Carcinogenicity of butyraldehyde in rats by a two-year inhalation study

Abstract

We conducted a two-year inhalation study of butyraldehyde using F344/DuCrlCrlj rats. The rats were exposed to 0, 300, 1,000 and 3,000 ppm (v/v) for 6 hr/day, 5 days/ week for 104 weeks using whole-body inhalation chambers. The incidence of squamous cell carcinoma of the nasal cavity was increased in the 3,000 ppm groups of both male and female rats, with Fisher’s exact test and the Peto test indicating that the incidence was significant. In addition to squamous cell carcinoma in the nasal cavity, in the 3,000 ppm groups one male had an adenosquamous carcinoma, one male had a carcinosarcoma, one male had a sarcoma NOS (Not Otherwise Specified), and one female had a squamous cell papilloma in the nasal cavity. The combined incidence of squamous cell carcinoma, adenosquamous carcinoma and carcinosarcoma was significantly increased in male rats and the combined incidence of squamous cell papilloma and carcinoma was significantly increased in female. Based on these results, we conclude that there is clear evidence of butyraldehyde carcinogenicity in male and female rats.

INTRODUCTION

Butyraldehyde is a clear colorless liquid with a pungent odor and a boiling point of 74.8°C. Butyraldehyde is soluble in water (7 g/100 mL), very soluble in acetone and benzene, and miscible with ethanol and ethyl ether. It is used to make other chemicals, rubber accelerators, solvents, synthetic resins, high polymers, and plasticizers (National Center for Biotechnology Information, 2023). Butyraldehyde is a chemical with high production volumes, with exports and imports in Japan (2020) totaling 7,887,898 kg (7,887 ton) and 5,103,858 kg (5,103 ton), respectively (Ministry of Economy, Trade and Industry, 2022).

A few studies on the toxicity in rodents of butyraldehyde have been reported, but almost all of them were in-house non-peer reviewed publications. Inhalation LC₅₀ values of 6,400 ppm (4 hr, strain/sex unknown) (US National Institute for Occupational Safety and Health Registry of Toxic Effects of Chemical Substances (RTECS) Database, 2016) have been reported for rats. In an inhalation repeated-dose toxicity study, rats (Alderley Park, male and female) were exposed to 1,000 ppm butyraldehyde by inhalation for 6 hr/day, with no effects (nasal examination was not performed) (Gage, 1970). In another study, rats (SD, male and female) were exposed to butyraldehyde at concentrations of 0, 125, 500, and 2,000 ppm for 13 weeks (6 hr/day, 5 days/week) (Texas Commission on Environmental Quality (TCEQ), 2014). One rat died at 2,000 ppm and nasal lesions (squamous cell necrosis) were observed at 125 ppm and above. No other data suggesting carcinogenicity was found in the available literature.

Genotoxicity testing in cultured cell lines showed positive results in a cytogenetic/chromosome aberration test using Chinese hamster ovary (CHO) cells (Galloway et al., 1987). The chromosome aberration test (CHL/IU cells) was positive with or without the S9 mix (JBRC, Report for the Ministry of Health Labor Welfare of Japan, 2005), and the micronucleus test (mouse peripheral blood) was negative (Witt et al., 2000). In the Ames test, butyraldehyde was highly cytotoxic to bacteria, and results were negative with or without S9 mix (Mortelmans et al., 1986, Dillon et al., 1998).

Because butyraldehyde is produced in large quantities, workers may be exposed for extended periods of time. Therefore, long-term toxicity information is needed to assess the risk to workers exposed for long periods of time. However, no long-term toxicity or carcinogenicity studies have been conducted. Consequently, the International Agency for Research on Cancer, the European Chemicals Bureau, and the U.S. Environmental Protection Agency have not evaluated the carcinogenicity of butyraldehyde. Therefore, in the present study, male and female F344 rats were exposed to butyraldehyde by whole-body inhalation for 2 years to provide basic data for health risk assessment of workers exposed to butyraldehyde.

