The Journal of Toxicological Sciences
Online ISSN : 1880-3989
Print ISSN : 0388-1350
ISSN-L : 0388-1350
Original Article
Carcinogenicity of quinoline by drinking-water administration in rats and mice
Michiharu Matsumoto , Hirokazu Kano, Masaaki Suzuki, Tadashi Noguchi, Yumi Umeda, Shoji Fukushima
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Keywords: Quinoline, Hemangioma, Hemangiosarcoma, Rats, Mice
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2018 Volume 43 Issue 2 Pages 113-127

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Abstract

The carcinogenicity of quinoline was examined by administrating quinoline in the drinking water to groups of 50 F344/DuCrj rats and 50 Crj: BDF1 mice of each sex. In rats, the doses of quinoline were 0, 200, 400, and 800 ppm for males and 0, 150, 300, and 600 ppm for females. In male rats, administration of quinoline was terminated at week 96 due to high mortality caused by tumors. There were significant increases of hepatocellular adenomas, hepatocellular carcinomas, hepatocellular adenomas and/or carcinomas (combined), and liver hemangiomas, hemangiosarcomas, hemangiomas and/or hemangiosarcomas (combined) in both male and female rats, and nasal esthesioneuroepitheliomas and sarcoma NOS (not otherwise specified) in males. In mice, doses of quinoline were 0, 150, 300 and 600 ppm for both males and females. Administration of quinoline was terminated at week 65 in males and at week 50 in females due to high mortality caused by tumors. There were marked increases of hemangiomas, hemangiosarcomas, and hemangiomas and/or hemangiosarcomas (combined) in the retroperitoneum, mesenterium, and liver in males, and in the retroperitoneum, mesenterium, peritoneum, and subcutis in females. Additionally, histiocytic sarcomas were statistically increased in the livers of female mice. Thus the present studies provided clear evidence of carcinogenic activity of quinoline administered in the drinking water in both rats and mice.

INTRODUCTION

Quinoline (CAS No. 91-22-5) is widely used as an intermediate in the manufacture of other products, extraction and purification in organic synthetic chemistry, and as a solvent and stabilizer in organic products. 900 tons of quinoline is produced annually in Japan (Chemical Daily, 2014). Quinoline is released as a byproduct of combustion from steel plants, waste incinerators, and gasoline- and diesel-powered vehicles. The total amount of quinoline transferred and released from various sectors of industry into the ambient air and public water in Japan in 2015 was 9.8 tons (Japan Ministry of the Environment 2017). In addition, exposure to quinoline can also occur from the inhalation of cigarette smoke (International Agency for Research on Cancer, 1985). It has been concluded that quinoline in the environment may constitute a danger to human life or health.

Quinoline has significant mutagenic activity in the Ames test using Salmonella Typhimurium and Escherichia coli with metabolic activation, and induces unscheduled DNA synthesis (UDS) in rat hepatocytes in vitro. Quinoline also induces chromosomal aberration in in vitro cultured cells. In vivo quinoline induces chromosomal aberration and sister chromatid exchange in the rat liver when administered by gavage, and quinoline induces gene mutations in the liver of the lacZ transgenic mouse (Muta Mouse). Discussion of other reports on the in vitro and in vivo genotoxicity of quinoline can be found in the U.S. Environmental Protection Agency's Toxicological Review of Quinoline (US EPA, 2001).

Administration of quinoline in the diet is reported to have hepatocarcinogenicity (hepatocellular carcinomas, and hemangioendotheliomas and/or hemangiosarcoma) in rats (Hirao et al., 1976; Shinohara et al., 1977; Hasegawa et al., 1989; Futakuchi et al., 1996), and mice (LaVoie et al., 1987, 1988), and intraperitoneal administration of quinoline is also reported to be carcinogenic (LaVoie et al., 1987, 1988). However, these studies had small sample sizes, examined only one sex, or were of short duration due to early mortality (US EPA, 2001). The studies on the mitogenicity and mutagenicity of quinoline and the dietary studies in rats led the EPA to classify quinoline as a Group L (Likely to be carcinogenic in human) compound (US EPA, 2001).

The object of the present study is to establish the carcinogenicity of quinoline in male and female rats and mice under Good Laboratory Practice (GLP).

MATERIALS AND METHODS

This study was conducted in accordance with Organisation for Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals 451 (Carcinogenicity studies) (OECD, 1981a) and OECD Principals of GLP (OECD, 1981b). The animals were cared for in accordance with guidelines for the care and use of laboratory animals (National Research Council (NRC), 1977). The present studies were approved by the ethics committee of the Japan Bioassay Research Center (JBRC).

Chemicals

Reagent grade quinoline (purity > 99.6%) was obtained from Tokyo Chemical Industry (Tokyo, Japan) in two lots and stored in sealed amber glass bottles at room temperature. The chemical was a colorless liquid and soluble in water (60 mg/mL). Each lot of the chemical was identified as quinoline by infrared spectrometry (FTIR-8200PC, Shimadzu Co., Kyoto, Japan) and mass spectrometry (M-80B, Hitachi Ltd., Ibaraki, Japan). The purity and stability of each lot was confirmed by analysis with gas chromatography (5890A, Hewlett Packard, Minneapolis, MN, USA) before and after use.

Preparation and treatment of quinoline

Quinoline was dissolved in deionized water at the target concentrations. The solutions were prepared once a week and were put into stainless steel containers (Volume: 20 L) of a closed and pressurized automatic watering system (Kano et al., 2002).

The concentration of quinoline in drinking water was examined at least once every 3 months, at the time of preparation, by high-performance liquid chromatography (HPLC: 1090, Hewlett Packard) and confirmed to be 95.3-101% of the target concentration for rats and 95.3-102% for mice. The stability of the quinoline in the drinking water was assessed by analysis of quinoline concentrations in dose formulations using HPLC. The doses of quinoline used in this study were stable in drinking water stored in the stainless steel containers under animal room conditions for 8 days.

Animals

Male and female F344/DuCrj rats (SPF) and Crj:BDF1 mice (SPF) were obtained at the age of 4 weeks from Charles River Japan, Inc. (Kanagawa, Japan). They were housed individually in stainless steel wire hanging cages in barrier system rooms in which temperature, relative humidity, and air change rate were maintained at 23 ± 2°C, 55 ± 10%, and 12 air changes/hr. Lighting was controlled automatically to give a 12-hr light/dark cycle. All rats and mice were given a commercial pellet diet (CRF-1, Oriental Yeast Co. Ltd., Tokyo, Japan) sterilized with gamma-radiation and assigned drinking water ad libitum. After quarantine and acclimation for 2 weeks they were used for the present study.

