Introduction

2,4-Dichloro-1-nitrobenzene (2,4-DCNB) is an organic solid which is used as a chemical intermediate for the synthesis of medical drugs, pesticides and pigments (BUA report 1991; OECD SIDS 1996). 2,4-DCNB is a High Production Volume (HPV) chemical (OECD 2004), and in order to protect human health and the environment from hazardous chemicals, the Organization for Economic Co-operation and Development (OECD) designated 2,4-DCNB a high priority chemical for initial assessment and a Screening Information Data Set (SIDS) Testing Plan was carried out (OECD SIDS 1996): The assessment report concluded that 2,4-DCNB showed strong toxicity toward daphnia and was genotoxic in the Ames test, but that environmental exposure was low, and the report recommended that 2,4-DCNB be given low priority for further study. No epidemiological data has been available for health risk assessments of workers exposed to 2,4-DCNB. No bioassay studies of rodent carcinogenicity or chronic toxicity of 2,4-DCNB have been reported. The carcinogenic potential and classification of 2,4-DCNB have not been evaluated by the International Agency for Research on Cancer (IARC), the American Conference of Governmental Industrial Hygienists (ACGIH) or the Japan Society for Occupational Health.

Several in vitro studies have shown that bacterial mutagenicity and mammalian clastogenicity were positive with S9 activation (OECD SIDS 1996; JETOC 1996), and, consequently, 2,4-DCNB has been evaluated as one of the existing chemical substances with positive mutagenicity. The Technical Guideline of the Japanese Industrial Safety and Health Law requires that occupational health countermeasures be taken to protect workers from exposure to mutagenic substances (Japan Industrial Safety and Health Association 2004). The present study was undertaken to provide dose–response data from long-term rodent carcinogenicity and chronic toxicity testing of 2,4-DCNB for health risk assessment of 2,4-DCNB-exposed workers. Carcinogenicity and chronic toxicity were examined by feeding F344 rats and BDF1 mice of both sexes diets containing different doses of 2,4-DCNB for 2 years.

Materials and methods

The present study was conducted with reference to the OECD Guideline for Testing of Chemicals 451 “Carcinogenicity Studies” (OECD 1981) and were carried out in conformity with the OECD Principle of Good Laboratory Practice (OECD 1998). The animals were cared for in accordance with the guidelines for the care and use of laboratory animals (NRC 1996), and the present study was approved by the ethics committee of the Japan Bioassay Research Center (JBRC).

Test substance

2,4-DCNB (CAS No. 611-06-3) of guaranteed grade (99.4 % pure) was obtained from Wako Pure Chemical Industries, Ltd (Osaka, Japan). The 2,4-DCNB was analyzed for purity and stability by gas chromatography (GC) (Hewlett Packard 6890, Agilent Technologies, Santa Clara, CA, USA) before and after its use. The test substance was stable throughout a 104-week period of storage. The lot used was found to contain 1,5-dichloro-2,3-dinitrobenzene and 1,2-dichloro-4,5-dinitrobenzene, and their concentrations were quantitated at 0.018 and 0.014 % by GC, respectively.

Animals and husbandry

Four-week-old F344/DuCrj rats (SPF) and Crj:BDF1 mice (SPF) of both sexes were obtained from Charles River Laboratories Japan, Inc (Kanagawa, Japan). After a 2-week quarantine and acclimation period, the animals were allocated by a stratified randomization procedure into 4 body-weight-matched groups, each comprising 50 rats or 50 mice of either sex. The animals were housed individually in stainless-steel wire hanging cages (170 mm [W] × 294 mm [D] × 176 mm [H] for rats and 112 mm [W] × 212 mm [D] × 120 mm [H] for mice) under controlled environmental conditions (temperature of 23 ± 2 °C and relative humidity of 55 ± 15 % with 15–17 air changes/h) in barrier controlled specific pathogen free (SPF) animal rooms. Fluorescent lighting was controlled automatically to provide a 12-h light/dark cycle. All animals had free access to filtered, UV-irradiation-sterilized water supplied by an automatic watering system.