MATERIALS AND METHODS

The present study was conducted in accordance with “Standards to be Observed by Testing Institutions” of the Ministry of Labour Japan, and in accordance with the Organisation for Economic Co-operation and Development (OECD) Good Laboratory Practice (OECD, 1997), and in accordance with the OECD Guideline for Testing of Chemicals 451 “Carcinogenicity Studies” (OECD, 2009). The rats were cared for in accordance with the Standards relating to the Care and Keeping and Reducing Pain of Laboratory Animals (Ministry of the Environment, 2013) and was reviewed and approved by the Institutional Animal Care and Use Committee of the Japan Bioassay Research Center (JBRC), Approved No. 0224.

Test Substance

Reagent grade butyraldehyde (purity >99.8%) was obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). The purity of each lot of the butyraldehyde used in the present study was verified by mass spectrometry. The stability of each lot the butyraldehyde used in the present study was confirmed by gas chromatography. No differences in the gas chromatography peaks of any of the lots of butyraldehyde prior to and after use were observed. The concentration of butyraldehyde in the chamber was monitored using gas chromatography, and no gas chromatographic peaks were detected except for butyraldehyde in the inhalation exposure chamber.

Animals

F344/DuCrlCrlj (SPF) rats of each sex were obtained at the age of 4 weeks from Charles River Japan, Inc. (Kanagawa, Japan). After 2 weeks of quarantine and acclimation, 50 rats of each sex were allocated through a stratified randomization procedure into body-weight-matched, butyraldehyde exposure and control clean air-exposure groups: three butyraldehyde-exposure groups and one control group. The animals were housed individually in stainless-steel wire hanging cages in 8.5 m³ stainless steel inhalation exposure chambers. Each exposure chamber was able to accommodate 100 rats (50 males and 50 females). The four exposure chambers were installed in a barrier system animal room. The chambers were maintained at a temperature of 23 ± 2°C and a relative humidity of 50 ± 20%, with 6 air changes per hour during the exposure periods and 12 air changes per hour during the non-exposure periods. There were 7–9 air changes per hour in the barrier system animal room. Fluorescent lighting in the animal room was automatically controlled to give a 12-hr light/dark cycle. All rats had free access to a γ-irradiation-sterilized commercial pellet diet (CR-LPF, Oriental Yeast Co., Ltd., Tokyo, Japan) and sterilized water.

Experimental design

Based on our previous 13-week inhalation study of butyraldehyde in rats (unpublished), the maximum exposure concentration for this 2-year study was set at 3,000 ppm. The 13-week study was conducted at concentrations of 0 (control group), 100, 300, 1,000, and 3,000 ppm (v/v). No deaths due to administration were observed in any group, and there were no notable changes in general condition. However, in the 3,000 ppm groups of both males and females, suppressed body weight gain and lower food intake were observed. The final body weight and food intake of males were 91% of the control group, while the final body weight and food intake of females were 96% and 98% of the control group, respectively. The degree of toxicity to the respiratory system was mild even in the highest dose group of 3,000 ppm, and it was judged that conducting a 104-week study at a concentration of 3,000 ppm would not cause severe changes affecting the survival of the animals. Based on the above, four groups of rats of each sex were exposed to butyraldehyde vapor at 0 (clean air control), 300, 1,000, and 3,000 ppm (v/v), for 6 hr/day, 5 days/week for 104 weeks.

Inhalation exposure to butyraldehyde

The inhalation exposure system of butyraldehyde is shown in Fig. 1. Airflows containing butyraldehyde vapor at the designated target concentrations were prepared using a vaporization technique. Saturated butyraldehyde vapor was generated by bubbling nitrogen through butyraldehyde liquid in a temperature-regulated glass flask (20ºC) and then cooling the mixture by passage through a thermostatic condenser at 15ºC: nitrogen was used for bubbling because butyraldehyde reacts with air and denatures. The cooled butyraldehyde vapor-nitrogen mixture was then introduced into a warming unit where it was warmed to 20ºC, completely vaporizing the butyraldehyde contained in the butyraldehyde-nitrogen mixture. The butyraldehyde vaper/nitrogen mixture was introduced into a spiral line mixer located above each concentration exposure chamber. Butyraldehyde concentrations in the chambers were monitored using gas chromatography every 15 min throughout the exposure period.

