In vivo Micronucleus Test of Cyclohexanone and Mutagenicity Classification According to a Globally Harmonized System

Cyclohexanone의 in vivo 소핵시험을 통한 GHS 변이원성 구분

Abstract

A micronucleus test of cyclohexanone has not yet been conducted. To classify the chemical hazard posed by cyclohexanone according to a globally harmonized system of classification and labeling of chemicals (GHS), we investigated its mutagenicity by micronucleus induction in ICR bone marrow cells of 7-weeek-old male mice. The mice were administered three dosages of the chemical for 24 hr via the oral route. After 24 hr, the mice were sacrificed, and their bone marrow cells were prepared for smearing slides. Based on counts of micronucleated polychromatic erythrocytes (MNPCEs) of 2,000 polychromatic erythrocytes, cyclohexanone did not inhibit bone marrow cell proliferation in any of the treated groups, but it resulted in micronucleus induction. According to the results of the mammalian bone marrow micronucleus test, this chemical is mutagenic and classified as category 2 in the GHS.

Cyclohexanone의 GHS 분류 기준에 따른 화학물질의 변이원성 구분을 위해, 다른 연구에서는 아직까지 수행된 바 없는 ICR계 마우스의 골수세포를 이용하는 in vivo 소핵시험을 수행하였다. 7주령의 수컷 ICR계 마우스에 동 시험물질의 3가지 농도를 투여하였으며, 경구투여 24시간 후에 도살, 골수세포를 채취하여 슬라이드 표본을 제작하였고, 2,000개의 다염성적혈구 중 소핵을 갖는 다염성 적혈구(MNPCE)를 계수하였다. 모든 투여군에서 cyclohexanone은 골수세포의 증식을 억제하지 않았으며, 소핵을 유발하였다. 이 골수세포를 이용한 소핵시험의 결과로 동 시험물질(cyclohexanone)은 GHS 분류기준에 의거, 변이원성 구분2로 분류하였다.

Introduction

Chemical substances may have various hazardous effects on human health or the environment. Some of them may be carcinogenic, mutagenic or reproductive toxins, which are categorized as CMRs [1]. In view of the hazards they present, these classified substances and their mixtures are subject to restrictive regulations, particularly in the workplace. The necessity for chemical hazard assessments has increased because the number of workers exposed to chemicals has risen with the development of many industries, and it is necessary to determine what these substances are and how they are regulated [7].

Carcinogens are substances and preparations that, if inhaled, ingested, or penetrated into the skin, may induce cancer or increase its incidence. Mutagens are substances that may induce heritable genetic defects or increase their incidence. They trigger permanent changes in the amount or structure of the genetic material in a cell, and these chemicals, in accordance with the UN globally harmonized system of the classification and labeling of chemicals (GHS), shall be considered mutagens for the purposes of this article [15].

To classify a chemical’s mutagenicity according to GHS, an in vivo micronucleus (MN) test was performed on mammalian bone marrow cells treated with cyclohexanone (CAS No. 108-94-1), for which the definitive information is insufficient for its mutagenicity. Although many toxicological studies have been conducted other than the MN test, the available genotoxic data on cyclohexanone are still controversial with and without mammalian metabolic activation (S9). Therefore, it was necessary for further study, according to the good laboratory practice (GLP) guidelines to secure a quality assurance of the test [9]. The purpose of this MN test is to screen the cytogenetic damage that results in the formation of micronuclei containing lagging chromosome fragments or whole chromosomes. Micronuclei were first used to quantify chromosomal damage and are now recognized as one of the most successful and reliable assays for genotoxic carcinogens [12].

Cyclohexanone is used as a metal-degreasing solvent for lacquers, resins, and insecticides [13]. It is also produced for use as a raw material in the production of the precursors to Nylon-6 and Nylon-6,6. Other important applications include its use as a cleaning agent and as a solvent in the production of pesticides, magnetic tapes, and paint [8]. The physicochemical and toxicological information regarding cy-clohexanone is shown in Table 1. In addition, the major findings related to the mutagenicity of cyclohexanone are shown in Table 2.

Table 1.Sourced by searching in ChemIDplus Advanced, US National Library of Medicine (http://chem.sis.nlm.nih.gov/chemidplus/rn/108-94-1). Rockville Pike, Bethesda, MD 20894, and Material Safety Data Sheet, Sigma-Aldrich (http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=C102180&brand=SIAL&PageToGoToURL=http％3A％2F％2Fwww.sigmaaldrich.com％2Fcatalog％2Fproduct％2Fsial％2Fc102180％3Flang％3Den), MO, USA. Searches were conducted using keywords chemical name AND/OR CAS number.

