2,3-Butanedione is commonly used by the flavor manufacturing industry for production of artificial flavor formulations. Examples of flavored food products include popcorn, cake mixes, flour, beer, wine, margarines and soft spreads, cheese, candy, bakery products, crackers, cookies, ice cream, frozen foods, and many other food and beverage products. 2,3-Butanedione was nominated by the United Food and Commercial Workers Union for long-term inhalation studies due to outbreaks of bronchiolitis obliterans in workers exposed to its vapors. Male and female Wistar Han [CRL:WI (Han)] rats and B6C3F1/N mice were exposed to 2,3-butanedione (greater than or equal to 98.5%) by inhalation for 3 months or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium and Escherichia coli, mouse bone marrow cells, and rat and mouse peripheral blood erythrocytes.
Three-month study in rats
Groups of 10 male and 10 female rats were exposed to 2,3-butanedione vapor by whole body inhalation at concentrations of 0, 6.25, 12.5, 25, 50, or 100 ppm, 6 hours plus T90 (10 minutes) per day, 5 days per week for 14 weeks. Additional groups of 10 male and 10 female rats were exposed to the same concentrations for 23 days for clinical pathology analyses. Two male rats in the 100 ppm group died before the end of the study. All other rats survived to the end of the study. The mean body weights of 100 ppm males and females were significantly less than those of the chamber control groups. Clinical observations, noted only in the 50 and 100 ppm groups, included abnormal breathing, sneezing, and lethargy.
On day 23 and at study termination, neutrophil counts were significantly increased in 100 ppm females and were consistent with the inflammation observed in the respiratory tract. Significant increases in the erythron occurred most consistently in the 100 ppm male and female groups on day 23 and at study termination. These erythron increases were consistent with dehydration or a secondary erythrocytosis.
2,3-Butanedione exposure resulted in a significant increase of nonneoplastic lesions in the respiratory tract of male and female rats, primarily in the 50 and 100 ppm groups. The highest number of lesions occurred in the nose and included suppurative inflammation; necrosis, regeneration, squamous metaplasia and hyperplasia of the respiratory epithelium; necrosis, degeneration, and respiratory metaplasia of the olfactory epithelium; atrophy of the turbinate; and hyperplasia of the lymphoid tissue. In the larynx, lesions included respiratory epithelium squamous metaplasia and squamous epithelium hyperplasia in males and females and epithelium necrosis and chronic active inflammation in females. In the trachea, necrosis and regeneration occurred in the epithelium of males and females and hyperplasia occurred in the epithelium of females. In the lung, significantly increased incidences of nonneoplastic lesions only occurred in the 100 ppm groups of males and females and included hyperplasia and regeneration of the bronchus epithelium and bronchiole epithelium hyperplasia; in addition, the incidences of histiocyte cellular infiltration and bronchus epithelium necrosis were significantly increased in 100 ppm males, and the incidence of atypical hyperplasia of the bronchus epithelium was significantly increased in 100 ppm females.
Three-month study in mice
Groups of 10 male and 10 female mice were exposed to 2,3-butanedione vapor by whole body inhalation at concentrations of 0, 6.25, 12.5, 25, 50, or 100 ppm, 6 hours plus T90 (10 minutes) per day, 5 days per week for 14 weeks. All mice survived to the end of the study. The mean body weights of males exposed to 50 or 100 ppm and females exposed to 12.5 ppm or greater were significantly less than those of the chamber control groups. Clinical observations in mice exposed to 50 or 100 ppm included sneezing and abnormal breathing.
Significant increases in neutrophil counts occurred in 50 and 100 ppm males and 100 ppm females and were consistent with inflammation. In all of these groups, mean cell volume and mean cell hemoglobin were significantly decreased, possibly indicating minimal alterations in iron metabolism or hemoglobin production.
Exposure-related significantly increased incidences of nonneoplastic lesions occurred in the respiratory tract of male and female mice, primarily in the 50 and 100 ppm groups. As in rats, the highest number of lesions occurred in the nose and included suppurative inflammation; necrosis, regeneration, and squamous metaplasia of the respiratory epithelium; necrosis and atrophy of the turbinate; and atrophy and respiratory metaplasia of the olfactory epithelium. In the larynx, lesions included necrosis of the epithelium; regeneration, hyperplasia, squamous metaplasia, and atypical squamous metaplasia of the respiratory epithelium; hyperplasia and atypical hyperplasia of the squamous epithelium; and chronic active inflammation. In the trachea, the incidences of atypical squamous metaplasia, hyperplasia, and degeneration in the epithelium were significantly increased in the 100 ppm groups, as were the incidences of chronic active inflammation. The incidences of regeneration of the tracheal epithelium were significantly increased in the 50 ppm groups. In the lung, the incidences of chronic inflammation and polymorphonuclear cellular infiltration of the bronchus were significantly increased in the 100 ppm groups. The incidences of atypical hyperplasia, atypical squamous metaplasia, and regeneration of the bronchus epithelium were significantly increased in the 100 ppm groups.