Dose level, diet preparation and feeding

The 2,4-DCNB dose levels (wt/wt) in the diet was 0, 750, 1,500 or 3,000 ppm for male and female rats; 0, 750, 1,500 or 3,000 ppm for male mice; and 0, 1,500, 3,000 or 6,000 ppm for female mice. The highest dose level was chosen so as to not exceed the maximum tolerated dose (MTD) obtained from our preliminary 13-week administration study of body weight gain and subchronic toxicity (unpublished data). The criteria of the MTD used in the present study are described in the guidelines of the National Cancer Institute (NCI) (Sontag et al. 1976) and IARC (Bannasch et al. 1986). A diet containing 2,4-DCNB was prepared by mixing 2,4-DCNB with γ-irradiation-sterilized CRF-1 powdered diet (Oriental Yeast Co., Ltd., Tokyo, Japan) in a spiral mixer for 20 min, and stored at 7 °C until use. The powdered diet containing 2,4-DCNB was prepared at intervals of 2 weeks throughout the 2-year administration period. The feeders in each cage filled with control or 2,4-DCNB-containing diet were exchanged once a week. 2,4-DCNB concentrations in the powdered diet were determined by GC, and were found to be 91.7–106 % of the target concentrations at the time of preparation. Initial concentrations decreased to 84.3–88.7 % on the 9th day after preparation, with the concentration at the time of preparation being taken as 100 %. The animals were fed the control or 2,4-DCNB-containing diets throughout the 2-year administration period, starting at the age of 6 weeks.

Clinical observations and analysis, and pathological examinations

The animals were observed daily for clinical signs and mortality. Body weight and food consumption were measured once a week for the first 14 weeks of the 2-year administration period and every 4 weeks thereafter. Urinary parameters were measured near the end of the 2-year administration period with Ames reagent strips (Multistix for rats and Uro-Labstix for mice: Bayer HealthCare, NY, USA), and the following parameters were determined: pH, protein, glucose, ketone, occult blood and urobilinogen (rats and mice) and bilirubin (rats). For hematology and blood biochemistry, blood was collected under etherization at the terminal necropsy after overnight fasting. Hematological parameters were measured with an Automatic Blood Cell Analyzer (ADVIA 120, Bayer HealthCare, NY, USA and MICROX HEG-120NA, Omron Co., Kyoto, Japan): red blood cell count (RBC), hemoglobin concentration (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), white blood cell count (WBC) and differential leukocyte count. The blood biochemical parameters were measured with an Automatic Analyzer (HITACHI 7080, Hitachi Ltd., Tokyo, Japan): total protein (TP), albumin (Alb), albumin/globulin ratio (A/G ratio), total bilirubin (T-Bil), glucose, total cholesterol (T-Cho), triglyceride (TG), phospholipid, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (γ-GTP), creatine kinase (CK), blood urea nitrogen (BUN), sodium, potassium, chloride, calcium and inorganic phosphorus (rats or mice), and creatinine (rats). A complete necropsy was performed on all animals, including those that were found dead or in a moribund state. Organs were removed, weighed and examined for macroscopic lesions. The tissues used for microscopic examination were fixed in 10 % neutral buffered formalin and embedded in paraffin. Tissue sections 5 μm in thickness were prepared and stained with hematoxylin and eosin (H & E).

Statistical analysis

Survival curves were plotted according to the method of Kaplan–Meier. The log-rank test was used to test for a statistically significant difference in survival rate between any 2,4-DCNB-fed group of either sex and the respective control group. Body weight, food consumption, organ weights and hematological and blood biochemical parameters were analyzed by Dunnett’s test. Incidences of pre- and non-neoplastic lesions and urinary parameters were analyzed by Chi-square test. Incidences of neoplastic lesions were analyzed for a dose response relationships by Peto’s test and for a statistically significant difference from the concurrent control group by Fisher’s exact test. Two-tailed testing was used for all statistical analyses except for Peto’s test. In all cases, statistical analysis with p values of 0.05 and 0.01 was performed and are indicated in the tables; a p value of 0.05 was used for statistical significance.

Results

Rat 2 year test

Survival, body weight, food consumption and clinical signs

The Kaplan–Meier survival analysis showed no significant reduction in the survival rate between any 2,4-DCNB-fed group of either sex and their respective control group. Growth rates were dose-dependently suppressed in all the 2,4-DCNB-fed groups (Fig. 1a, b). In males, terminal body weights of the 750, 1,500 and 3,000 ppm-fed groups were significantly decreased by 7, 11 and 15 %, and in females, terminal body weights of the 1,500 and 3,000 ppm-fed groups were significantly decreased by 8 and 14 % (Table 1). Slightly decreased food consumption was observed sporadically in all the 2,4DCNB-fed groups of both sexes during the initial 2 months of the 2-year administration period. The estimated amounts of 2,4-DCNB intake were proportionally-increased with an increase in the dietary concentration of 2,4-DCNB (Table 1). Yellow-colored urine was observed in all the 2,4-DCNB-fed groups of both sexes throughout the 2-year administration period.