Table 2.Findings related to the mutagenicity of cyclohexanone

 

Materials and Methods

Chemicals and animal feeding conditions

In this in vivo MN test, cyclohexanone (Sigma-Aldrich, 99.8％, MO, USA, Cat. No. C8930) was used to test the chemicals. Olive oil (Sigma-Aldrich, MO, USA, Cat. No. C1514) was used as a solvent according to the results of the solubility test. The positive control used mitomycin C (MMC) (Sigma-Aldrich, MO, USA, Cat. No. M4287).

Animals and experimental design

The mouse (Mus musculus) bone marrow MN test was carried out according to the Organisation for Economic Co-operation and Development (OECD) test guideline (TG) 474 [9] as well as Hayashi [2] and Heddle [3]. Groups of specific pathogen-free male Institute for Cancer Research (ICR) mice were treated with the test substance at three dosage levels, the highest dosage level being the estimated maximum tolerated dose or the standard limit dose for the MN test, whichever is least. Concurrent negative and positive control groups were also treated. In this study, it was performed using 7-week-old male ICR mice at 300, 600, and 1,200 mg/kg dosages. At 24 hr post-treatment, with cyclohexanone administered orally, there were normally six male animals per group. An animal ethics committee approved the animal studies to ensure appropriate care before the animals were obtained for research (Approval No. IACUC-12-03).

Bone marrow preparation and MN test

Bone marrow cells were obtained from the femurs immediately following euthanization. Immature erythrocytes could be differentiated using a variety of staining techniques that rely on their relatively high content of residual DNA. Mature erythrocytes with low nucleic acid content appeared pinkish-orange, while 5％ Giemsa was used for mouse bone marrow, for peripheral blood, and for staining immature erythrocytes blue. Based on the cell cycles and maturation times of the erythrocytes, the bone marrow was harvested after 24 hr. The bone marrow was flushed from the femurs and spread onto slides. The slides were air-dried, fixed, and stained with a fluorescent DNA-specific stain that easily illuminates any micronuclei that may be present. The 2,000 polychromatic erythrocytes (PCEs, reticulocytes; immature erythrocytes) were scored per animal regarding the frequency of micronucleated cells in each of the six animals per dosage group. In addition, the percentage of PCEs among the 500 erythrocytes in the bone marrow was recorded for each dosage group as an indicator of chemically induced toxicity.

The presence of micronucleated PCEs was visually scored (at least 2,000 cells per mouse) by optical microscopy using a fluorescence microscope (Optiphot-2, Nikon, Tokyo, Japan) with a BA-2 filter. Cells were considered micronucleated when they neatly contained defined chromatin corpuscles with a diameter of less than one-third the diameter of the cell nucleus as well as when they were stained an equal or lighter shade than the nucleus of the cell from which the micronucleated cell was developed.

Statistical analysis

Data were presented as the mean number of micronucleated cells per 2,000 cells for each treatment group. The conclusion of the MN test was determined in consideration of the results of the statistical analyses. The experimental and control MN frequency for each specimen within and between the different mice strains were compared with a one-way ANOVA test and a Kruskal-Wallis test (α=0.05) using SigmaStat v. 3.11.

 

Results and Discussion

There were no specific symptoms among the animals orally exposed to cyclohexanone. The ranges of the body weights of the animals exposed to this chemical was 35.33 to 39.02 g (Table 3). The preliminary tests were performed to determine the maximum dosage. The inhibition of the proliferation in the bone marrow cells was not observed in this test. The frequencies of erythrocytes with MN inductions were 0.18±0.03％, 0.21±0.09％, 0.22±0.05％, and 0.35±0.07％ in the negative control group and 300, 600, and 1,200 mg/kg in the cyclohexanone-treated group, respectively. The ratios of PCEs within the total number of erythrocytes were 52.21±15.05％, 51.84±8.61％, 49.86±6.04％, and 54.29±12.47％ in the negative control group and 300, 600, and 1,200 mg/kg in the cyclohexanone-treated group, respectively (Table 4).