Experimental design

Groups of 50 rats and 50 mice of each sex were exposed to targeted concentrations of quinoline in their drinking water. The drinking water concentrations of quinoline were 0 (control), 200, 400, and 800 ppm (wt/wt) for male rats; 0, 150, 300, and 600 ppm for female rats; and 0, 150, 300, 600 ppm for male and female mice. The highest doses used in the present carcinogenicity studies were selected based on the results of our previous 13-week studies using doses of 158, 237, 355, 533, and 800 ppm in rats and 237, 355, 533, 800, and 1200 ppm in mice. In the 13-week studies, all rats and mice survived to the end of the study. The final body weights of the 800 ppm group of male rats were 93% of male controls. In female rats, the final body weights of the 800 ppm and 553 ppm groups were 83% and 89%, respectively, of the controls. In mice, the final body weights of the 533 ppm male and female groups were 88% and 98%, respectively, of their controls. No biochemical or hematological changes in any of quinoline-administered animals were observed. In histopathology, only single cell vacuolic changes in the liver were increased in male rats administered 533 ppm and 800 ppm. From these data, we determined the MTD as 800 ppm for male rats and 600 ppm for female rats and male and female mice (unpublished data).

The initial design was to expose rats and mice to quinoline in their drinking water for up to 104 weeks; however, dose-related decreases in survival occurred in rats and mice of both sexes necessitating early termination of the experiment in male rats and male and female mice. All male rats administrated 800 and 400 ppm quinoline died by the end of weeks 76 and 95, respectively, and there were only 19 surviving animals in the 200 ppm group; therefore, this arm of the experiments was terminated at week 96. All female rats administered 600 ppm quinoline died by the end of week 88, however, the other groups had a fairly high number of surviving animals; therefore, administration of quinoline to female rats was continued for 104 weeks. All male mice administered 600 and 300 ppm quinoline died by the end of weeks 55 and 65 respectively, and there were only 15 surviving animals in the 150 ppm group; therefore, this arm of the experiments was terminated at week 65. All female mice administered 600 ppm quinoline died by the end of week 44, and there were only 6 surviving animals in the 150 ppm group; therefore, this arm of the experiments was terminated at week 50.

Clinical observations, food and water consumptions

The animals were carefully observed daily for clinical signs and mortality. Animals found in a moribund state were euthanized under anesthesia. Body weight, food and water consumption were measured weekly for the first 14 weeks of the administration period and every 2 weeks thereafter. Daily chemical intake (mg/kg body weight/day) was calculated as the concentration of quinoline in the drinking water and multiplied by the volume of drinking water consumed.

Pathological examinations

Organs were removed, weighed and examined for macroscopic lesions at necropsy. All organs and tissues designated in the OECD test guideline 451 (OECD, 1981a), including the entire respiratory tract, were examined for histopathology. The organs and tissues were fixed in 10% neutral buffered formalin. The nasal cavity was decalcified in a formic acid formalin solution prior to trimming, and was transversely trimmed at three levels according to the procedure described previously (Nagano et al., 1997). The organs and tissues were prepared for staining with hematoxylin and eosin (H&E). Histopathological diagnosis was performed by pathologists certified by the Japanese Society of Toxicologic Pathology and peer reviewed by outside board-certified pathologists.

Statistics and data analysis

Body and organ weight and food consumption were analyzed following the algorithm of decision tree methods (Hamada et al., 1998). Bartlett's test was used to test whether the variance was homogeneous. When the variance was homogeneous, one-way ANOVA was used. When the variance was not homogeneous, the Kruskal-Wallis rank sum test was used. Statistical differences in the means among the groups were analyzed by Dunnett's multiple comparison test when the variance was homogeneous and Dunnett's multiple comparison test by rank when the variance was not homogeneous. Incidences of non-neoplastic lesions were analyzed by Fisher's exact test. Incidences of neoplastic lesions were analyzed for a dose-response relationship by Peto et al. (1980) and for a statistically significant difference from the concurrent control group by Fisher's exact test. A biologically meaningful increase in the incidence for rare tumors was evaluated by whether or not the observed incidence exceeded the maximum tumor incidence in the JBRC historical control data compiled from 2-year studies of rodent carcinogenicity conducted by JBRC over the past 15 years. Two-tailed testing was used for all statistical analyses except for Peto's test. A p-value of 0.05 was considered statistically significant.

RESULTS

Rat study

Survival, body weights, food and water consumption and chemical intake

All of the 800 ppm and 400 ppm dosed male rats died by the end of week 76 and week 95, respectively. Due to the low number of surviving animals in the 200 ppm group, this arm of the experiments was terminated at week 96. The survival rates of the control and 200 ppm dosed males at week 96 were 98% (49/50) and 38% (19/50), respectively (Fig. 1A, B). All of the 600 ppm dosed female rats died by the end of week 88. The survival rates of the control and 150 and 300 ppm dosed females at the end of the 104 week administration period were 82% (41/50), 34% (17/50), and 4% (2/50), respectively. The mean survival times of males and females was shortened dose-dependently (Table 1). The decreased survival rates in male and female rats was attributed to deaths due to liver tumors.

Fig. 1

Survival curves (A and B) and body weight changes (C and D) of rats administered 0 (control), 200, 400, and 800 ppm quinoline (males) or 0 (control), 150, 300, and 600 ppm quinoline (females) in the drinking water for up to 2 years.

