Assessment of the Genotoxicity of 1,2-Dichloropropane and Dichloromethane after Individual and Co-exposure by Inhalation in Mice

Abstract

Objective: Occurrence of cholangiocarcinoma was recently reported at a high incidence rate among the employees working for an offset printing company in Osaka, Japan. 1,2-Dichloropropane (1,2-DCP) and dichloromethane (DCM) are suspected to be the causes of the cancer, as they had been used as ink cleaners in large amounts. However, it is not clear whether these chlorinated organic solvents played a role in the occurrence of cholangiocarcinoma or why the incidence rate is so high among the workers in this industry. To provide possible evidence for this severe occupational problem, we investigated the genotoxic effects of 1,2-DCP and DCM. Methods: Male B6C3F1 and gpt Delta C57BL/6J mice were exposed by inhalation to the individual solvents or both solvents at multiple concentrations including the levels that were possibly present in the workplaces. The genotoxicity was analyzed by Pig-a gene mutation and micronuclei assays in peripheral blood and gpt mutation and comet assays in the livers of mice after repeated inhalation of 1,2-DCP or/and DCM. Results: The Pig-a mutant frequencies and micronuclei incidences were not significantly increased by exposure of either 1,2-DCP or/and DCM at any concentration, suggesting there was no genotoxic potential in bone marrow for both solvents. In the liver, DNA damage, as measured by the comet assay, was dose dependently increased by 1,2-DCP but not by DCM. The gpt mutant frequency was 2.6-fold that of the controls in the co-exposure group. Conclusions: These results indicate that 1,2-DCP showed stronger genotoxicity in the liver and that the genotoxic effects were greatly enhanced by simultaneous exposure to DCM.

(J Occup Health 2014; 56: 205-214)

Introduction

Recently, the occurrence of cholangiocarcinoma cases was reported among the employees and former employees of an offset printing company in Osaka, Japan¹⁾. So far, 11 cancer patients have been diagnosed among 62 workers conducting offset color proof printing in this factory, and this surely shows an extremely high incidence rate of this kind of cancer compared with the national average during the same period. In addition, the ages of the workers at diagnosis were 25–45 years, and this is obviously much younger that in the general population. Later, more cases were found in factories, including other offset printing companies, in a national survey sponsored by the occupational health authorities²⁾. The occurrence of cholangiocarcinoma in this industry is therefore suspected to be related to some factors in the workplaces.

1,2-Dichloropropane (1,2-DCP) and dichloromethane (DCM) were the substances suspected of causing the cancer, since they were used as the ink cleaners in a large amounts and can easily evaporate into the air, meaning that the workers were therefore exposed to these chlorinated organic solvents at high concentrations. Our colleagues carried out an experiment to reproduce the working environment of the proof-printing room of this company in Osaka and concluded that the exposure concentrations might have been as high as several hundreds ppm for either 1,2-DCP or DCM under the conditions of ordinary work amount³⁾, and the latter solvent showed 2–3 fold higher concentration than the former solvent because of the difference in evaporation rate. Other components in the cleaner included 1,1,1-trichloroethane, petroleum-hydrocarbons, and surface-active agents, but they were used only for a short period and in small amounts, and therefore were hardly responsible for the cancer occurrences.

There have only been a few reports about the carcinogenesis of 1,2-DCP. A recent report showed that inhalation of the solvent in mice and rats increased the incidence of Harderian gland adenoma and nasal cavity tumor, respectively^{4, 5)}. By gavage administration, 1,2-DCP induced hepatocellular adenomas in mice but not in rats⁶⁾. In in vitro studies, 1,2-DCP exhibited weak mutagenicity at high concentrations in the Ames test^7–9) and induced sister chromatid exchange and chromosome aberration in CHO cells^{10, 11)}. There is an unpublished report showing that the micronucleus in bone marrow cells was negative in mice administered 1,2-DCP by gavage¹²⁾. However, the evidence of genotoxicity in the liver, where it is metabolized, is still lacking.

Inhalation of DCM by mice and rats significantly raised the incidences of liver and lung tumors and benign mammary gland tumor, respectively¹³⁾. Genotoxicity is one of the important steps with relevance to cancer development. DCM exhibited genotoxic effects in bacteria¹⁴⁾ and mammalian cells¹⁵⁾ and induced DNA damage in the liver and lung in mice and rats^{16, 17)}. While chromosome aberration and micronucleus tests in bone marrow cells^{14, 18)} and the unscheduled DNA synthesis (UDS) assay in hepatocytes showed negative results in mice and rats administered DCM via gavage¹⁹⁾, DCM inhalation induced chromosome aberration in bone marrow and pulmonary cells and micronuclei (MNs) in peripheral blood erythrocytes²⁰⁾.