Fig. 1

Inhalation exposure system for butyraldehyde.

The butyraldehyde concentration in the inhalation exposure chambers (four chambers) for the 104 week study period is shown in Fig. 2. Butyraldehyde concentrations were maintained at 0 ppm for control, 299.9 ± 3.0 (mean ± SD) ppm in the 300 ppm chamber, 1000.9 ± 5.7 ppm in the 1,000 ppm chamber, and 2998.2 ± 37.8 ppm in the 3,000 ppm chamber of rats. The butyraldehyde concentration in the inhalation exposure chambers was maintained with high accuracy at the target concentrations.

Fig. 2

Butyraldehyde concentrations throughout the 104 week experimental period. Butyraldehyde vapor was confirmed to be generated at each target concentration throughout the exposure period (6 hr/day, 5 days/week, 104 weeks) in the rat inhalation exposure chambers.

Clinical observations and analysis, and pathological examinations

The animals were observed daily for clinical signs and mortality. Moribund animals were promptly euthanized. Body weight and food consumption were measured once a week for the first 14 weeks and once every 4 weeks thereafter. All animals, including those found dead or moribund, received a complete necropsy. At the terminal necropsy, blood was collected for hematology and blood biochemistry from the abdominal aorta under isoflurane anesthesia after overnight fasting.

All organs were removed and tissues including the entire respiratory tract including nasal cavity, pharynx and larynx were examined histopathologically. The organs and tissues were fixed in 10% neutral buffered formalin. The nasal cavity was decalcified by immersion in formic acid-formalin solution prior to trimming and was transversely trimmed at three levels according to the procedure described in our previous paper (Nagano et al., 1997): at the level of the posterior edge of the upper incisor teeth (Level 1), at the incisive papilla (Level 2), and at the level of the anterior edge of the upper molar teeth (Level 3). The tissues were embedded in paraffin and 3 µm-thick sections were prepared and stained with Hematoxylin and Eosin (H&E). Nasal lesions were diagnosed with reference to the criteria set by the International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice (Bach et al., 2010; Renne et al., 2009).

Histopathological diagnosis was performed by pathologists certified by the Japanese Society of Toxicologic Pathology and peer reviewed by outside pathologists.

Statistical analysis

Survival curves were plotted according to the Kaplan-Meier method, (Kaplan and Meier, 1958). Two-tailed tests were used for all statistical analyses except for Peto’s test and Fisher’s exact test. P values less than 0.05 were considered to be statistically significant. Body weight, organ weight and food consumption were analyzed by Bartlett's test to test whether the variance was homogeneous. When the variance was homogeneous, one-way ANOVA was used to test for statistical differences between groups, and when the variance was not homogeneous, the Kruskal-Wallis rank sum test was used. Statistical difference from the control group was analyzed by the Dunnett's multiple comparison test when the variance was homogeneous and Dunnett's multiple comparison test by rank when the variance was not homogeneous. The incidences of neoplastic lesions were statistically analyzed by Fisher’s exact test. The incidences of non-neoplastic lesions were statistically analyzed by Chi-square Test. Positive trends for neoplastic incidence were analyzed using Peto’s test (Peto et al., 1980). Tumor incidence was also evaluated by comparison with incidences of the same tumor type from the Japan Bioassay Research Center (JBRC) historical control data. The use of historical control data for evaluation of tumor induction is described by Haseman et al. (1984, 1985 and 1995).