Table 1. Survival, water consumption, food consumption and chemical intake of rats administered quinoline.
Male / Dose (ppm) Control 200 400 800
Number of rats examined 50 50 50 50
Week that study terminated 96 96 95 76
Mean survival time
(week)		 95.8 88.4 70.7 49.6
Water consumption
(g/day) a)		 19.1 15.4 12.9 11.8
Food consumption
(g/day)		 15.9 ± 0.8 15.5 ± 0.8** 15.1 ± 1.0** 14.5 ± 0.9**
Quinoline intake
(mg/kg bw/day)		 0 8.8 15.4 30.5
Female / Dose
(ppm)		 Control 150 300 600
Number of rats examined 50 50 50 50
Week that study terminated 104 104 104 88
Mean survival time
(week)		 101.6 95.4 80.9 64.9
Water consumption
(g/day) a)		 17.6 13.8 11.6 9.5
Food consumption
(g/day)		 11.5 ± 0.9 11.1 ± 0.9** 10.2 ± 0.7** 9.8 ± 0.7**
Quinoline intake
(mg/kg bw/day)		 0 10.0 18.8 32.5

Values are expressed as mean ± S.D.

* and **: Significantly different from control at p < 0.05 and p < 0.01 by Dunnett's test, respectively.

a): Statistical analysis not applied.

The growth rates of all exposed males and the 300 and 600 ppm exposed females were generally less than the controls throughout the study period (Fig. 1 C, D). Average food and water consumption decreased in an apparent dose-dependent manner (Table 1). The average daily intake of quinoline per kg body weight increased approximately in proportion to the dosing levels (Table 1).

Histopathology

Tumors developed in the liver of male and female rats treated with quinoline, and tumors developed in the nasal cavity of male rats (Table 2a, 2b). In all exposed groups of each sex the incidences of hepatocellular adenomas, hepatocellular carcinomas, hepatocellular adenomas and/or carcinomas (combined), and hepatic hemangiosarcomas were significantly increased (Table 2a, 2b, Fig. 2A, 2B); the incidences of hepatic hemangiosarcomas increased dose-dependently. In all exposed groups of both sexes, hepatocellular carcinomas and hepatic hemangiosarcomas metastasized to the lung. The earliest deaths due to malignant tumors were observed at weeks 22, 37, and 75 in the male 800 ppm, 400 ppm, and 200 ppm groups, respectively, and at weeks 40, 33, and 68 in the female 600 ppm, 300 ppm, and 150 ppm groups, respectively (Table 3). Overall, the time to the development of lethal tumors was dose-dependent (Table 3).

Table 2a. Numbers of male rats with selected lesions induced by quinoline administration.
Dose (ppm) Control 200 400 800 Peto test
Organs Number of animals examined 50 50 50 50
Neoplastic lesion
Liver Hepatocellular adenoma 1
(2) a)		 10
(20)**		 10
(20)**		 9
(18)**		 ↑
Hepatocellular carcinoma 0
(0)		 22
(44)**		 24
(48)**		 18
(36)**		 ↑↑
Hepatocellular adenoma /
carcinoma (combined)		 1
(2)		 31
(62)**		 29
(58)**		 23
(46)**		 ↑↑
Hemangiosarcoma 0
(0)		 25
(50)**		 34
(68)**		 43
(86)**		 ↑↑
Nasal cavity Hemangioma 0
(0)		 0
(0)		 1
(2)		 0
(0)
Sarcoma NOS 0
(0)		 1
(2)		 5
(10)*		 1
(2)		 ↑↑
Esthesioneuroepithelioma 0
(0)		 0
(0)		 1
(2)		 6
(12)*		 ↑↑
Lung Hemangiosarcoma 0
(0)		 0
(0)		 2
(4)		 1
(2)
Adenosquamous carcinoma 0
(0)		 0
(0)		 1
(2)		 0
(0)
Metastasis from liver b) 0
(0)		 22
(44)		 27
(54)		 25
(50)
Mediastinum Sarcoma NOS 0
(0)		 1
(2)		 2
(4)		 3
(6)		 ↑↑
Mesenterium Hemangiosarcoma 0
(0)		 0
(0)		 2
(4)		 2
(4)		 ↑
Peritoneum Hemangiosarcoma 0
(0)		 0
(0)		 0
(0)		 1
(2)
Adipose tissue Hemangiosarcoma 0
(0)		 2
(4)		 0
(0)		 3
(6)		 ↑↑
All organs Hemangiosarcoma 0
(0)		 26
(52)**		 36
(72)**		 45
(90)**		 ↑↑
Non-neoplastic lesion
Liver Acidophilic cell foci 6
(12)		 14
(28)*		 4
(8)		 1
(2)
Basophilic cell foci 8
(16)		 24
(48)**		 12
(24)		 9
(18)
Clear cell foci 3
(6)		 15
(30)**		 4
(8)		 0
(0)
Central necrosis 0
(0)		 10
(20)**		 22
(44)**		 11
(22)**
Focal necrosis 0
(0)		 7
(14)**		 17
(34)**		 16
(32)**
Nasal cavity Basal cell hyperplasia : olfactory epithelium 0
(0)		 0
(0)		 0
(0)		 10
(20)**
Atrophy: olfactory epithelium 0
(0)		 2
(4)		 6
(12)*		 21
(42)**
Bone marrow Increased hematopoiesis 4
(8)		 25
(50)**		 38
(76)**		 38
(76)**
Spleen Extramedullary hematopoiesis 2
(4)		 21
(42)**		 40
(80)**		 33
(66)**
Kidney Tubular necrosis 1
(2)		 18
(36)**		 23
(46)**		 20
(40)**
Deposit of hemosiderin 1
(2)		 6
(12)*		 13
(26)**		 7
(14)*

* and **: Significantly different from control at p < 0.05 and p < 0.01 by Fisher's exact test, respectively.

↑ and↑↑: Significantly increased at p < 0.05 and p < 0.01 by Peto's test, respectively.

a): Parenthesis shows percent of incidence.

b) : Statistical analysis not applied.