Most of the cancer cases were found to be exposed to both 1,2-DCP and DCM in the workplace, though a few were only exposed to 1,2-DCP. These organic solvents were known to be metabolized by the same oxidizing enzyme, i.e., CYP2E1^{21, 22)}. Thus, the genotoxic effects of 1,2-DCP and DCM could be enhanced by competitive inhibition or induction of the metabolizing enzymes under a condition of co-exposure, and this may have contributed to the high incidence of cancer. Therefore, it is important to examine what happens with regard to genotoxicity when a subject is exposed simultaneously to the two solvents.

In this study, we investigated the dose response of the genotoxic effects of 1,2-DCP and DCM in mice by inhalation exposure to either a single solvent or co-exposure to the two solvents simultaneously. Different stages of genotoxic effects such as early DNA damage, clastogenicity/aneugenicity and mutagenicity were included in the assessment, and the target was not only the liver but also bone marrow, which represents a different tissue with regard to the metabolizing capacity. We found that 1,2-DCP showed a stronger genotoxic effect than DCM in the liver, and this effect was enhanced by simultaneous exposure to the two solvents.

Materials and Methods

Animals and reagents

During the experiment, we followed the guidelines for the care and use of laboratory animals set forth by the Institutional Animal Care and Use Committee of Japan National Institute of Occupational Safety and Health. B6C3F1 and gpt Delta C57BL/6J mice were purchased from Charles River Laboratories Japan (Yokohama, Japan) and Japan SLC (Hamamatsu, Japan), respectively. They were housed under specific pathogen-free conditions with a 12-h light-dark cycle and given tap water and CE-2 pellets (CLEA Japan Inc.) ad libitum. 1,2-DCP (>98.5% in purity) and DCM (99.5% in purity) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) and Wako Pure Chemical Industries (Osaka, Japan), respectively.

Inhalation exposure to 1,2-DCP and/or DCM

Eight to ten 8-week-old male B6C3F1 mice were used in each of the eight exposure groups and in the control group. Inhalation exposures were conducted in stainless steel chambers (Sibata Scientific Technology, Tokyo, Japan). The exposure concentrations of 1,2-DCP or DCM were set at 150, 300 and 600 ppm or 400, 800 and 1,600 ppm. Co-exposure groups were exposed to 150 + 400 ppm and 300 + 800 ppm of 1,2-DCP + DCM. The control group was exposed to filtered air only. The selected exposure concentrations were based on a reproductive experiment in the factory³⁾. The target exposure concentrations of 1,2-DCP and DCM were monitored using a gas chromatograph (Shimadzu GC-7A) and adjusted with flowmeters to a constant target value ± 5% throughout exposure. B6C3F1 mice were exposed for 6 hours per day, 5 consecutive days per week, for 6 weeks. The duration of exposure was designed taking into consideration that the life-span of erythrocytes is about 40 days. B6C3F1 mice were euthanized under anesthesia 18 hours after the last exposure.

To examine the mutagenicity in the liver, five 8-week-old male gpt Delta C57BL/6J mice were used in each of the three exposure groups and in the control group. The exposure concentrations of 1,2-DCP, DCM and 1,2-DCP + DCM were 300, 800 and 300 + 800 ppm, respectively. The control group was exposed to filtered air only. The mice were exposed for 6 hours per day, 5 consecutive days per week, for 4 weeks. The duration of exposure was determined according to the recommendation in OECD guideline TG488²³⁾. The mice were euthanized under anesthesia 7 days after the final inhalation to fix mutation.

Pig-a gene mutation assay

Blood was collected from each animal in weeks 3 and 6 after inhalation of 1,2-DCP and/or DCM. The Pig-a mutation assay was conducted as previously described²⁴⁾. A flow cytometer (Epics XL-MCL; Beckman Coulter) and the EXPO32 Analysis software were used for data acquisition. After gating for the single cell population, about 1,000,000 TER-119-positive cells were analyzed to determine the frequency of CD24-negative red blood cells (RBCs).

Micronucleus assay

The frequencies of micronucleated reticulocytes (MN-RETs) and micronucleated normochromatic erythrocytes (MN-NCEs) were determined in blood specimens collected in week 6 using an Epics XL-MCL flow cytometer, and following the protocol for the In Vivo Mouse MicroFlow PLUS Kit (Litron Laboratories, Rochester, NY, USA). The frequencies of MN-RETs and MN-NCEs were determined by acquisition of about 20,000 RETs and about 1,000,000 NCEs for each animal.