RESULTS

Survival, body weight, food consumption and clinical observations

Survival rates are shown in Fig. 3. As shown in Fig. 3A, in males, there was no difference in survival between any of the exposure groups and the control group, but in females, survival was reduced in the 3,000 ppm group (Fig. 3B). Survival numbers and rates at the end of the 2-year exposure period in the 0 (Control), 300, 1,000 and 3,000 ppm exposed male groups was 32/50 (64%), 36 /50 (72%), 34/50 (68%) and 31/50 (62%), and were 33/50 (66%), 39/50 (78%), 38/50 (76%) and 22/50 (44%) in the female groups, respectively.

Fig. 3

Survival curves of male (A) and female (B) rats exposed to butyraldehyde or clean air as a control for 2 years.

Body weights are shown in Fig. 4. As shown in Fig. 4A, males in the 3,000 ppm group significantly lost weight from week 1 to week 104. Body weights 3,000 ppm group were less than 90% of the control group from week 18 to week 104. In the 1,000 ppm group, significant suppression of body weight was observed from week 34 to week 54 and from week 62 to week 90. In the 300 ppm group, significant suppression of body weight was observed from week 18 to week 78. In females, the 3,000 ppm group showed significant suppression of body weight from week 1 to week 104 (Fig. 4B). Body weights in the 3,000 ppm group were less than 90% of the control group from week 58 to week 104. The 1,000 ppm group showed significant suppression of body weight from week 78 to week 104 (Fig. 4B). The final body weights of the 300, 1,000 and 3,000 ppm-groups at the end of the 2-year exposure period were 99 (394 g), 99 (393 g), and 76% (304 g) of the controls (398 g) for males and 101 (259 g), 95 (245 g), and 81% (207 g) of the controls (257 g) for females.

Fig. 4

Body weights of male (A) and female (B) rats exposed to butyraldehyde or clean air as a control for 2 years.

Abnormal breath sounds were observed in male rats in the 3,000 ppm group for the final 65 weeks of exposure, and the number of such observations increased with the number of days of exposure. Abnormal breath sounds were present in 19/43 males at 88 weeks and in 21/31 males at 104 weeks. No abnormal breath sounds were observed in the 300 ppm or 1,000 ppm groups. In females, abnormal breath sounds were observed in the 3,000 ppm group for the final 70 weeks of exposure. Abnormal breath sounds were observed in 15/25 females at 99 weeks, 16/25 females at 100 weeks, and 14/24 females at 101 weeks. Abnormal breath sounds were observed in only 4/22 females at 104 weeks. No abnormal breath sounds were observed in the 300 ppm or 1,000 ppm groups.

Food consumption is shown in Fig. 5. As shown in Fig. 5A, males in the 3,000 ppm group had significantly lower food consumption from week 1 to week 104. Food consumption was about 90% of the control group for most of the experimental period. Significantly lower levels of food consumption was also observed in the 1,000 and 300 ppm groups for most of the experimental period. In females, significantly lower food consumption was observed in the 3,000 ppm group from week 1 to week 11, during week 14, and from week 86 to week 104 (Fig. 5B). Food consumption was less than 90% of the control group from week 98 to week 104. The 1,000 ppm group had significantly higher food consumption during week 42 week (106% of the control group) and significantly lower food consumption from week 90 to week 102 (96% of the control group). The 300 ppm group had significantly higher food consumption during week 42, 82, and 86. The average daily food consumption during the 2-year exposure period was 18.0, 17.8, 17.7, and 16.2 g for males and 12.5, 12.8, 12.4, and 11.9 g for females in the 0 (control), 300, 1,000, and 3,000 ppm groups, respectively.

Fig. 5

Food consumptions by male (A) and female (B) rats exposed to butyraldehyde or clean air as a control for 2 years.