Table 3.a)b.w.: body weight

Table 4.a)PCE: polychromatic erythrocyte. b)MNPCE: micronucleated polychromatic erythrocyte. c)NCE: normochromatic erythrocyte. d)b.w.: body weight. All values are expressed as mean ± SD. *: compare with negative control (0 mg/kg b.w.), p<0.05.

The presence of micronucleated PCEs was visually scored by optical microscopy using a fluorescence microscope. Cells were considered micronucleated when they neatly contained defined chromatin corpuscles with a diameter of less than one-third the diameter of the cell nucleus as well as when they were stained an equal or lighter shade than the nucleus of the cell from which the micronucleated cell was developed (Fig. 1).

Fig 1.The presence of micronucleated polychromatic erythrocytes. Micronucleated mouse bone marrow cells with Giemsa staining by optical microscopy. Arrows show true MN: (A) Magnification 400×, (B) Magnification 1,000×.

Cyclohexanone did not inhibit bone marrow cell proliferation in all the treated groups, though it did initiate MN induction. Based on the results of these tests, it is concluded that this chemical is mutagenic, as demonstrated by the mammalian bone marrow MN test, and it can be classified as a category 2 in mutagen, according to the GHS [14, 15]. In short, according to OECD test guideline [9], we used MMC as a positive control, and we determined as a positive result which is said that “There are several criteria for determining a positive result, such as a dose-related increase in the number of micronucleated cells or a clear increase in the number of micronucleated cells in a single dose group at a single sampling time. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results.”. Also we classified the test chemical as a category 2 in mutagen according to the UN GHS which is said that “Positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from: (a) Somatic cell mutagenicity tests in vivo, in mammals; or (b) Other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays.” [15].

There are three tests typically used to classify the the mutagenicity, with the GHS category used for industrial chemicals, reverse mutation (Ames) tests, in vitro chromosomal aberration tests, and in vivo MN tests. The objective of an in vivo MN test is to evaluate the test article for in vivo clastogenic activity and/or disruptions of the mitotic apparatus by detecting micronuclei in the PCE of mouse bone marrow [3, 4, 11]. A MN assay is now recognized as one of the most successful and reliable assays for genotoxic carcinogens due to the MN formation results, which are from either chromosome breakage (clastogenicity) or aneuploidy. By using pancentromeric probes, it is possible to draw conclusions if the MN is formed because of chromosomal breakage (clastogenicity) or aneuploidy [5].

In this study, we performed in vivo MN tests based on the results of a dose range-finding assay; the maximum dose was estimated to be 1,200 mg/kg, as based on regulatory guidelines. Bone marrow was extracted, and at least 2,000 PCEs per animal were analyzed for the frequency of micronuclei. Cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCEs) in at least the first 500 erythrocytes for each animal. Cyclohexanone did not induce signs of clinical toxicity in the animals treated at the highest dose level (as based on regulatory guidelines). Cyclohexanone did induce statistically significant increases in the micronucleated PCEs at all doses. In addition, it was not cytotoxic to the bone marrow (i.e., it did not produce statistically significant decreases in the PCE:NCE ratio) at any dose.

When a bone marrow erythroblast develops into a PCE, the main nucleus is extruded. Any MN that has been formed may remain behind in the otherwise anucleated cytoplasm. Visualization of the micronuclei is facilitated in these cells because they lack a main nucleus. An increase in the frequency of MNPCEs in treated animals is an indication of induced chromosomal damage.

Rodent bone marrow is routinely used in this test since PCEs are produced in that tissue. The measurement of micronucleated, immature (polychromatic) erythrocytes in peripheral blood is equally acceptable in any species in which the inability of the spleen to remove micronucleated erythrocytes has been demonstrated, or which has shown an adequate sensitivity to detect agents that cause structural or numerical chromosomal aberrations. There are several criteria for determining a positive result, such as a dose-related increase in the number of micronucleated cells or a clear increase in the number of micronucleated cells in a single dose group at a single sampling time. The biological relevance of the results should be considered first. Statistical methods may be used as aids in evaluating the test results [6, 10]. Statistical significance should not be the only determining factor for a positive response; positive results in a MN test indicate that a substance induces micronuclei that are the result of chromosomal damage or damage to the mitotic apparatus in the erythroblasts of the test species.

We recommend that additional studies focusing on lung exposure and the long-term effects of these low-level contaminants are also needed to improve the assessment of hazardous effects to predict risks for human health.