Alkaline comet assay

A portion of the middle lobe of the liver was removed and minced with a pair of scissors to release the cells in 1 mL of mincing buffer (20 mM EDTA-2Na and 10% DMSO in Hank's balanced salt solution (Ca²⁺ and Mg²⁺ free), pH 7.5) and stored frozen at −80°C until analysis. The comet assay was conducted by using the protocol recommended by the Japanese Center for the Validation of Alternative Methods²⁵⁾, except for using a 20-well CometSlide (Trevigen, Gaithersburg, MD, USA). After electrophoresis, the cells were stained with SYBR gold, and the comets were analyzed by using a fluorescence microscope (ECLIPSE E600; Nikon) with a Comet IV capture system (LMS). For each sample, at least 100 cells were scored. The tail intensity (TI) was measured for each nucleus scored.

gpt Mutation assay

High molecular weight genomic DNA was extracted from portions of middle liver lobe by the standard phenol/chloroform method²⁶⁾. We rescued lambda EG10 phages using Transpack Packaging Extract (Agilent Technologies) and conducted the gpt mutation assay as previously described²⁷⁾, calculating the gpt mutant frequency by dividing the number of 6-thioguanine-resistant colonies after clonal correction by the number of colonies with rescued plasmids. Approximately 1.5 to 3.7 million colonies were obtained from each sample. All mutant colonies obtained were verified by replating on 6-thioguanine-containing agar plates and further by gpt DNA sequencing as previously described²⁷⁾.

Statistical analysis

Statistical significance in the results of the Pig-a assay was examined by the Kruskal-Wallis test with the Steel post hoc test. The Dunnett's test was used for comparison of statistically significant differences in the results of micronucleus, comet and gpt mutation assays between the control and each exposed group. The existence of dose dependence was assessed with the linear regression analysis when significant differences were detected in the Dunnett's test. The slope factor of linear regression was checked for statistical significance. The Tukey-Kramer test was used for comparison of statistically significant differences in the results of the comet assay for the 1,2-DCP-, DCM- and 1,2-DCP + DCM-exposed groups at the same concentration. For statistical analysis of mutation spectra, Fisher's exact test was used with a Bonferroni correction as a multiple comparison²⁸⁾. Levels of p<0.05 were considered to be significant.

Results

Pig-a gene mutant frequency

The Pig-a mutant frequencies in total RBCs in weeks 3 and 6 after initial inhalation are shown in Fig. 1. The mutations induced by the three different doses of 1,2-DCP (150, 300 and 600 ppm) or DCM (400, 800 and 1,600 ppm) were not statistically different from those in the control at weeks 3 and 6. In addition, simultaneous inhalation of 1,2-DCP and DCM at two concentrations did not increase the mutant frequency at either 3 or 6 weeks. A few mice were intraperitoneally administered a single dose of with N-ethyl-N-nitrosourea at 70 mg/kg, and the Pig-a mutant frequencies at 3 and 6 weeks were significantly higher than those of the control (222 ± 81 × 10⁻⁶ at 3 weeks, p=0.0076; 265 ± 312 × 10⁻⁶ at 6 weeks, p=0.0102, the Steel test, n=3). These results suggest that 1,2-DCP and DCM may have no mutagenic potential in hematopoietic stem cells.

Fig. 1.

Pig-a mutant frequency of erythrocytes in mice exposed to 1,2-DCP or/and DCM at week 3 (A) and 6 (B). Pig-a mutant frequencies are shown in the box plot. The variance of the results was not significant in the Kruskal-Wallis test (p=0.332 at 3 weeks, p=0.519 at 6 weeks).

Micronuclei incidence in peripheral blood

To examine the clastogenicity/aneugenicity of 1,2-DCP or/and DCM in bone marrow, micronuclei in RETs and NCEs were analyzed in week 6 after inhalation (Fig. 2A). The MN incidences in RETs and NCEs were not significantly increased by inhalation of 1,2-DCP (150, 300 and 600 ppm), DCM (400, 800 and 1,600 ppm) or co-exposures (1,2-DCP + DCM, 150 + 400 and 300 + 800 ppm) in comparison with the filtered air control. In addition, the %RETs was also not significantly different among the groups (Fig. 2B). These results indicate that 1,2-DCP and DCM did not display clastogenicity/aneugenicity or adverse effects on hematopoiesis in bone marrow cells.