Pathology

Neoplastic lesions, the number of occurrences by tumor type and the results of statistical analysis (Fisher’s exact test, Peto test) are shown in Table 1. In males, squamous cell carcinoma of the nasal cavity occurred in 0 animals in the control, 300, and 1,000 ppm groups and in 19 of 50 males (38%) in the 3,000 ppm group. The incidence of squamous cell carcinoma in the 3,000 ppm group was significantly increased compared to the control group by Fisher’s exact test, and the incidence had an increased trend in the Peto’s test (mortality, prevalence, mortality + prevalence): Figure 6A and 6B show a typical squamous cell carcinoma of the nasal cavity. Squamous cell carcinoma showed invasive and destructive growth, and tumor growth destroyed the nasal bone. Adenosquamous carcinoma, carcinosarcoma, and sarcoma NOS were each observed in one animal (2%), male in the 3,000 ppm group. The combined incidence of squamous cell carcinoma, adenosquamous carcinoma, and carcinosarcoma was 21 of 50 (42%) in the 3,000 ppm group and 0 in the control, 300 ppm, and 1,000 ppm groups. The combined incidence in the 3,000 ppm group showed a significant increase by Fisher’s exact test, and an increased trend in the Peto test. In the larynx, squamous cell carcinoma occurred in one animal (2%) in the 3,000 ppm group.

Table 1. Number of tumor-bearing F344 rats exposed to butyraldehyde for 2 years.

Fig. 6

Butyraldehyde induced neoplastic and non-neoplastic lesions of the nasal cavity, and non-neoplastic lesions of the trachea. (A) Squamous cell carcinoma in the nasal cavity of a female rat in the 3,000 ppm group. The tumor showed invasive and destructive growth. Tumor growth destroyed the nasal bone. (B) High magnification of A. (C) Squamous cell hyperplasia (squamous epithelium with dysplasia) in the nasal cavity of a male rat in the 3,000 ppm group. (D) Rhinitis of the olfactory epithelium in the nasal cavity of a male rat in the 3,000 ppm group. Inflammatory cell infiltration of the lamina propria and respiratory metaplasia of the gland. (E) Basal cell hyperplasia and atrophy of the olfactory epithelium in nasal cavity of a male rat in the 3,000 ppm group. (F) Squamous cell hyperplasia in the trachea of a male rat in the 3,000 ppm group.

In females, squamous cell carcinoma of the nasal cavity occurred in 0 animals in the control, 300 ppm, and 1,000 ppm groups and in 8 of 50 females (16%) in the 3,000 ppm group. The 3,000 ppm group showed a significant increase by Fisher’s exact test, and the incidence had an increased trend in the Peto test. Squamous cell papilloma occurred in one animal (2%) in the 3,000 ppm group. The combined incidence of squamous cell carcinoma and squamous cell papilloma was 0 in the control, 300 ppm, and 1,000 ppm groups and 9 of 50 (18%) in the 3,000 ppm group. The combined incidence showed a significant increase by Fisher’s exact test, and an increased trend in the Peto test.

Non-neoplastic lesions in the nasal cavity, larynx, trachea, lung, and eye, the number of occurrences by lesion type and the results of statistical analysis (chi-square test) are shown in Table 2. In males, the incidence of squamous cell metaplasia of the respiratory epithelium in the nasal cavity was increased in the 1,000 ppm and 3,000 ppm groups. These lesions were distributed in the respiratory epithelium of the nasal septum in the anterior part of the nasal cavity (level 1) or in the nasal and maxillary turbinate protruding into the nasal lumen. Squamous cell hyperplasia of the respiratory epithelium was found only in the 3,000 ppm group. Squamous cell hyperplasia had cellular atypia (Fig. 6C). Rhinitis increased in the 1,000 ppm and 3,000 ppm groups. Rhinitis was characterized mainly by inflammatory cell infiltration in the lamina propria, edema, respiratory metaplasia with mild hyperplasia of the glands, goblet cell hyperplasia of the respiratory epithelium, respiratory metaplasia of the olfactory epithelium, and inflammatory cell infiltration throughout the entire nasal cavity (Fig. 6D). Basal cell hyperplasia and atrophy of the olfactory epithelium were observed in the 1,000 ppm 3,000 ppm groups (Fig. 6E), and adhesions of the nasal turbinate were increased in the 3,000 ppm group. Significantly increased squamous cell hyperplasia in the larynx, a few squamous cell hyperplasia in the trachea (Fig. 6F), and foreign body inflammation in the lungs were found in the 3,000 ppm group. In the eyes, keratitis was significantly increased in the 300 ppm, 1,000 ppm, and 3,000 ppm groups. Corneal degeneration was increased in the 300 ppm group. In females, lesions similar to those found in males were observed in the nasal cavity, larynx, trachea, lungs, and eyes. In the female lungs, there was an increase in edema, but no increase in foreign body inflammation in the 3,000 ppm group.