Fig. 1
figure 1

Growth curves of rats (a, b) and mice (c, d) fed 2,4-DCNB-containing diets or control diet for 2 years

Full size image
Table 1 Chemical intake, terminal body weight and organ weight of rats administered 2,4-DCNB in the diet for 2 years
Full size table

Organ weights and macroscopic findings

Absolute and relative liver weights were significantly increased in all the 2,4-DCNB-fed males and in the 1,500 and 3,000 ppm fed females (Table 1). Absolute kidney weight was significantly increased in all the 2,4-DCNB-fed groups, and relative kidney weight was significantly increased in all the 2,4-DCNB-fed males and in the 1,500 and 3,000 ppm fed females. Grayish white nodules were observed in the kidneys of the 3,000 ppm-fed male and females: 1–15 mm in diameter in males and 1–7 mm in diameter in females.

Hematology, blood biochemistry and urinalysis

Plasma levels of T-Cho and phospholipids were increased in the 2,4-DCNB-fed groups of both sexes. γ-GTP was increased in all the 2,4-DCNB-fed males. Statistically significant increases in BUN was noted in all the 2,4-DCNB-fed males and in the 1,500 and 3,000 ppm-fed females. There were no biologically significant changes in any other parameter assayed (data not shown).

Histopathological findings

Incidences of renal cell adenomas and carcinomas were increased dose-dependently in both sexes, as indicated by a significant positive trend by Peto’s test (Table 2). The incidences of renal cell adenomas and carcinomas were significantly increased in the 3,000 ppm-fed groups of both sexes. Since the incidences of renal cell adenomas in the 1,500 ppm-fed males (6 %) and females (6 %) exceeded the maximum incidences of the JBRC historical control data (2 cases in 1,749 male rats with a maximum incidence of 2 % in a single study, and 2 cases in 1,597 female rats with a maximum incidence of 2 % in a single study), the renal cell adenomas occurring in the 1,500 ppm-fed males and females are judged to be compound-related. The renal cell carcinomas were solid lesions showing a lobular architecture with central degeneration and necrosis and some outlying tubular differentiation. Loss of nuclear polarity can be seen in the area adjacent to the central necrotic region (Fig. 2a insert). None of the renal carcinomas metastasized to any other organ. Renal cell adenoma was first observed in the 76th week in a 3,000 ppm-fed, moribund male rat. Atypical tubule hyperplasias at the proximal tubule, a proliferative pre-neoplastic lesion (Montgomery and Seely 1990; Hard et al. 1995), were noted in all the 2,4-DCNB-fed groups (Table 2). Eosinophilic droplets in the proximal tubule were also increased in all the 2,4-DCNB-fed groups. Granular surfaces on the kidneys were diagnosed as indicative of chronic progressive nephropathy (CPN) (Kawai 1980). The incidences of marked and severe grades of CPN with were significantly increased in a dose-related manner in the 2,4-DCNB-fed males, and the incidences of CPN were increased in the 750 and 1,500 ppm-fed females. The incidences of urothelial hyperplasia in the pelvis and mineralization in the papilla were increased in all the 2,4-DCNB-fed males (Table 2).

Table 2 Incidences of neoplastic, pre-neoplastic and non-neoplastic lesions in rats administered 2,4-DCNB in the diet for 2 years
Full size table
Fig. 2
figure 2

The principal neoplasms induced by 2-year dietary administration of 2,4-DCNB. Each section was stained by H&E. Bar indicates 200 μm. a Renal cell carcinoma in the kidney of a male rat fed 3,000 ppm 2,4-DCNB. Solid tumor growth can be seen including a necrotic area. Insert shows massive growth of tumor cells and the loss of nuclear polarity. b Hepatocellular carcinoma in the liver of a male mouse fed 3,000 ppm 2,4-DCNB. Solid growth pattern (lower part) and island-like structures (upper part) of thickened hepatic plate can be seen. c Hepatoblastoma in the liver of a male mouse fed 3,000 ppm 2,4-DCNB. A basophilic area composed of anaplastic hepatocytes can be seen adjacent to the hepatocellular carcinoma. d Hemangiosarcoma in the peritoneum around the uterus of a female mouse fed 6,000 ppm 2,4-DCNB

Full size image

In the preputial glands, adenomas occurred dose-dependently in the males, and the incidence of adenomas was significantly increased in the 3,000 ppm-fed males (Table 2).