Fig. 2.

Micronucleus frequency in reticulocytes (white bar) and normochromatic erythrocytes (gray bar) (A) and ratio of reticulocytes in whole red blood cells (B) in mice exposed to 1,2-DCP or/and DCM. Micronucleus frequency and %RETs are shown as means ± SD. The statistical significance between the control and each exposed group was examined by the Dunnett's test. The p-values for the micronucleus frequency in reticulocytes: DCP150, p=0.825; DCP300, p=1.00; DCP600, p=1.00; DCM400, p=1.00; DCM800, p=0.999; DCM1600, p=0.990; DCP150 + DCM400, p=0.976; DCP300 + DCM800, p=0.968. The p-values for the micronucleus frequency in normochromatic erythrocytes: DCP150, p=0.996; DCP300, p=1.00; DCP600, p=0.991; DCM400, p=0.508; DCM800, p=0.575; DCM1600, p=0.531; DCP150 + DCM400, p=1.00; DCP300 + DCM800, p=0.947. The p-values for the %RET: DCP150, p=0.983; DCP300, p=1.00; DCP600, p=0.241; DCM400, p=0.994; DCM800, p=0.997; DCM1600, p=0.964; DCP150 + DCM400, p=1.00; DCP300 + DCM800, p=0.967.

Level of DNA damage in the liver

The level of DNA damage in the liver was examined by alkaline comet assay (Fig. 3). The TI values were dose-dependently increased by exposure to 1,2-DCP, there were statistically significant differences between the medium and high concentrations, and the controls and the slope factor value in the 1,2-DCP-exposed groups was significant (p=7.4 × 10⁻⁴). On the other hand, the TI values in mice exposed to DCM were not significantly different from that in the control. In addition, the TI values in the co-exposure groups were further increased, and in the co-exposure group administered a lower concentration, the TI value was significantly higher even than the respective individual exposure at the same concentration. These results suggested that 1,2-DCP may induce DNA damage in the liver and that the damage could be enhanced by DCM.

Fig. 3.

Induction of the DNA damage by exposure to 1,2-DCP or/and DCM in the liver. Tail intensity values are shown as means ± SD. *p<0.05 vs. control (Dunnett's test); #p<0.05 vs. 1,2-DCP or DCM individual exposure group at the same concentration (Tukey-Kramer test). The p-values for the Dunnett's test: DCP150, p=0.871; DCP300, p=0.035*; DCP600, p=0.028*; DCM400, p=1.00; DCM800, p=0.160; DCM1600, p=0.959; DCP150 + DCM400, p=2.4 × 10⁻⁶*; DCP300 + DCM800, p=4.5 × 10⁻⁶*. The p-values for the Tukey-Kramer test: DCP150 vs. DCP150 + DCM400, p=4.1 × 10^−5#; DCM400 vs. DCP150 + DCM400, p=4.0 × 10^−4#; DCP300 vs. DCP300 + DCM800, p=0.0556; DCM800 vs. DCP300 + DCM800, p=0.212^#.

gpt Mutant frequency in the liver

To evaluate the mutagenicity of 1,2-DCP and DCM in the liver, the mutant frequency was determined in gpt Delta transgenic mice (Fig. 4). In comparison with the control mice treated with filtered air, the gpt mutant frequencies in the liver were not increased by exposure to 1,2-DCP at 300 ppm and DCM at 800 ppm. On the other hand, in the co-exposure group (1,2-DCP + DCM, 300 + 800 ppm), the gpt mutant frequency was 2.6-fold that of the controls, showing a significant increase. In addition, the ratio of mutations in the A:T pair was significantly increased in the co-exposure group in comparison with the control group (Table 1, p=0.032), suggesting that the occurrence of mutations in the treatment group is occurs via a different mechanism from that in the controls, in which mutations are considered to occur spontaneously.

Fig. 4.

The gpt mutant frequency induced by 1,2-DCP or/and DCM in the liver. For the gpt assay, more than 1,500,000 colonies derived from the rescued phages per liver per mouse were analyzed. Mutant frequencies are shown as means ± SD. *p<0.05 vs. control (Dunnett's test). The p-values for the Dunnett's test: DCP300, p=0.899; DCM800, p=0.999; DCP300 + DCM800, p=0.034*.