Table 2. Selected non-neoplastic lesions in the nasal cavity, larynx, trachea, lung and eye in F344 rats exposed to butyraldehyde for 2 years.

DISCUSSION

In this study, 3,000 ppm was selected as the highest concentration because in a 13-week preliminary study in which rats were exposed to concentrations of 0 (control group), 100, 300, 1,000, and 3,000 ppm (volume ratio v/v) the groups exposed to the highest concentration of 3,000 ppm showed no changes in the general condition either males or females, the suppression of body weight gain was 9% for males and 4% for females compared to the control group, and histopathological examination showed squamous cell metaplasia of the respiratory epithelium of the nasal cavity and larynx of both males and females: unpublished report prepared for the Ministry of Health & Welfare of Japan, 2019. Therefore, in the 13-week subacute study, the maximum tolerated dose (MTD) was determined to be 3,000 ppm (Sontag et al., 1976; Bannasch et al., 1986). In the carcinogenicity study, the concentration of 3,000 ppm was determined to be appropriate as the highest concentration of exposure. Exposure to 3,000 ppm did not induce toxic effects that caused weight gain inhibition greater than 10% at 90 days. In the carcinogenicity study, after 13 weeks, the weight of male and female were 89.7% and 93.2%, respectively, of the control group, nearly meeting the 90th percentile weight for the MTD. While the reduction in body weight for most of the study was greater than at 90 days, the MTD that is determined by subacute studies is used to predict maximum levels of exposure that are not excessively toxic, and it has been concluded that in the absence of data indicating excessive toxicity in the present study, a 10% lower body weight relative to controls should not be taken as an indication of excessive toxicity (van Berlo et al., 2022). Moreover, as can be seen in Fig. 3A, there was no difference in survival between any of the male groups, indicating that 3,000 ppm was not excessively toxic. In contrast to the male rat groups, the survival rate of females was only 44% at 3000 ppm. However, when decreased survival rate is due to tumors and tumor-related lesions, the decreased survival rate should not be considered to be due to excessively high doses of the test material (Haseman, 1985). Both van Berlo et al. and Haseman also note that the MTD criteria should not be too restrictive, leading to under-estimation of the hazardous properties of the materials being tested.

In males, squamous cell carcinoma in the nasal cavity was increased in the 3,000 ppm group, and the Fisher’s exact test and Peto tests showed that the increase was significant. Adenosquamous carcinoma, carcinosarcoma and sarcoma NOS in the nasal cavity were each found in one animal in the 3,000 ppm group. The combined incidence of squamous cell carcinoma, adenosquamous carcinoma, and carcinosarcoma was significantly increased (Fisher’s exact test and the Peto test). Spontaneous occurrence of nasal cavity tumors in male F344 rats is extremely rare, and nasal cavity tumors did not occur in control male F344 rats from the last 10 studies conducted at JBRC (Table 3). In females, squamous cell carcinoma in the nasal cavity was also increased in the 3,000 ppm group, and the Fisher’s exact test and Peto tests showed that the increase was significant. Squamous cell papilloma in the nasal cavity was found in one animal in the 3,000 ppm group. The combined incidence of squamous cell carcinoma and squamous cell papilloma was significantly increased (Fisher’s exact test and Peto tests). Spontaneous occurrence of nasal cavity tumors in female F344 rats is extremely rare, and squamous cell carcinoma and squamous cell papilloma of the nasal cavity have not occurred in the control female F344 rats from the last 10 studies conducted at JBRC (Table 3). Therefore, the increased incidences of tumors in the nasal cavity in both males and females indicate that butyraldehyde has carcinogenic activity in male and female rats.