Two-year study in rats
Groups of 50 male and 50 female rats were exposed to 2,3-butanedione vapor by whole body inhalation at concentrations of 0, 12.5, 25, or 50 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 105 weeks. Survival of 50 ppm males was significantly less than that of the chamber control group. Survival was moderately reduced in 25 ppm females. At the end of the study, mean body weights of both sexes exposed to 50 ppm were decreased relative to the respective chamber control groups, with more of an effect in males (81% of chamber controls) than in females (91% of chamber controls). Exposure-related clinical observations included thinness, abnormal breathing, eye abnormality, and nasal/eye discharge in males and eye abnormality and abnormal breathing in females.
Three squamous cell carcinomas and one squamous cell papilloma of the nasal mucosa occurred in male rats exposed to 50 ppm, and three squamous cell carcinomas of the nasal mucosa occurred in females exposed to 50 ppm. No squamous cell carcinomas or papillomas of the nose occurred in the concurrent male or female chamber controls, and none are recorded in the NTP historical control database.
A spectrum of nonneoplastic lesions of the nose occurred in both the respiratory and olfactory epithelium, primarily in the 25 and 50 ppm groups. Nasal lesions with incidences significantly greater than the chamber control incidences included suppurative inflammation; respiratory epithelium hyperplasia and squamous metaplasia; olfactory epithelium atrophy, respiratory metaplasia, and necrosis (males); turbinate hyperostosis; and fibrosis of the lamina propria.
In the larynx, incidences of chronic active inflammation in the 50 ppm groups, hyperplasia of the squamous epithelium in the 25 and 50 ppm groups, focal areas of ulceration of the squamous epithelium in the 50 ppm groups, and squamous metaplasia of the respiratory epithelium in the 50 ppm groups were significantly greater than the chamber control incidences.
In the trachea, the incidences of chronic active inflammation, epithelium hyperplasia, and submucosa fibrosis were significantly increased in 50 ppm males and females. In 50 ppm males, the incidences of epithelium squamous metaplasia, regeneration, atrophy, and necrosis were significantly increased. The incidence of epithelium regeneration was also significantly increased in 25 ppm males.
In the lung, the incidences of suppurative inflammation in 50 ppm males, granulomatous inflammation in 50 ppm females, and peribronchial chronic active inflammation in 50 ppm males and females were significantly increased. Significantly increased lesion incidences in the airways included bronchial and bronchiolar epithelium hyperplasia and bronchial epithelium atrophy in 50 ppm males and females and bronchial epithelium regeneration and submucosa fibrosis in 50 ppm males. The incidence of bronchiolar epithelium hyperplasia was also significantly increased in 25 ppm females. Lesions occurring in the lung parenchyma included histiocytic cellular infiltration in the alveolar spaces, alveolar epithelium hyperplasia, and interstitium fibrosis. The incidences of these lesions were significantly increased in 50 ppm rats, except the incidence of alveolar epithelium hyperplasia in females.
In the eye, chronic active inflammation of the cornea in 25 and 50 ppm rats, suppurative inflammation of the anterior chamber in 25 ppm rats, acute inflammation of the iris in 25 ppm females, cornea epithelium hyperplasia in 25 ppm females and 50 ppm males and females, cornea epithelium ulcer in 50 ppm rats, lens cataract in 25 ppm males and females and 50 ppm females, and unilateral phthisis bulbi in 50 ppm females occurred with significantly increased incidences.
Two-year study in mice
Groups of 50 male and 50 female mice were exposed to 2,3-butanedione vapor by whole body inhalation at concentrations of 0, 12.5, 25, or 50 ppm, 6 hours plus T90 (12 minutes) per day, 5 days per week for 105 weeks. Survival of 50 ppm males and females was significantly less than that of the chamber control groups. At the end of the study, the mean body weights of the 50 ppm groups were reduced to 65% (males) and 62% (females) of those of the respective chamber control groups. Clinical observations were most prominent in the 50 ppm groups and included abnormal breathing, thinness, sneezing, and eye abnormality in both sexes.
In the nose, adenocarcinomas occurred in two 50 ppm females. No nasal adenocarcinomas have been recorded in the NTP historical control database.