Table 1 Classification of the gpt mutations recovered from the livers of control, 1,2-DCP-treated or/and DCM-treated mice^a

	Control	1,2-DCP	DCM	DCM+1,2-DCP
Transition
A:T → G:C	1 (10)	3 (21)	0 (0)	4 (25)
G:C → A:T	6 (60)	6 (43)	6 (50)	4 (25)
Transversion
A:T → T:A	0 (0)	0 (0)	3 (25)	3 (19)
A:T → C:G	0 (0)	1 (7.1)	1 (8.3)	0 (0)
G:C → T:A	1 (10)	1 (7.1)	1 (8.3)	4 (25)
G:C → C:G	0 (0)	2 (14)	0 (0)	1 (6.3)
Frameshift (−1)	1 (10)	1 (7.1)	0 (0)	0 (0)
Deletion	0 (0)	0 (0)	1 (8.3)	0 (0)
Other	1b (10)	0 (0)	0 (0)	0 (0)
Mutation at A:T pair	1 (10)	4 (29)	4 (33)	7 (44)*
Mutation at G:C pair	7 (70)	7 (64)	9 (58)	9 (56)
Total	10 (100)	14 (100)	12 (100)	16 (100)

^a  All data are presented as cases found (%).

^b  This represents CC → AA.

*  p<0.05 (Fisher's exact test with a Bonferroni correction).

Discussion

The Pig-a mutant frequencies and MNs in RBCs were not significantly increased by exposure to 1,2-DCP and/or DCM. These results indicate that 1,2-DCP and DCM may have no mutagenic and clastogenic/aneugenic potential in hematopoietic lineage cells. It is considered that these chemicals are metabolized in the liver via oxidation and conjugation reactions, and it is the reactive metabolites produced in the liver that may cause toxicities. The metabolism of the chemicals may count for the no observed genotoxic effects in bone marrow under the concentrations used in this study. There is one report about MN induction in bone marrow cells after inhalation of DCM²⁰⁾, but DCM was used in that report at 4,000 and 8,000 ppm, a concentration much higher than that we used. Additionally, there have been a few reports of negative results for MNs in bone marrow^{14, 18)}, and the results were consistent with those of the present study.

Exposure to 1,2-DCP increased the level of DNA damage in liver in a dose-dependent manner. In addition, the gpt mutant frequency in mice exposed to 1,2-DCP at 300 ppm was increased by 32%, though without statistical significance. These results suggest that 1,2-DCP may have genotoxic potential in liver. 1,2-DCP is known to be metabolized by CYP2E1 and GSH conjugation^{29, 30)}. While the metabolic pathways and the metabolites responsible for the hepatotoxicity and genotoxicity have yet to be elucidated for 1,2-DCP, episulfonium ions produced by GST-dependent bioactivation are especially suspected to react to DNA and then cause genotoxicity like other haloalkanes^31–33). Both positive and negative results of Ames tests have been reported^{6–9, 34, 35)}. Most positive results were obtained at quite higher doses than those recommendation by OECD guideline TG471³⁶⁾, and obvious growth inhibition was found at these doses^{34, 35)}. Thus, the positive results of the Ames test are unreliable. 1,2-DCP induced chromosomal aberrations and sister chromatid exchanges in mammalian cells but could not induce MNs in bone marrow of mice^10–12). These in vivo studies are consistent with our negative results in the Pig-a assay and MN test in the erythrocytes. However, bone marrow is not the target organ for the carcinogenesis of the solvent^4–6). In this study, we found that 1,2-DCP induced DNA damage in liver tissue, suggesting that 1,2-DCP is metabolized in the liver and that it is the active metabolites that display genotoxicity.

In the liver, DNA damage and gpt mutant frequency were not significantly increased in mice exposed to DCM up to 1,600 ppm. In previous reports, DCM was considered to cause DNA single-strand breaks (SSBs) in the mouse liver^{37, 38)}. However, the concentrations of DCM at which genotoxicity was detected in those reports were more than 4,000 ppm and higher than the highest dose used in our study. A positive result was obtained in the alkaline comet assay in the livers of mice administered DCM by gavage, but the dosage was substantially high¹⁷⁾. In contrast, UDS activity and alkylation of DNA were negative in the livers of mice administered 4,000 ppm DCM by inhalation^{19, 39)}. Thus, DNA damage in the livers of mice administered DCM was only induced at a substantially high dose. The mutagenicity of DCM is well established in Ames tests, but this is not necessarily reflected in mammalian cells^{14, 40–43)} and animals^{19, 39)}. Therefore, our negative result for DCM in the comet assay is consistent with other reports^{19, 37–39)}.