Table 2b. Numbers of female rats with selected lesions induced by quinoline administration.
Dose (ppm) Control 150 300 600 Peto test
Organs Number of animals examined 50 50 50 50
Neoplastic lesion
Liver Hepatocellular adenoma 1
(2)a)		 30
(60)**		 31
(62)**		 33
(66)**		 ↑↑
Hepatocellular carcinoma 0
(0)		 5
(10)*		 16
(32)**		 21
(42)**		 ↑↑
Hepatocellular adenoma /
carcinoma (combined)		 1
(2)		 32
(64)**		 38
(76)**		 42
(84)**		 ↑↑
Hemangiosarcoma 0
(0)		 15
(30)**		 27
(54)**		 41
(82)**		 ↑↑
Nasal cavity Sarcoma NOS 0
(0)		 0
(0)		 1
(2)		 1
(2)
Lung Hemangiosarcoma 0
(0)		 2
(4)		 0
(0)		 0
(0)
Metastasis from liver b) 0
(0)		 13
(26)		 19
(38)		 27
(54)
Ovary Hemangioma 0
(0)		 1
(2)		 0
(0)		 0
(0)
Retroperitoneum Hemangiosarcoma 0
(0)		 0
(0)		 0
(0)		 1
(2)
Peritoneum Hemangiosarcoma 0
(0)		 0
(0)		 1
(2)		 0
(0)
Adipose tissue Hemangiosarcoma 0
(0)		 0
(0)		 2
(4)		 0
(0)
All organs Hemangiosarcoma 0
(0)		 17
(34)**		 28
(56)**		 42
(84)**		 ↑↑
Non-neoplastic lesion
Liver Acidophilic cell foci 1
(2)		 6
(12)		 9
(18)**		 5
(10)
Basophilic cell foci 21
(42)		 6
(12)**		 7
(14)**		 8
(16)**
Clear cell foci 1
(2)		 5
(10)		 6
(12)		 7
(14)*
Central necrosis 1
(2)		 10
(20)**		 14
(28)**		 11
(22)**
Focal necrosis 0
(0)		 18
(36)**		 15
(30)**		 17
(34)**
Nasal cavity Basal cell hyperplasia : olfactory epithelium 0
(0)		 0
(0)		 0
(0)		 2
(4)
Atrophy: olfactory epithelium 0
(0)		 0
(0)		 5
(10)*		 20
(40)**
Bone marrow Increased hematopoiesis 7
(14)		 18
(36)*		 29
(58)**		 40
(80)**
Spleen Extramedullary hematopoiesis 8
(16)		 16
(32)		 27
(54)**		 36
(72)**
Kidney Tubular necrosis 3
(6)		 13
(26)**		 19
(38)**		 20
(40)**
Deposit of hemosiderin 0
(0)		 3
(6)		 4
(8)		 8
(16)**

* and **: Significantly different from control at p < 0.05 and p < 0.01 by Fisher's exact test, respectively.

↑ and↑↑: Significantly increased at p < 0.05 and p < 0.01 by Peto's test, respectively.

a): Parenthesis shows percent of incidence.

b) : Statistical analysis not applied.

Fig. 2

(A) Hepatocellular carcinoma of a male rat administered 800 ppm quinoline. Bar indicates 200 µm. H&E stain. (B) Hemangiosarcoma in the peritoneum of a male rat administered 800 ppm quinoline. Bar indicates 100 µm. H&E stain. (C) Esthesioneuroepithelioma in the nasal cavity of a male rat administered 800 ppm quinoline. Bar indicates 1000 µm. H&E stain. (D) Higher magnification of Fig. 2 (C) Bar indicates 100 µm. H&E stain.

Table 3. Time-dependent tumor occurrence in rats administered quinoline.
Male Administration period (week) Terminal (week) First tumor
Group No. of rats 0- 25- 45- 65- 85-96 97 death (wk)
Control Rats examined 0 0 0 1 0 49
Rats with tumor 0 0 0 0 0 47 97
   Benign tumor 0 0 0 0 0 47
   Malignant tumor 0 0 0 0 0 6
200 ppm Rats examined 0 1 1 10 19 19
Rats with tumor 0 0 0 10 19 19 75
   Benign tumor 0 0 0 7 15 12
   Malignant tumor 0 0 0 9 18 18
400 ppm Rats examined 0 2 13 30 5 -
Rats with tumor 0 2 13 30 5 - 37
   Benign tumor 0 0 8 19 3 -
   Malignant tumor 0 2 12 29 5 -
800 ppm Rats examined 1 13 33 3 - -
Rats with tumor 1 13 32 3 - - 22
   Benign tumor 0 4 9 1 - -
   Malignant tumor 1 13 32 3 - -
Female Administration period (week) Terminal (week) First tumor
Group No. of rats 0- 25- 45- 65- 85-104 105 death (wk)
Control Rats examined 0 0 1 2 6 41
Rats with tumor 0 0 0 2 6 29 77
   Benign tumor 0 0 0 1 3 27
   Malignant tumor 0 0 0 1 4 3
150 ppm Rats examined 0 0 0 5 28 17
Rats with tumor 0 0 0 5 27 17 68
   Benign tumor 0 0 0 5 22 16
   Malignant tumor 0 0 0 2 18 9
300 ppm Rats examined 0 1 6 24 17 2
Rats with tumor 0 1 5 24 17 2 33
   Benign tumor 0 0 3 17 13 2
   Malignant tumor 0 1 5 19 17 1
600 ppm Rats examined 0 3 19 27 1 -
Rats with tumor 0 2 18 27 1 - 40
   Benign tumor 0 1 13 23 0 -
   Malignant tumor 0 2 17 27 1 -

In the nasal cavity, the incidences of nasal esthesioneuroepitheliomas in the 800 ppm exposed males (Fig. 2C, 2D) and sarcomas NOS (not otherwise specified) in the 400 ppm exposed males were significantly increased, but the incidences of these tumors was not increased in any of the female treatment groups (Table 2a, 2b). In addition to the liver, sporadic development of hemangiosarcomas was observed in some other organs.

The incidences of non-neoplastic lesions (acidophilic, basophilic, and clear cell foci) in the liver in the 200 ppm exposed males were significantly higher than the control group, but were not significantly increased in the higher dosed groups (Table 2a). In females, basophilic foci in the liver were decreased in all exposed groups, but acidophilic and clear cell foci in liver were increased in some exposed groups (Table 2b). In the nasal cavity, the incidence of basal cell hyperplasia in the olfactory epithelium was significantly increased in the 800 ppm exposed males, and significantly increased incidences of atrophy in the olfactory epithelium were observed in the 400 and 800 ppm exposed males and 600 ppm exposed females (Table 2a, 2b). Other observed non-neoplastic lesions were enhanced hematopoiesis in the bone marrow, extramedullary hematopoiesis in the spleen, central and focal necrosis in the liver, and tubular necrosis and deposition of hemosiderin in the kidney. Specific exposure-related lesions were not observed in other organs.