Although other types of tumors were observed in both 2,4-DCNB-fed and control rats, there were no dose-related differences in the incidences of these tumors between any of the 2,4-DCNB-fed groups and the control group.

Mouse 2 year test

Survival, body weight, food consumption and clinical signs

The Kaplan–Meier survival analysis showed a significant difference in the survival rate between the 3,000 ppm-fed males and the 3,000 and 6,000 ppm-fed females and their respective controls. The decreased survival rates were attributed to the increased number of death due to hepatic tumors in the males and to hepatic tumors and peritoneal tumors in the females. Growth rates were dose-dependently suppressed in all the 2,4-DCNB-fed male and female groups except for the 750 ppm-fed male group (Fig. 1c, d). In males, terminal body weights of the 1,500 and 3,000 ppm-fed groups were significantly decreased by 10 and 27 %, and in females, terminal body weights of the 1,500, 3,000 and 6,000 ppm-fed groups were significantly decreased by 8, 24 and 40 % (Table 3). Food consumption was decreased in the 3,000 ppm-fed males and in the 6,000 ppm-fed females during the first year of the 2-year administration period. The estimated amounts of 2,4-DCNB intake were proportionally-increased with an increase in the dietary concentration of 2,4-DCNB (Table 3). Yellow-colored urine was observed in all the 2,4-DCNB-fed groups of both sexes throughout the 2-year administration period.

Table 3 Chemical intake, terminal body weight and organ weight of mice administered 2,4-DCNB in the diet for 2 years
Full size table

Organ weights and macroscopic findings

Absolute and relative liver weights were increased in the 1,500 and 3,000 ppm-fed males and in all the 2,4-DCNB-fed females (Table 3). The incidence of liver nodules and the sizes of the nodules were increased dose-dependently in the 2,4-DCNB-fed groups of both sexes. The incidence of peritoneal nodules was increased dose-dependently in the 2,4-DCNB-fed groups of both sexes; these nodules were found predominantly in the peritoneum around the pelvic viscera.

Hematology, blood chemistry and urinalysis

Decreased RBC and Hb, and increased MCV were noted in the 3,000 ppm-fed males. T-Cho was increased in all the 2,4-DCNB-fed groups of both sexes. Phospholipid was increased in all the 2,4-DCNB-fed groups of both sexes, except the 750 ppm-fed males. ALP was increased in all the 2,4-DCNB-fed mice of both sexes, and AST and ALT were increased in all the 2,4-DCNB-fed mice of both sexes, except the 1,500 ppm-fed females. γ-GTP was increased in the 3,000 ppm-fed males and 3,000- and 6,000 ppm-fed females. LDH and CK were increased in all the 2,4-DCNB-fed groups of both sexes, except the 750 ppm-fed males. BUN was increased in the 3,000- and 6,000 ppm-fed females. T-Bil was increased in the 1,500 and 3,000 ppm-fed males and in the 6,000 ppm-fed females. There were no biologically significant changes in any other parameter assayed (data not shown).

Histopathological findings

Incidences of hepatocellular adenomas, hepatocellular carcinomas (Fig. 2b) and hepatoblastomas (Fig. 2c) were increased dose-dependently in both sexes, as indicated by a significant positive trend by Peto’s test (Table 4). Incidences of hepatocellular adenomas were significantly increased in all the 2,4-DCNB-fed male and female groups; in particular, the incidences of hepatocellular carcinomas were significantly increased in the 3,000 ppm-fed males and in the 3,000 and 6,000 ppm-fed females. Incidences of hepatoblastomas were significantly increased in the 1,500 and 3,000 ppm-fed males and in the 3,000 and 6,000 ppm-fed females. Since the incidences of hepatoblastomas in the 750 ppm-fed males and in the 1,500 ppm fed females (10 and 4 %, respectively) exceeded the respective maximum incidences of the JBRC historical control data (10 cases in 1,496 male mice with a maximum incidence of 6 %, 0 cases in 1,498 female mice), the hepatoblastomas occurring in the 750 ppm-fed males and in the 1,500 ppm-fed females are judged to be compound-related. In addition, combined incidences of hepatocellular adenomas, hepatocellular carcinomas and/or hepatoblastomas were significantly increased in all the 2,4-DCNB-fed groups of both sexes. The hepatocellular carcinomas and hepatoblastomas metastasized predominantly to the lung, followed by the peritoneum, lymph node, stomach (whole layer infiltration), ovary and pancreas. Incidences of acidophilic cell foci were increased dose-dependently in the 3,000 and 6,000 ppm-fed females (Table 4). Incidences of centrilobular hypertrophy of hepatocytes were increased in all the 2,4-DCNB-fed males and in the 6,000 ppm-fed females (Table 4).