Table 3. Historical Control Data of selected Neoplastic lesions in Japan Bioassay Research Center: F344/DuCrlCrlj Rat

Non-neoplastic lesions in the nasal cavity and other organs increased in both sexes. In the nasal cavity, squamous cell hyperplasia with squamous cell metaplasia of the respiratory epithelium is considered to be a pre-neoplastic lesion of the nasal cavity. Other lesions in the nasal cavity that were not considered to be pre-neoplastic lesions were rhinitis, basal cell hyperplasia, atrophy of the olfactory epithelium, and adhesions of the nasal turbinate. These non-neoplastic lesions in the nasal cavity are considered to be toxic effects of butyraldehyde.

It is very important to investigate the outcome pathway of chemical-induced carcinogenesis. As a part of this investigation, we examined the pathogenesis of nasal cavity carcinogenesis induced by butyraldehyde. In the nasal cavity: (1) Almost all of the squamous cell tumors were malignant (squamous cell carcinoma) in both males and females. Only one benign squamous cell tumor developed, a squamous cell papilloma developed in a female rat. Consequently, transition from benign to malignant tumors, which is observed in non-genotoxic carcinogenesis, was not observed. (2) Squamous cell hyperplasia, a pre-neoplastic lesion, showed dysplasia. Therefore, it is not likely that the squamous cell papilloma that developed in the female rat could transform into a squamous cell carcinoma. Therefore, the carcinogenesis pathway observed in this carcinogenicity study of butyraldehyde, a genotoxic substance, is de novo carcinogenesis that develops as a malignant event.

In addition to the present study on butyraldehyde, JBRC has conducted inhalation carcinogenicity studies on two other aldehydes, crotonaldehyde (JBRC, Report for the Ministry of Health Labor Welfare of Japan, 2001), and acrolein (JBRC, Report for the Ministry of Health Labor Welfare of Japan, 2016). Both crotonaldehyde and acrolein caused tumors in the nasal cavity. The doses of crotonaldehyde (2-butenal) and acrolein (2-propenal) were 0, 3, 6, and 12 ppm, and 0, 0.1, 0.5, and 2 ppm, respectively. The ratio of the highest concentrations of these aldehydes administered by inhalation is 250-fold for butyraldehyde/crotonaldehyde and 1500-fold for butyraldehyde/acrolein. Aldehydes are classified as compounds with a molecular formula of R-C=O. Butyraldehyde has a molecular formula of C₄H₈O (C-C-C-C=O), crotonaldehyde has a molecular formula of C₄H₆O (C-C=C-C=O), and acrolein has a molecular formula of C₃H₄O (C=C-C=O). As can be seen, crotonaldehyde and butyraldehyde are similar in chemical structure, with crotonaldehyde being an unsaturated aldehyde with a double bond, whereas butyraldehyde is a saturated aldehyde without a double bond. The double bond in the chemical structural formula resulted in a 250-fold increase in the carcinogenic concentration (crotonaldehyde < butyraldehyde). The administration of butyraldehyde in this study resulted in mostly squamous cell carcinomas, whereas adenomas and rhabdomyosarcomas were characteristic for crotonaldehyde and squamous cell carcinomas and rhabdomyosarcomas for acrolein. Adenomas and squamous cell carcinomas were also observed in one to two cases in the highest concentration groups of crotonaldehyde and acrolein. Finally, a very rare rhabdomyosarcoma (1/1199 males and 0/1097 females in the JBRC historical control data) was observed in one male in the 12 ppm crotonaldehyde exposure group and in four females in the 2 ppm acrolein exposure group. Both substances were evaluated as showing evidence of carcinogenicity.