Simultaneous exposure to the two solvents increased DNA damage and the gpt mutant frequency in the liver in comparison with individual exposure, suggesting that the mutagenic potential of 1,2-DCP may be enhanced by DCM. However, the mutagenicity in the liver was examined at a single concentration, and further studies under various concentrations and long periods are needed to clarify the mutagenicity of 1,2-DCP and DCM and the effects of co-exposure in the liver. Dose dependence was not observed in the results of the comet assay in the co-exposure groups. The TI value may reach a plateau by saturation of the distribution or metabolic process. Further analyses at lower concentrations are required to examine the dose dependence. Both 1,2-DCP and DCM are metabolized by CYP2E1^{21, 22)}. In addition, both solvents are known to have glutathione conjugation reactions in their metabolism, and this metabolic pathway is suspected to be related to their toxicities. DCM is mainly metabolized by GSTT1-1, but GST molecular species involved in 1,2-DCP metabolism have not been identified. On the other hand, many haloalkanes are activated by conjugation of glutathione mediated by GSTT1-1^{44, 45)}, and 1,2-DCP could share the same isozyme in its metabolism. Simultaneous exposure is suspected to elicit competitive inhibition in the metabolic processes. In fact, the amounts of 1,2-DCP and DCM in the liver at the end of the 6-h inhalation period were 1.9-fold and 2.5-fold increased by co-exposure in comparison with individual exposure to 1,2-DCP at 300 ppm and DCM at 800 ppm, respectively (unpublished data). Therefore, the genotoxic effects in the liver may be enhanced by an increase in active metabolites of 1,2-DCP and DCM. Even though the concentration of DCM in the liver of the co-exposure group greatly increased greatly, it may not reach as high as that corresponding to inhalation at 4,000 ppm, the level at which a genotoxic effect was observed. Moreover, DNA damage by 1,2-DCP was induced at a considerably lower dose in comparison with DCM. These results suggest that DNA damage induced by 1,2-DCP could be enhanced by co-exposure of DCM. Induction or suppression of metabolizing enzymes after subchronic exposure to the two solvents may also contribute to enhancement of the genotoxic effect in the liver, and this needs to be investigated further.

Overall, our study showed that 1,2-DCP displayed a genotoxic effect in the liver of mice at concentrations that were present in the workplaces, and this effect was enhanced by DCM. To our knowledge, this constitutes the first evidence regarding the genotoxic potential of 1,2-DCP in vivo. Our findings suggest that 1,2-DCP, not DCM, may be the main, if not the only, chemical causing the occurrence of cholangiocarcinoma in the offset printing factories, and provide some possible explanations for the extremely high incidence of the cancer by showing the enhancement of genotoxicity with co-exposure of the two solvents. However, further studies including studies of the metabolic interaction between 1,2-DCP and DCM, genotoxicity and other toxic effects in cholangiocytes are required to address the reasons for the high incidence of cholangiocarcinoma among the offset printing workers.

Acknowledgments: We are deeply grateful to Dr. Kunugida and Dr. Ohtani for helpful advices regarding the Pig-a assay and Dr. Miura and Dr. Hojo for their valuable cooperation in our experiments.