Mouse study

Survival, body weights, food and water consumption and chemical intake

All of the 600 ppm and 300 ppm dosed male mice died by week 55 and week 65, respectively. Due to the low number of surviving animals in the 150 ppm group, this arm of the experiments was terminated at week 65. The survival rates of the control and 150 ppm dosed males at week 65 were 92% (46/50) and 30% (15/50), respectively (Fig. 3A). The mean survival time of males was shortened dose-dependently (Table 4). All of the 600 ppm dosed female mice died by week 44. Due to the low number of surviving animals in the 150 ppm group, this arm of the experiments was terminated at week 50. The survival rates of the control and 150 and 300 ppm dosed females at week 50 were 98% (49/50), 40% (20/50), and 12% (6/50), respectively (Fig. 3B). The decreased survival rates in male and female mice were attributed to deaths due to hemangiomas and/or hemangiosarcomas of the retroperitoneum, mesenterium, and subcutis.

Fig. 3

Survival curves (A and B) and body weight changes (C and D) of mice administered 0 (control), 150, 300, and 600 ppm quinoline for 65 (males) or 50 (females) weeks.

Table 4. Survival, water consumption, food consumption and chemical intake of mice administered quinoline.
Male / Dose (ppm) 0 (Control) 150 300 600
Number of mice examined 50 50 50 50
Week that study terminated 65 65 65 55
Mean survival time
(week)		 63.0 58.9 53.7 40.7
Water consumption
(g/day)		 4.3 4.2 3.6 2.9
Food consumption
(g/day)		 4.4 ± 0.4 4.3 ± 0.3 4.2 ± 0.2** 3.8 ± 0.4**
Quinoline intake
(mg/kg bw/day)		 0 16.5 29.1 54.1
Female / Dose
(ppm)		 0 (Control) 150 300 600
Number of mice examined 50 50 50 50
Week that study terminated 50 50 50 44
Mean survival time
(week)		 49.6 46.9 43.4 36.9
Water consumption
(g/day)		 5.8 5.5 4.3 3.4
Food consumption
(g/day)		 4.1 ± 0.2 4.1 ± 0.2 3.9 ± 0.3** 3.7 ± 0.2**
Quinoline intake
(mg/kg bw day)		 0 30.5 46.6 74.2

* and **: Significantly different from control at p < 0.05 and p < 0.01 by Dunnett's test, respectively.

The growth rates of all exposed males and the 600 ppm exposed females were generally less than the controls throughout the study period (Fig. 3C, D). Average food and water consumption decreased in an apparent dose-dependent manner (Table 4). The average daily intake of quinoline per kg body weight increased approximately in proportion to the dosing levels in male mice, but was somewhat less than proportional in female mice (Table 4).

Histopathology

Quinoline increased the incidences of hemangiosarcomas and hemangiomas and/or hemangiosarcomas (combined) in all male exposed groups; quinoline increased the incidence of hemangiomas in males exposed to 300 and 600 ppm; and quinoline increased the incidences of hemangiomas, hemangiosarcomas, and hemangiomas and/or hemangiosarcomas (combined) in all female exposed groups (Table 5a, 5b). The incidences of hemangiosarcomas were greater than those of hemangiomas (Table 5a, 5b). These tumors were observed at high incidences in the retroperitoneum, mesenterium, peritoneum, subcutis, and liver (Fig. 4A, 4B, Table 5a, 5b). Notably, the incidences of hemangiosarcomas in the retroperitoneum and mesenterium were 70% and 38% in males and 54% and 36% in females at the lowest exposure dose of 150 ppm. The earliest malignant tumor deaths were observed at weeks 32, 40, and 36 in the male 600 ppm, 300 ppm, and 150 ppm groups, respectively, and at weeks 27, 28, and 33 in the female in 600 ppm, 300 ppm, and 150 ppm groups, respectively (Table 6). Overall, the time to the development of lethal tumors was dose-dependent (Table 6).

Table 5a. Numbers of male mice with selected lesions induced by quinoline administration.
Dose (ppm) Control 150 300 600 Peto test
Organ Number of animals examined 50 50 50 50
Neoplastic lesion
Liver Hepatocellular adenoma 4
(8)a)		 4
(8)		 3
(6)		 0
(0)
Hepatocellular carcinoma 0
(0)		 4
(8)		 0
(0)		 1
(2)		 ↑↑
Histiocytic sarcoma 0
(0)		 0
(0)		 3
(6)		 1
(2)		 ↑↑
Hemangioma 1
(2)		 1
(2)		 1
(2)		 1
(2)
Hemangiosarcoma 0
(0)		 2
(4)		 1
(2)		 12
(24)**		 ↑↑
Subcutis Hemangioma 0
(0)		 0
(0)		 1
(2)		 0
(0)
Hemangiosarcoma 0
(0)		 2
(4)		 2
(4)		 3
(6)		 ↑↑
Retroperitoneum Hemangioma 0
(0)		 0
(0)		 0
(0)		 3
(6)		 ↑↑
Hemangiosarcoma 0
(0)		 35
(70)**		 38
(76)**		 35
(70)**		 ↑↑
Mesenterium Hemangioma 0
(0)		 1
(2)		 1
(2)		 2
(4)
Hemangiosarcoma 0
(0)		 19
(38)**		 22
(44)**		 16
(32)**		 ↑↑
Mediastinum Hemangiosarcoma 0
(0)		 2
(4)		 0
(0)		 1
(2)
Peritoneum Hemangiosarcoma 0
(0)		 0
(0)		 0
(0)		 1
(2)
All organs Hemangioma 1
(2)		 2
(4)		 3
(6)		 7
(14)*		 ↑↑
Hemangiosarcoma 0
(0)		 43
(86)**		 47
(94)**		 43
(86)**		 ↑↑
Hemangioma / Hemangiosarcoma(Combined) 1
(2)		 44
(88)**		 47
(94)**		 46
(92)**		 ↑↑
Non-neoplastic lesion
Liver Central necrosis 0
(0)		 3
(6)		 7
(14)**		 5
(10)*
Focal necrosis 1
(2)		 6
(12)		 10
(20)**		 13
(26)**
Central degeneration 0
(0)		 10
(20)**		 18
(36)**		 7
(14)**
Extramedullary hematopoiesis 0
(0)		 12
(24)**		 4
(8)		 6
(12)*
Accumulation of immature blood cells 0
(0)		 30
(60)**		 41
(82)**		 28
(56)**
Mobilization of Kuppfer cell 0
(0)		 2
(4)		 8
(16)**		 8
(16)**
Angiectasis 0
(0)		 0
(0)		 1
(2)		 3
(6)
Nasal cavity Eosinophilic change in respiratory epithelium 8
(16)		 5
(10)		 2
(4)*		 3
(6)
Respiratory metaplasia in gland 9
(18)		 2
(4)*		 3
(6)		 0
(0)**
Lung Inflammatory infiltration 2
(4)		 23
(46)**		 34
(68)**		 32
(64)**
Accumulation of immature blood cells 0
(0)		 13
(26)**		 21
(42)**		 22
(44)**
Bone marrow Increased erythropoiesis 0
(0)		 23
(46)**		 36
(72)**		 27
(54)**
Spleen Extramedullary hematopoiesis 0
(0)		 40
(80)**		 48
(96)**		 44
(88)**
Kidney Deposit of hemosiderin 0
(0)		 8
(16)**		 5
(10)*		 4
(8)