Oral administration of another aldehyde, glyoxal, caused a slight increase in the incidence of endometrial stromal polyps in females, but no increase in tumor development in the nasal cavity. This indicates that glyoxal was not metabolized in the liver to a nasal carcinogen, and suggests that in the inhalation studies, butyraldehyde, crotonaldehyde, and acrolein acted as direct carcinogens in the nasal cavity. Metabolism of butyraldehyde in the rat nasal cavity may be primarily mediated by aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) (Bogdanffy, 1990). It is thought to be oxidized by ALDH to butyric acid and reduced by ADH to 1-butanol (Marchitti et al., 2008). In addition, the CYP enzyme system is also present in the epithelial cells of the nasal cavity and may be involved in the oxidative metabolism of butyraldehyde (Bogdanffy, 1990). No reports indicating carcinogenicity were found for the main metabolites, butyric acid and 1-butanol. Although the mechanism of carcinogenesis of butyraldehyde has not yet been elucidated, it has been suggested that the carcinogenic mechanism of butyraldehyde may include errors in repairing genetic damage originating from stimulation-induced cytotoxicity, depletion of metabolic systems due to high concentrations and prolonged administration, and, like formaldehyde, a typical aldehyde, cross-linking of DNA and proteins, modification of proteins, and accumulation of denatured proteins (Bolt, 2003; Bolt and Degen, 2004).

In vitro genotoxicity studies found that butyraldehyde is highly cytotoxic to bacteria, and that the results of the Ames test with or without the S9 mix was negative (Mortelmans et al., 1986; Dillon et al., 1998). Because of the high antimicrobial activity of aldehyde compounds, the mutagenicity of aldehyde compounds is unlikely to be detected in the Ames test using bacteria as the test strain. However, using Chinese hamster ovary (CHO) cells, Galloway et al. reported positive results in cytogenetic/chromosome aberration tests (Galloway et al., 1987). Since the levels of butyraldehyde that were tested had little or no cytotoxicity in cultured cells, the genotoxic effects are considered to be reliable. The chromosome aberration test (CHL/IU cells) was also positive with or without the S9 mix in a study performed by JBRC (JBRC, Report for the Ministry of Health Labor Welfare of Japan, 2005), however, the micronucleus test (mouse peripheral blood) was negative (Witt et al., 2000). Based on the JBRC data, the genotoxicity of aldehyde compounds in cultured cells may include two types of chromosomal aberration effects. The first is an action that causes structural aberrations of chromosomes, and the second is an action that alters the number of chromosomes. In structural aberrations, chromosomes are directly damaged, whereas in numerical aberrations, errors in the number of chromosomes sets lead to instability in cell division, which may lead to oncogenic promoter-like effects.

In conclusion, inhalation of butyraldehyde was carcinogenic in F344/DuCrlCrlj male and female rats. The rats were exposed to 0, 300, 1,000 and 3,000 ppm (v/v) using whole-body inhalation chambers. The study was carried out in accordance with GLP principles and OECD test guidelines. The incidence of squamous cell carcinoma of the nasal cavity was increased in the 3,000 ppm groups of both male and female rats, with Fisher’s exact test and the Peto test indicating that the increases were significant. In addition to squamous cell carcinoma in the nasal cavity, in the 3,000 ppm group one male had an adenosquamous carcinoma, one male had a carcinosarcoma, one male had a sarcoma NOS, and one female had a squamous cell papilloma in the nasal cavity. The combined incidence of nasal tumors was significantly increased in male and female rats. Based on these results, we conclude that there is clear evidence of butyraldehyde carcinogenicity in male and female F344/DuCrlCrlj rats.

ACKNOWLEDGMENT

The present study was contracted and supported by the Ministry of Health, Labour and Welfare, Japan. We also acknowledge the efforts of all members of JBRC.

Conflict of interest

The authors declare that there is no conflict of interest.

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