References

• 1)   Kumagai  S,  Kurumatani  N,  Arimoto  A,  Ichihara  G. Cholangiocarcinoma among offset colour proof-printing workers exposed to 1,2-dichloropropane and/or dichloromethane. Occup Environ Med 2013; 70: 508-10.
• 2)  Ministry of Health, Labour, and Welfare, Japan Web site. [Online]. 2013 [cited 2013 Mar 14]; Available from: URL: http://www.mhlw.go.jp/stf/houdou/2r9852000002x6at-att/2r9852000002x6zy.pdf (in Japanese)
• 3)  Ministry of Health, Labour, and Welfare, Japan web site. [Online]. 2012 [cited 2012 Aug 31]; Available from: URL: http://www.mhlw.go.jp/stf/houdou/2r9852000002ioeh-att/2r9852000002ioik.pdf (in Japanese)
• 4)   Umeda  Y,  Matsumoto  M,  Aiso  S, et al. Inhalation carcinogenicity and toxicity of 1,2-dichloropropane in rats. Inhal Toxicol 2010; 22: 1116-26.
• 5)   Matsumoto  M,  Umeda  Y,  Take  M,  Nishizawa  T,  Fukushima  S. Subchronic toxicity and carcinogenicity studies of 1,2-dichloropropane inhalation to mice. Inhal Toxicol 2013; 25: 435-43.
• 6)  National Toxicology Program. NTP Toxicology and Carcinogenesis Studies of 1,2-Dichloropropane (Propylene Dichloride) (CAS No. 78-87-5) in F344/N Rats and B6C3F1 Mice (Gavage Studies). Natl Toxicol Program Tech Rep Ser 1986; 263: 1-182.
• 7)   De Lorenzo  F,  Degl'Innocenti  S,  Ruocco  A,  Silengo  L,  Cortese  R. Mutagenicity of pesticides containing 1,3-dichloropropene. Cancer Res 1977; 37: 1915-7.
• 8)   Haworth  S,  Lawlor  T,  Mortelmans  K,  Speck  W,  Zeiger  E. Salmonella mutagenicity test results for 250 chemicals. Environ Mutagen 1983; 5 (Suppl 1): 1-142.
• 9)   Principe  P,  Dogliotti  E,  Bignami  M, et al. Mutagenicity of chemicals of industry and agricultural relevance in Salmonella, Streptomyces and Aspergillus. J Sci Food Agric 1981; 32: 826-32.
• 10)   von der Hude  W,  Scheutwinkel  M,  Gramlich  U,  Fissler  B,  Basler  A. Genotoxicity of three-carbon compounds evaluated in the SCE test in vitro. Environ Mutagen 1987; 9: 401-10.
• 11)   Galloway  SM,  Armstrong  MJ,  Reuben  C, et al. Chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells: evaluations of 108 chemicals. Environ Mol Mutagen 1987; 10 (Suppl 10): 1-175.
• 12)  OECD. Screening Information Dataset (SIDS) Initial Assessment Report, 1,2-dichloropropane. Paris: Organisation for Economic Cooperation and Development.
• 13)  National Toxicology Program. NTP Toxicology and Carcinogenesis Studies of Dichloromethane (Methylene Chloride) (CAS No. 75-09-2) in F344/N Rats and B6C3F1 Mice (Inhalation Studies). Natl Toxicol Program Tech Rep Ser 1986; 306: 1-208.
• 14)   Gocke  E,  King  MT,  Eckhardt  K,  Wild  D. Mutagenicity of cosmetics ingredients licensed by the European Communities. Mutat Res 1981; 90: 91-109.
• 15)   Thilagar  AK,  Kumaroo  V. Induction of chromosome damage by methylene chloride in CHO cells. Mutat Res 1983; 116: 361-7.
• 16)   Kitchin  KT,  Brown  JL. Biochemical effects of three carcinogenic chlorinated methanes in rat liver. Teratog Carcinog Mutagen 1989; 9: 61-9.
• 17)   Sasaki  YF,  Saga  A,  Akasaka  M, et al. Detection of in vivo genotoxicity of haloalkanes and haloalkenes carcinogenic to rodents by the alkaline single cell gel electrophoresis (comet) assay in multiple mouse organs. Mutat Res 1998; 419: 13-20.
• 18)   Sheldon  T,  Richardson  CR,  Elliott  BM. Inactivity of methylene chloride in the mouse bone marrow micronucleus assay. Mutagenesis 1987; 2: 57-9.
• 19)   Trueman  RW,  Ashby  J. Lack of UDS activity in the livers of mice and rats exposed to dichloromethane. Environ Mol Mutagen 1987; 10: 189-95.
• 20)   Allen  J,  Kligerman  A,  Campbell  J, et al. Cytogenetic analyses of mice exposed to dichloromethane. Environ Mol Mutagen 1990; 15: 221-8.
• 21)   Ohtani  S,  Unno  A,  Ushiyama  A,  Kimoto  T,  Miura  D,  Kunugita  N. The in vivo Pig-a gene mutation assay is useful for evaluating the genotoxicity of ionizing radiation in mice. Environ Mol Mutagen 2012; 53: 579-88.
• 22)   Nohmi  T,  Suzuki  T,  Masumura  K. Recent advances in the protocols of transgenic mouse mutation assays. Mutat Res 2000; 455: 191-215.
• 23)  OECD. Test guideline 488: OECD guideline for the testing of chemicals. Transgenic rodent somatic and germ cell gene mutation assays. 2011. Paris: Organisation for Economic Cooperation and Development.
