those observed in the squamous epithelium. In the 2-year mouse study, inflammation and squamous metaplasia of the respiratory epithelium were also observed. In rats, degeneration of the olfactory epithelium occurred. Nasal lesions appeared to progress in severity with continued exposure to glutaraldehyde. In addition, the incidence of the lesions increased more in the anterior than in the posterior section of the nose.
No nasal neoplasms were observed in male or female F344/N rats or B6C3F1 mice in the current 2-year studies or in B6C3F1 mice exposed to 100-ppb in a 78-week inhalation study (Zissu et al., 1998). This is in contrast to exposure to the structural analogues formaldehyde and acetaldehyde, which has been shown to result in squamous cell carcinomas in the nasal cavities of rats after long-term exposure by inhalation (Kerns et al., 1983
; Monticello et al., 1996
; Woutersen et al., 1986
).
Although the survival in female rats was decreased at the highest exposure concentration and some male rats were removed early in the study, it was felt that sufficient animals survived to test the carcinogenic potential under the conditions of the study, since squamous cell carcinomas of the nasal cavity were detected relatively early with formaldehyde, i.e., after about a year, in both male and female rats (Kerns et al., 1983; Swenberg et al., 1980
).
Decreases in neoplastic lesions of the mammary and pituitary gland in female rats and hepatocellular adenomas in mice were considered to be related to the decrease in body weight rather than to a direct effect of exposure to glutaraldehyde (Haseman, 1995; Haseman and Johnson, 1996
; Rao et al., 1987
, 1990
; Seilkop, 1995
; Turturro et al., 1995
).
Exposure to glutaraldehyde, formaldehyde, or acetaldehyde by inhalation results in tissue damage that is confined to the upper respiratory tract. However, the location of the major non-neoplastic lesions differs for each of these chemicals. Glutaraldehyde had a more profound effect on the most anterior portion of the nasal cavity that is lined by squamous epithelium. Just caudal to this region, respiratory epithelium predominates and was significantly affected by both glutaraldehyde and formaldehyde. However, in that area, changes described as squamous hyperplasia and squamous papillary hyperplasia with foci or cellular atypia were described in the study of formaldehyde (Kerns et al., 1993; Monticello et al., 1996) which were not observed in the current study with glutaraldehyde. These changes were thought to be precursor lesions to squamous cell neoplasia in the formaldehyde study.
Acetaldehyde, an aldehyde with an additional methyl-group in comparison to formaldehyde, resulted in nasal lesions (including squamous cell carcinomas) which were mainly located in the olfactory epithelium (Woutersen et al., 1986). The non-carcinogenic isobutyraldehyde that is a larger molecule than acetaldehyde resulted mainly in non-neoplastic lesions of the respiratory and olfactory epithelium (Abdo et al., 1998
). The extreme anterior location of glutaraldehyde-induced lesions suggests that glutaraldehyde is more reactive and does not penetrate as far into the nasal cavity as do acetaldehyde and isobutyraldehyde.
An approach to comparing the toxicity of formaldehyde with acetaldehyde and also glutaraldehyde in inhalation and oral studies was previously presented by Morris and co-workers (1996). Glutaraldehyde was 6 to 8 times more potent than formaldehyde in its ability to produce DNAprotein crosslinks and about 10 times more potent than formaldehyde in producing tissue damage after instillation into the nose (Kuykendall and Bogdanffy, 1992; St. Clair et al., 1990
), whereas genotoxicity was generally observed at similar concentrations (Galloway et al., 1985
; Valencia et al., 1989
). This is in agreement with the results of the current 2-year study in male rats using hyperplasia and squamous metaplasia of the respiratory epithelium as an endpoint. The respiratory epithelium was the area in which squamous cell carcinomas were observed after exposure to formaldehyde (Kerns et al., 1983
; Monticello et al., 1996
). For both hyperplasia and metaplasia of the respiratory epithelium, the no-observable-adverse-effect level (NOAEL) and the lowest-observable-adverse-effect level (LOAEL) after exposure to glutaraldehyde were 250 and 500 ppb, respectively. Exposure to formaldehyde for 2 years resulted in increased incidences of hyperplasia and squamous metaplasia of the respiratory epithelium in rats administered 10 ppm or about 6 ppm, respectively (Table 3
). Comparison of the NOAEL and LOAEL for endpoints for glutaraldehyde and formaldehyde in 2-year inhalation studies in F344/N rats indicated that glutaraldehyde is 3 to 7 times more potent than formaldehyde (Table 4
).
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In summary, exposure to glutaraldehyde for 2 years by inhalation resulted in various non-neoplastic lesions in the nose in rats and mice. In contrast to the nasal carcinogen formaldehyde, no neoplastic lesions were related to glutaraldehyde exposure.
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ACKNOWLEDGMENTS |
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NOTES |
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Presented in part at the 38th Annual Meeting of the Society of Toxicology, New Orleans, March 1999.
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