Table 4 Incidences of neoplastic, pre-neoplastic and non-neoplastic lesions in mice administered 2,4-DCNB in the diet for 2 years
Full size table

In the peritoneum, hemangiosarcomas (Fig. 2d) occurred dose-dependently in the males and females, and the incidences of hemangiosarcomas was significantly increased in the 3,000 and 6,000 ppm-fed females (Table 4). Since the incidences of peritoneal hemangiosarcomas in the 3,000 ppm-fed males and in the 1,500 ppm-fed females (10 and 6 %, respectively) exceeded the maximum incidence of the JBRC historical control data (3 cases in 1,496 male mice with a maximum incidence of 4 %, and 6 cases in 1,498 female mice with a maximum incidence of 4 %), the hemangiosarcomas occurring in the 3,000 ppm-fed males and in the 1,500 ppm-fed females are judged to be compound-related. Hemangiosarcomas were found in the peritoneum around the pelvic viscera (e.g., urinary bladder, uterus, and male accessory sex gland).

In the nasal cavity, the incidences of deposition of brown pigment and respiratory metaplasia in the olfactory epithelium and submucosal gland were increased in males and females, and the incidences of eosinophilic globules in the olfactory and respiratory epithelia were increased in females. Deposition of brown pigment was only observed in the respiratory epithelial cells and submucosal glands, and this change was not seen our preliminary 13-week examination and was very rare in the examination conducted by the JBRC. Increased incidence of eosinophilic globules in the nasopharynx occurred in the 3,000 ppm-fed males and in all the 2,4-DCNB-fed females.

Although other types of tumors were observed in both 2,4-DCNB-fed and control mice, there were no dose-related differences in the incidences of those tumors between any 2,4-DCNB-fed groups and the control group.

Discussion

We previously reported the effect of 2,4-DCNB on bacterial mutagenicity and mammalian chromosome aberration (JETOC 1996): The bacterial mutagenicity of 2,4-DCNB was positive with Salmonella typhimurium TA98 and TA100, when metabolically activated with S9; the chromosomal aberration assay with Chinese hamster lung cells (CHL/IU) was positive for structural aberration with S9 activation. Importantly, 2,4-DCNB was mutagenic only when metabolically activated with S9.

In the present study, a 2-year dietary administration of 2,4-DCNB was found to be carcinogenic in male and female rats and mice. Renal carcinogenicity of 2,4-DCNB in rats was evidenced by dose-related increases in the incidence of renal cell adenomas and carcinomas and in the incidence of atypical tubular hyperplasia, a pre-neoplastic lesion, in the proximal tubule (Montgomery and Seely 1990). α2u-Globulin-induced renal tumors have been reported to occur in male F344 rats exposed to a variety of chemicals due to excessive accumulation of α2u-globulin in the proximal tubular epithelial cells of male rats (Anden et al. 1984; MacFarland et al. 1984; Charbonneau et al. 1989; NTP 1990). The dose-dependent increase in the incidence of renal tumors noted in female rats in the present study, however, strongly suggest that 2,4-DCNB induction of renal tumors is by some other mechanism. 1,4-Dichloro-2-nitrobenzene (1,4-DCNB), a positional isomer of 2,4-DCNB, is metabolised to N-acetyl-S-(4-chloro-3-nitrophenyl)-L-cysteine by β-lyase in the kidney and excreted in the urine (Ohnishi et al. 2004), and since cysteine conjugates made from γ-glutamyltranspeptitase and β-lyase are nephrotoxic (Vambakas et al. 1988; Elfarra et al. 1986; Dekant et al. 1986, 1988), it is likely that N-acetyl-S-(4-chloro-3-nitrophenyl)-L-cysteine is responsible for the renal tumors induced in rats by 2-year dietary administration 1,4-DCNB (Yamazaki et al. 2006). Similarly to 1,4-DCNB, 2,4-DCNB is metabolized to N-acetyl-S-(5-chloro-2-nitrophenyl)-L-cysteine by β-lyase in the kidney and excreted in the urine (Ohnishi et al. 2009). These data, taken in conjunction with the fact that 2,4-DCNB is mutagenic only when metabolically activated by S9, lead to the premises that genotoxic metabolites of 2,4-DCNB, such as N-acetyl-S-(5-chloro-2-nitrophenyl)-L-cysteine, are responsible for the renal carcinogenesis observed in the present study.