• 24)   Carr  GJ,  Gorelick  NJ. Mutational spectra in transgenic animal research: data analysis and study design based upon the mutant or mutation frequency. Environ Mol Mutagen 1996; 28: 405-13.
• 25)  Japanese Center for the Validation of Alternative Methods. (JacVAM). International validation of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens (Version 14.2). [Online]. 2009. Available from: URL: http://cometassay.com/JaCVAM.pdf
• 26)   Sambrook  J,  Russell  DW. Molecular cloning, a laboratory manial. 3rd ed. New York: Cold Spring Harbor Laboratory Press; 1989.
• 27)   Guengerich  FP,  Kim  DH,  Iwasaki  M. Role of human cytochrome P-450 IIE1 in the oxidation of many low molecular weight cancer suspects. Chem Res Toxicol 1991; 4: 168-79.
• 28)   Guengerich  FP,  Shimada  T,  Raney  KD, et al. Elucidation of catalytic specificities of human cytochrome P450 and glutathione S-transferase enzymes and relevance to molecular epidemiology. Environ Health Perspect 1992; 98: 75-80.
• 29)   Guengerich  FP. Activation of dihaloalkanes by thiol-dependent mechanisms. J Biochem Mol Biol 2003; 36: 20-7.
• 30)   Anders  MW. Chemical toxicology of reactive intermediates formed by the glutathione-dependent bioactivation of halogen-containing compounds. Chem Res Toxicol 2008; 21: 145-59.
• 31)   Anders  MW. Glutathione-dependent bioactivation of haloalkanes and haloalkenes. Drug Metab Rev 2004; 36: 583-94.
• 32)   Graves  RJ,  Coutts  C,  Eyton-Jones  H,  Green  T. Relationship between hepatic DNA damage and methylene chloride-induced hepatocarcinogenicity in B6C3F1 mice. Carcinogenesis 1994; 15: 991-6.
• 33)   Graves  RJ,  Coutts  C,  Green  T. Methylene chloride-induced DNA damage: an interspecies comparison. Carcinogenesis 1995; 16: 1919-26.
• 34)   Stolzenberg  SJ,  Hine  CH. Mutagenicity of 2- and 3-carbon halogenated compounds in the Salmonella/mammalian-microsome test. Environ Mutagen 1980; 2: 59-66.
• 35)  Ministry of Health, Labour, and Welfare, Japan web site. [Online]. Available from: URL: http://anzeninfo.mhlw.go.jp/user/anzen/kag/pdf/B/B78-87-5.pdf (in Japanese)
• 36)  OECD. Test guideline 471: OECD guideline for the testing of chemicals. Bacterial Reverse Mutation Test. Paris: Organisation for Economic Cooperation and Development; 1997.
• 37)   Gargas  ML,  Clewell  HJ, 3rd,  Andersen  ME. Metabolism of inhaled dihalomethanes in vivo: differentiation of kinetic constants for two independent pathways. Toxicol Appl Pharmacol 1986; 82: 211-23.
• 38)   Casanova  M,  Deyo  DF,  Heck  H. Dichloromethane (methylene chloride): metabolism to formaldehyde and formation of DNA-protein cross-links in B6C3F1 mice and Syrian golden hamsters. Toxicol Appl Pharmacol 1992; 114: 162-5.
• 39)   Green  T,  Provan  WM,  Collinge  DC,  Guest  AE. Macromolecular interactions of inhaled methylene chloride in rats and mice. Toxicol Appl Pharmacol 1988; 93: 1-10.
• 40)   Andrae  U,  Wolff  T. Dichloromethane is not genotoxic in isolated rat hepatocytes. Arch Toxicol 1983; 52: 287-90.
• 41)   Jongen  WM,  Lohman  PH,  Kottenhagen  MJ,  Alink  GM,  Berends  F,  Koeman  JH. Mutagenicity testing of dichloromethane in short-term mammalian tests systems. Mutat Res 1981; 81: 203-13.
• 42)   Thilagar  AK,  Kumaroo  PV. Induction of chromosome damage by dichroromethane in cultured human peripheral lymphocytes, CHO cells and mouse lymphoma L5178Y cells. Environ Mutagenesis 1984; 6: 422.
• 43)   Thilagar  AK,  Back  AM,  Kirby  PE, et al. Evaluation of dichloromethane in short term in vitro genetic toxicity assays. Environ Mutagen 1984; 6: 418-9.
• 44)   Gisi  D,  Leisinger  T,  Vuilleumier  S. Enzyme-mediated dichloromethane toxicity and mutagenicity of bacterial and mammalian dichloromethane-active glutathione S-transferases. Arch Toxicol 1999; 73: 71-9.
• 45)   Oda  Y,  Yamazaki  H,  Thier  R,  Ketterer  B,  Guengerich  FP,  Shimada  T. A new Salmonella typhimurium NM5004 strain expressing rat glutathione S-transferase 5-5: use in detection of genotoxicity of dihaloalkanes using an SOS/umu test system. Carcinogenesis 1996; 17: 297-302.