Compared to the chamber control group incidences, nonneoplastic lesions of the nose that were significantly increased in 50 ppm mice included suppurative inflammation; respiratory epithelium squamous metaplasia, hyperplasia (males), and necrosis; respiratory metaplasia of the Steno’s glands; regeneration of the mucosa epithelium; olfactory epithelium atrophy, respiratory metaplasia, and necrosis; turbinate atrophy and necrosis; perforation of the nasal septum; and fibrosis of the lamina propria. Most of these lesion incidences were also significantly increased in 25 ppm mice and sometimes in 12.5 ppm mice.
In the larynx, incidences of chronic active inflammation; lumen exudate (females); respiratory epithelium squamous metaplasia, hyperplasia (males), necrosis, and regeneration; and squamous epithelium hyperplasia were significantly increased in 50 ppm mice. Incidences of chronic active inflammation, squamous epithelium hyperplasia, and respiratory epithelium necrosis were also significantly increased in the 25 ppm groups. The incidence of squamous epithelium hyperplasia was significantly increased in 12.5 ppm females.
Incidences of tracheal lesions that were significantly increased in the 50 ppm groups included chronic active inflammation; lumen exudate; necrosis; epithelium regeneration, hyperplasia (males), and squamous metaplasia (males); submucosa fibrosis; and carina submucosa fibrosis, mineralization, and chronic active inflammation (males). The incidence of epithelium regeneration was also increased in 25 ppm females.
In the lung of 50 ppm mice, the most common bronchial lesion in both males and females was bronchus epithelium regeneration. Bronchus submucosa fibrosis occurred in five 50 ppm females. Incidences of suppurative inflammation of the lung, pleura, and mediastinum were significantly increased in 50 ppm females. Incidences of chronic active inflammation of the mediastinum were significantly increased in 50 ppm males and females.
In the cornea of the eye, incidences of acute inflammation, mineralization, and epithelium hyperplasia in 25 ppm females and 50 ppm males and females; epithelium ulcer in 25 and 50 ppm females; and necrosis in 50 ppm females were significantly greater than the chamber control incidences. The incidence of suppurative inflammation of the anterior chamber was increased in 50 ppm males.
Genetic toxicology
2,3-Butanedione was mutagenic in two independent bacterial mutagenicity assays. In the initial assay, conducted with a different lot of the chemical than was tested in the NTP rodent studies, a weak positive response was observed in S. typhimurium strain TA97 with and without exogenous metabolic activation (S9 mix). No clear mutagenic activity was observed in any of the other strains tested (TA98, TA100, and TA1535). In the second bacterial mutation assay, conducted with the same lot of 2,3-butanedione that was used in the 2-year rodent bioassay, mutagenic activity was seen in S. typhimurium strain TA97a in the absence of S9 and in the E. coli strain WP2 uvrA/pKM101 with and without S9 mix. As with the initial test, no clear mutagenic activity was seen in any of the other strains tested (TA100, TA98) with or without S9.
To assess chromosomal damage, the frequency of micronucleated polychromatic erythrocytes (PCEs) was measured in bone marrow samples obtained from male B6C3F1/N mice following intraperitoneal injection of 2,3-butanedione once daily for 3 days; no increases in micronucleated PCEs were observed at doses up to 500 mg 2,3-butanedione/kg body weight per day.
At the end of the 3-month inhalation studies, peripheral blood samples were obtained from male and female rats and mice and analyzed by flow cytometry for the frequency of micronucleated PCEs and mature (normochromatic) erythrocytes (NCEs). No increases in micronucleated PCEs or NCEs were seen in either sex or species. The percentage of PCEs among circulating red blood cells was unaffected by exposure to 2,3-butanedione, suggesting the chemical had no effect on erythropoiesis.
Conclusions
Under the conditions of these 2-year inhalation studies, there was some evidence of carcinogenic activity of 2,3-butanedione in male Wistar Han rats based on the combined incidences of squamous cell papilloma and squamous cell carcinoma of the nose. There was some evidence of carcinogenic activity of 2,3-butanedione in female Wistar Han rats based on the incidences of squamous cell carcinoma of the nose. There was no evidence of carcinogenic activity of 2,3-butanedione in male B6C3F1/N mice exposed to 12.5, 25, or 50 ppm. There was equivocal evidence of carcinogenic activity of 2,3-butanedione in female B6C3F1/N mice based on the occurrences of adenocarcinoma of the nose.
Exposure to 2,3-butanedione resulted in increased incidences of nonneoplastic lesions of the nose, larynx, trachea, lung, and eye in male and female rats and mice.
National Toxicology Program (NTP). 2018. NTP technical report on the toxicology and carcinogenesis studies of 2,3-butanedione (CASRN 431-03-8) in Wistar Han [Crl:WI (Han)] rats and B6C3F1/N mice (inhalation studies). Research Triangle Park, NC: National Toxicology Program. Technical Report 593. https://doi.org/10.22427/NTP-TR-593