Hepatocarcinogenicity of 2,4-DCNB for male and female mice was clearly evidenced by dose-related increases in the incidences of hepatocellular adenomas, hepatocellular carcinomas and hepatoblastomas in male and female mice. A striking feature of 2,4-DCNB-induced hepatocarcinogenicity was the induction of historically rare hepatoblastomas; hepatoblastomas have a morphological structure completely different from that of hepatocellular carcinomas (Frith et al. 1994). The liver tumors induced by 2,4-DCNB were highly malignant as evidenced by metastasis of the hepatocellular carcinomas and hepatoblastomas to the lung, peritoneum, lymph node and stomach. Although neither hypertrophy of hepatocytes nor necrotic or regenerative changes were histopathologically observed in the 1,500 or 3,000 ppm female mouse livers of the present study, liver tumors were induced in the 1,500 and 3,000 ppm 2,4-DCNB-fed females. Since it is well known that centrilobular hypertrophy relates to a non-genotoxic mechanism of hepatocarcinogenesis in mice, a genotoxic mode of action is therefore likely to be responsible for the observed 2,4-DCNB-induced hepatocarcinogenesis.

Induction of carcinogenic peritoneal tumors by 2,4-DCNB was clearly evidenced by dose-related increases in the incidences of malignant peritoneal hemangiosarcomas in male and female mice. The incidence of hemangiosarcomas in the 1,500 ppm-fed females exceeded the maximum incidence of the JBRC historical control data and was significantly increased in the 3,000 and 6,000 ppm-fed females. In males, while the increase in the incidence of peritoneal hemangiosarcomas was not statistically significant by Fisher’s exact test, the incidences of peritoneal hemangiosarcomas in the 3,000 ppm-fed males exceeded the maximum incidence of the JBRC historical control data.

In addition, 2,4-DCNB also induced preputial gland adenomas as shown by a significant increase in the incidence of benign preputial gland adenomas in the 3,000 ppm-fed male rats and a slight increase in tumor incidence over the upper range of the JBRC historical control data.

Chronic toxicity of dietary 2,4-DCNB was seen in both rats and mice. 2,4-DCNB-induced renal toxicity was evidenced by increased severity and progression of CPN as shown by a dose-dependent increase in the incidences of marked and severe grades of CPN in the 2,4-DCNB-fed male rats and an increase in the incidence of CPN in the 750 and 1,500 ppm-fed female rats. In addition, papillary mineralization and urothelial hyperplasia in the renal pelvis were noted in the male rats. Moreover, eosinophilic droplets in the proximal tubule and the serum levels of BUN were increased in the male and female rats.

Although the 2,4-DCNB was administered by the oral route, it is noteworthy that the 2,4-DCNB-induced pathological changes were observed in the upper respiratory tract in mice: The incidences of pigment deposition and respiratory metaplasia in the olfactory epithelium and submucosal gland were increased in males and females, and the incidences of eosinophilic globules in the olfactory and respiratory epithelia were increased in females. In addition, the incidence of eosinophilic globules in the nasopharynx were increased in males and females.

2,4-DCNB is a genotoxic carcinogen, and the hepatocellular adenomas and the combined incidence of total hepatic tumors were increased at the lowest dose level in male and female mice. Therefore, we used a non-threshold approach to calculate the benchmark dose associated with 10 % risk over background (BMDL10). Based on the dose–response relationships between 2,4-DCNB-intake and incidences of tumors obtained in the present study, a 95 % lower confidence limit of the BMDL10 was calculated using the linearized multistage model for total hepatic tumors of male and female mice with US EPA’s benchmark dose software (Ver. 2.1.1) (US EPA 2009). The BMDL10 value for the endpoint of total hepatic tumors in male mice was 12.3 mg/kg per day and in female mice was 23.5 mg/kg per day.