* and **: Significantly different from control at p < 0.05 and p < 0.01 by Fisher's exact test, respectively.

↑ and↑↑: Significantly increased at p < 0.05 and p < 0.01 by Peto's test, respectively.

a): Parenthesis shows percent of incidence.

Table 5b. Numbers of female mice with selected lesions induced by quinoline administration.
Dose (ppm) Control 150 300 600 Peto test
Organ Number of animals examined 50 50 50 50
Neoplastic lesion
Liver Hepatocellular adenoma 0
(0)		 0
(0)		 2
(4)		 1
(2)
Histiocytic sarcoma 0
(0)		 2
(4)		 6
(12)*		 4
(8)		 ↑↑
Hemangioma 0
(0)		 1
(2)		 2
(4)		 5
(10)*		 ↑↑
Hemangiosarcoma 0
(0)		 0
(0)		 0
(0)		 2
(4)
Subcutis Hemangioma 0
(0)		 0
(0)		 7
(14)**		 15
(30)**		 ↑↑
Hemangiosarcoma 0
(0)		 4
(8)		 15
(30)**		 33
(66)**		 ↑↑
Ovary Hemangioma 1
(2)		 0
(0)		 0
(0)		 0
(0)
Hemangiosarcoma 0
(0)		 1
(2)		 4
(8)		 1
(2)		 ↑↑
Retroperitoneum Hemangioma 0
(0)		 5
(10)*		 1
(2)		 1
(2)		 ↑
Hemangiosarcoma 0
(0)		 27
(54)**		 36
(72)**		 32
(64)**		 ↑↑
Mesenterium Hemangioma 0
(0)		 2
(4)		 2
(4)		 2
(4)
Hemangiosarcoma 0
(0)		 18
(36)**		 18
(36)**		 11
(22)**		 ↑↑
Mediastinum Hemangioma 0
(0)		 0
(0)		 0
(0)		 1
(2)
Hemangiosarcoma 0
(0)		 2
(4)		 3
(6)		 1
(2)		 ↑↑
Peritoneum Hemangioma 0
(0)		 2
(4)		 6
(12)*		 2
(4)		 ↑↑
Hemangiosarcoma 0
(0)		 3
(6)		 6
(12)*		 15
(30)**		 ↑↑
All organs Hemangioma 1
(2)		 9
(18)**		 16
(32)**		 24
(48)**		 ↑↑
Hemangiosarcoma 0
(0)		 43
(86)**		 48
(96)**		 49
(98)**		 ↑↑
Hemangioma / Hemangiosarcoma(combined) 1
(2)		 45
(90)**		 48
(96)**		 50
(100)**		 ↑↑
Non-neoplastic lesion
Liver Central necrosis 0
(0)		 0
(0)		 2
(4)		 0
(0)
Focal necrosis 1
(2)		 6
(12)		 9
(18)**		 12
(24)**
Central degeneration 0
(0)		 6
(12)*		 2
(4)		 11
(22)**
Extramedullary hematopoiesis 0
(0)		 3
(6)		 10
(20)**		 6
(12)*
Accumulation of immature blood cells 0
(0)		 36
(72)**		 47
(94)**		 46
(92)**
Mobilization of Kuppfer cell 0
(0)		 6
(12)*		 8
(16)**		 10
(20)**
Angiectasis 0
(0)		 3
(6)		 3
(6)		 7
(14)**
Nasal cavity Eosinophilic change in respiratory epithelium 20
(40)		 29
(58)**		 36
(72)**		 46
(92)**
Respiratory metaplasia in gland 1
(2)		 2
(4)		 3
(6)		 10
(20)**
Lung Inflammatory infiltration 0
(0)		 22
(44)**		 31
(62)**		 39
(78)**
Accumulation of immature blood cells 0
(0)		 14
(28)**		 25
(50)**		 29
(58)**
Bone marrow Increased erythropoiesis 0
(0)		 27
(54)**		 33
(66)**		 35
(70)**
Spleen Extramedullary hematopoiesis 0
(0)		 36
(72)**		 47
(94)**		 50
(100)**
Kidney Deposit of hemosiderin 0
(0)		 11
(22)**		 13
(26)**		 13
(26)**

* and **: Significantly different from control at p < 0.05 and p < 0.01 by Fisher's exact test, respectively.

↑ and↑↑: Significantly increased at p < 0.05 and p < 0.01 by Peto's test, respectively.

a): Parenthesis shows percent of incidence.

Fig. 4

(A) Hemangioma in the mesenterium of a male mouse administered 150 ppm quinoline. Bar indicates 100 µm. H&E stain. (B) Hemangiosarcoma in the retroperitoneum of a male mouse administered 150 ppm quinoline. Bar indicates 100 µm. H&E stain.

Table 6. Time-dependent tumor occurrence in mice administered quinoline.
Male Administration period (week) Terminal (week) First tumor
Group No of mice 0- 35- 45- 55- 65 66 death (wk)
Control Mice examined 1 1 0 2 46 57
Mice with tumor 0 0 0 1 8
   Benign tumor 0 0 0 0 6
   Malignant tumor 0 0 0 1 3
150 ppm Mice examined 0 3 10 22 15 36
Mice with tumor 0 3 10 21 14
   Benign tumor 0 0 2 2 5
   Malignant tumor 0 3 10 21 11
300 ppm Mice examined 0 3 27 20 - 40
Mice with tumor 0 3 27 20 -
   Benign tumor 0 0 4 3 -
   Malignant tumor 0 3 27 20 -
600 ppm Mice examined 8 25 17 - - 32
Mice with tumor 4 25 17 - -
   Benign tumor 0 4 3 - -
   Malignant tumor 4 23 17 - -
Female Administration period (week) Terminal (week) First tumor
Group No. of mice 0- 25- 35- 45- 50 51 death (wk)
Control Mice examined 1 0 0 0 49 51a)
Mice with tumor 0 0 0 0 10
   Benign tumor 0 0 0 0 5
   Malignant tumor 0 0 0 0 4
150 ppm Mice examined 1 3 7 19 20 33
Mice with tumor 0 3 7 18 17
   Benign tumor 0 0 0 4 16
   Malignant tumor 0 3 7 18 9
300 ppm Mice examined 0 3 25 16 6 28
Mice with tumor 0 3 25 16 6
   Benign tumor 0 0 7 7 3
   Malignant tumor 0 3 25 16 6
600 ppm Mice examined 0 16 34 - - 27
Mice with tumor 0 16 34 - -
   Benign tumor 0 7 19 - -
   Malignant tumor 0 16 33 - -

a) : At terminal sacrifice

Exposure to quinoline did not cause an increase in the development of hepatocellular adenomas or carcinomas in any of the treated mouse groups; although, Peto's trend test suggests that quinoline may have caused an increase in hepatocellular carcinomas in male mice. Quinoline did cause a statistically significant increase in the incidence of hepatic histocytic sarcomas (300 ppm females), hemangiomas (600 ppm females), and hemangiosarcomas (600 ppm males) (Table 6). Peto's trend test suggests that quinoline induced an increase in hepatic histiocytic sarcomas and hemangiosarcomas in male mice and hepatic histiocytic sarcomas and hemangiomas in female mice.

Although no exposure-related increases in incidences of neoplasms were observed in the nasal cavity, eosinophilic change in the respiratory epithelium and respiratory metaplasia in gland were observed in females administered 600 ppm quinoline.

DISCUSSION

The present carcinogenicity study of quinoline was conducted with four groups of male and female rats and four groups of male and female mice. Test animals were administered quinoline in their drinking water for 50 to 104 weeks. Administration of quinoline was terminated early in male rats and male and female mice due to high mortality caused by quinoline-induced tumors.

Quinoline administration to rats resulted in a clear increase in the incidence of hepatocellular adenomas, hepatocellular carcinomas, hepatocellular adenomas and/or carcinomas (combined), and hepatic hemangiosarcomas in both males and females. In addition, increased incidences of esthesioneuroepitheliomas and sarcomas NOS in the nasal cavity were observed in male rats: nasal tumors in rats are extremely rare spontaneous lesions. Other than liver, low incidences of hemangioma and hemangiosarcoma were observed in adipose tissue, mesenterium, lung, and nasal cavity in males, and adipose tissue, peritoneum, retroperitoneum, lung, and ovary in females. Peto' s test suggests that quinoline induced hemangiosarcomas in adipose tissue and mesenterium in males, but had no apparent affect on the development of non-hepatic hemangiosarcomas in females. The sum of hemangiosarcomas of all organs was increased in both males and females administered the lowest dose of quinoline. Non-neoplastic lesions of basophilic cell foci, acidophilic cell foci, and clear cell foci were statistically increased in the liver in males, and atrophy and basal cell hyperplasia of the olfactory epithelium in the nasal cavity in males were also increased. These results are clear evidence of carcinogenicity of quinoline in male and female rats.

Quinoline administration to mice resulted in a clear increase in the incidence of hemangiomas and/or hemangiosarcomas in various tissues in both males and females. The high mortality caused by these quinoline-induced hemangiosarcomas necessitated termination of quinoline administration to male mice at week 65 and to female mice at week 50. Hemangiosarcomas were most prominently induced in the retroperitoneum, mesenterium, and liver in males, and in the subcutis, retroperitoneum, mesenterium, and peritoneum in females. In addition, in the liver, there was evidence of a possible increase in the incidence of histiocytic sarcomas and hepatocellular carcinomas in males and histiocytic sarcomas in females. These results are clear evidence of carcinogenicity of quinoline in male and female mice. However, in the present carcinogenicity studies, quinoline administration was terminated at week 50 in female mice, week 65 in male mice, and week 96 in male rats due to high mortality from quinoline-induced hemangioma and hemangiosarcoma in mice and liver tumors in rats.

Several carcinogenicity studies have reported liver tumors in rats and mice following oral administration of quinoline in the diet, and the carcinogenic responses of the rats and mice in the present study were consistent with these reports. Hirao et al. (1976) demonstrated that hepatocellular carcinomas and hemangioendotheliomas and/or hemangiosarcomas were found in the liver of SD rats fed diets containing 0.05, 0.10, and 0.25% quinoline for approximately 40 weeks. The average survival period of the 0.25% quinoline fed rats was 20 weeks, while that of the control group was 35.5 weeks. Most of the rats treated with high concentrations of quinoline died due to the toxic effects of quinoline or rupture of vascular tumors of the liver. Shinohara et al. (1977) examined species differences in the carcinogenicity of quinoline using male and female ddy mice, Wistar rats, Hartley guinea pigs, and Syrian golden hamsters fed a diet containing 0.2% quinoline for 30 weeks. Quinoline induced hemangioendotheliomas and hepatocellular carcinomas in both sexes of mice and rats but did not induce liver tumors in either guinea pigs or hamsters. Hasegawa et al. (1989) studied the toxic effects of quinoline by feeding male Wistar rats a dose of 0.25% quinoline in the diet for 4, 8, 12, 16, and 20 weeks; rats were killed immediately or 4, 8, 12, or 16 weeks after termination of quinoline treatment: overall time 20 weeks. Effects of quinoline, such as an increase in relative liver weight and increased SGOT (serum glutamic oxaloacetic transaminase) activity were found after 4 weeks, and liver lesions, such as megalocytosis, hepatic endothelial dysplasia, and hemangioendotheliomas were observed after 12 weeks. Futakuchi et al. (1996) examined species differences in the carcinogenicity of quinoline using male spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) fed a diet containing 0.2% quinoline for 32 weeks. Induction of hepatic hemangiosarcomas was observed in WKY rats, but not in SHR rats, and a few hepatic hyperplastic nodules were observed in both strains (Futakuchi et al., 1996). These studies used higher doses of quinoline and shorter administration periods than the present study. Notably, in the Hasegawa study (1989), hepatic hemangioendothelioma was induced within 12 weeks of the beginning of quinoline administration. Overall in these diet studies, quinoline primarily targeted the liver and the induction of hepatocellular carcinomas and hepatic hemangiosarcomas (same as hemangioendothelioma) in rats and hepatic hemangiosarcomas in mice closely matched the results of the present study. However, these animal studies used only a small number of animals and/or one-sex and were not in compliance with GLP.

In 550 carcinogenicity studies by NTP, 290 studies were positive in rats or mice, and 25 of these studies reported an increased incidence of vascular neoplasm:19 of the 25 studies reported an increase in mice and 3 of the 25 studies reported and increase in rats (Nyska et al., 2004; Choen et al., 2009). The chemicals that induced hemangiosarcoma above a 75% incidence in rats and/or mice were riddelliine (US NTP, 2003), tetrafluoroethylene (US NTP, 1997a), cupferron (US NCI, 1978), o-nitrotoluene (US NTP, 2002), and 2-methyl-1-nitroanthraquinone (Murthy et al., 1979). Of these five chemicals, riddelliine, a pyrrolizidine alkaloid, was the most potent endothelial cell carcinogen in male and female rats and male mice. Administration of 1 mg/kg riddelliine by gavage to rats induced hemangiosarcomas in 86% of the males by the 43rd week of the study and 76% of the females by the 50th week of the study. By week 72 all of the males had died due to tumor-mediated mortality, and by week 97 all of the females had died due to tumor-mediated mortality. Administration of 1 mg/kg riddelliine by gavage to mice induced hemangiosarcomas in 62% of the males by the 78th week of the study, but induced hemangiosarcomas in only one female (US NTP, 2003). Our results using administration of quinoline in the drinking water suggests that compared to riddelliine, quinoline seems to show a higher potential for inducing hemangiosarcomas in both male and female rats and mice.

The present study demonstrated that oral exposure of male rats to quinoline in the drinking water induced esthesioneuroepitheliomas and sarcomas NOS in the nasal cavity of male rats. Previous carcinogenicity studies of quinoline did not demonstrate an increased incidence of esthesioneuroepitheliomas or sarcomas NOS in the nasal cavity of male rats. Nasal tumors are not spontaneously observed in the JBRC historical control data of 1199 male rats. Thus, while the incidence of nasal tumors in male rats in the present study was much lower than the incidence of liver tumors, when coupled with the JBRC historical control data, induction of nasal tumors in male rats exposed to quinoline is judged to be compound-related. Only one other study conducted by JBRC, the study by Kano et al. (2009), induced tumors in the nasal cavity in rats administered a carcinogen orally: in the Kano study, administration of 1, 4-dioxane in the drinking water resulted in the induction of a low level of nasal tumors in male and female rats. The induction of esthesioneuroepitheliomas and sarcomas NOS in the nasal cavity of male rats in the present study may be attributed to circulatory or exhalatory influence of quinoline or its metabolites on nasal tissue after gastrointestinal absorption.

Quinoline has significant mutagenic activity in Salmonella Typhimurium (TA98 and TA100) and Escherichia coli (WP2uvrA) after metabolic activation by the rat liver S9 fraction (Nagao et al., 1977; Takahashi et al., 1988; LaVoie et al., 1991; Willems et al., 1992; JETOC, 1996) and induces unscheduled DNA synthesis in rat hepatocytes in vitro (LaVoie et al., 1991). Positive mammalian clastogenicity of quinoline on cultured Chinese hamster lung cells after metabolic activation by the rat liver S9 fraction has also been reported (JETOC, 1996). In vivo, Suzuki et al. (1998, 2000) showed that quinoline induced gene mutation in the liver of lacZ transgenic mice (Muta Mouse). Quinoline also induces chromosomal aberration and sister chromatid exchange in rat hepatocytes after oral administration (Asakura et al., 1997). In a recent in vivo study, oral administration of quinoline induced an increase in the frequency of micronucleated hepatocytes (Uno et al., 2015). Thus the genotoxicity of quinoline is supported by a number of genotoxicity assays (in vitro and in vivo). Since, quinoline induces tumor development in the early period of administration, the carcinogenic mode of action of quinoline is thought to be genotoxic.

In addition to oral administration, quinoline is also carcinogenic when administered by intraperitoneal injection to new born CD-1 mice (LaVoie et al., 1987, 1988; Weyand et al., 1993) and when administered by skin painting (LaVoie et al., 1984), indicating that quinoline is carcinogenic without metabolic activation in the liver. Several substituted quinolines have also been examined for carcinogenic potential in rats and mice. Dermal application of the quinoline derivative 1,2-dihydro-2,2,4-trimethylquinoline monomer on rats for 2 years resulted in an increased incidence of renal tubule adenoma (US NTP, 1997b). Similarly, dermal application 4-nitoquinoline 1-oxide on mice was carcinogenic (Nakahara et al., 1957). 8-nitroquinoline administered in the diet to rats (Fukushima et al., 1981) was also demonstrated to have carcinogenic activity. In contrast, 6-methylquinoline, 8-methyluqinoline, 5,7-dibromoquinoline, and 8-hydroquinoline in the diet were not carcinogenic in rats (Fukushima et al., 1981; US NTP, 1985).

In conclusion, the present studies demonstrated clear evidence of carcinogenic activity of quinoline when administered in the drinking water to rats and mice, resulting in high incidences of hepatic tumors in rats and high incidences of hemangiosarcoma in rats and mice accompanied by reduced lifespan due to high mortality caused by quinoline-induced tumors.

ACKNOWLEDGMENTS

The present study was contracted and supported by the Ministry of Labour, Japan (the present Ministry of Health, Labour and Welfare, Japan). We wish to express our thanks to Dr. David B. Alexander, Nanomaterial Toxicology Project Laboratory, Nagoya City University, for proofreading this manuscript.

Conflict of interest

The authors declare that there is no conflict of interest.

REFERENCES
 
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