Front. Public Health Frontiers in Public Health Front. Public Health 2296-2565 Frontiers Media S.A. 10.3389/fpubh.2017.00288 Public Health Review A Novel Strategy to Predict Carcinogenicity of Antiparasitics Based on a Combination of DNA Lesions and Bacterial Mutagenicity Tests Liu Qianying 1 Lei Zhixin 2 Zhu Feng 2 Ihsan Awais 3 Wang Xu 4 * Yuan Zonghui 1 2 4 * 1MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, China 2National Reference Laboratory of Veterinary Drug Residues (HZAU) and MAO Key Laboratory for Detection of Veterinary Drug Residues, Wuhan, China 3Department of Biosciences, COMSATS Institute of Information Technology, Sahiwal, Pakistan 4Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety, Wuhan, China

Edited by: Alberto Mantovani, Istituto Superiore di Sanità, Italy

Reviewed by: Iddya Karunasagar, Nitte University, India; Monica Catarina Botelho, Istituto Nacional de Saúde, Portugal

 *Correspondence: Xu Wang, wangxu@mail.hzau.edu.cn; Zonghui Yuan, yuan5802@mail.hzau.edu.cn

Specialty section: This article was submitted to Environmental Health, a section of the journal Frontiers in Public Health

 09 11 2017 2017 5 288 21 03 2017 16 10 2017 Copyright © 2017 Liu, Lei, Zhu, Ihsan, Wang and Yuan. 2017 Liu, Lei, Zhu, Ihsan, Wang and Yuan

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Genotoxicity and carcinogenicity testing of pharmaceuticals prior to commercialization is requested by regulatory agencies. The bacterial mutagenicity test was considered having the highest accuracy of carcinogenic prediction. However, some evidences suggest that it always results in false-positive responses when the bacterial mutagenicity test is used to predict carcinogenicity. Along with major changes made to the International Committee on Harmonization guidance on genotoxicity testing [S2 (R1)], the old data (especially the cytotgenetic data) may not meet current guidelines. This review provides a compendium of retrievable results of genotoxicity and animal carcinogenicity of 136 antiparasitics. Neither genotoxicity nor carcinogenicity data is available for 84 (61.8%), while 52 (38.2%) have been evaluated in at least one genotoxicity or carcinogenicity study, and only 20 (14.7%) in both genotoxicity and carcinogenicity studies. Among 33 antiparasitics with at least one old result in in vitro genotoxicity, 15 (45.5%) are in agreement with the current ICH S2 (R1) guidance for data acceptance. Compared with other genotoxicity assays, the DNA lesions can significantly increase the accuracy of prediction of carcinogenicity. Together, a combination of DNA lesion and bacterial tests is a more accurate way to predict carcinogenicity.

 genotoxicity carcinogenicity antiparasitics risk evaluation DNA lesions 2017YFD0501405 and 2017YFD0501401 National Key Research and Development Program of China Introduction

Antiparasitics are used widely throughout the world in humans and animals to kill or eliminate parasites in vivo and in vitro, and in public health to control diseases and prevent the spread of parasitism from livestock to humans. According to the pharmacological effects and the target parasite species, antiparasitics can be divided into three main groups: anthelmintics, antiprotozoal agents, and insecticides. Chemically based treatment remains the most frequently chosen tool to control parasitism. Unfortunately, the use of antiparasitics does not always result in the expected therapeutic success. The toxic effects were found to be responsible for the therapeutic failure of drug treatment (1). In the 1970s of the last century, it was reported that the chemicals had the capacity to cause cancer in both animals and humans (2, 3). Genetic and carcinogenic damage was found to have important health implications for the induction of diseases, such as lung cancer (4), pancreatic cancer (5), bladder cancer (6), leukemia (7–9), and non-Hodgkin’s lymphoma (10). Therefore, the regulatory agencies of Europe, the USA and Japan suggested that genotoxicity and carcinogenicity studies should be conducted to learn the benefit/risk ratio before commercial approval of pharmaceuticals.

It was recommended by the regulatory agencies that genotoxicity testing, which was considered to be a fundamental part of the carcinogenic risk assessment, should be performed prior to commercialization. It was forbidden to use compounds with proven genotoxic properties on humans except in rare cases with adequate justifications (11). According to the present guidelines for genotoxicity testing of pharmaceuticals (12–15), a standard test battery contains: (a) a test for gene mutations in bacteria, (b) an in vitro test with cytogenetic evaluation of chromosomal damage using mammalian cells or an in vitro mouse lymphoma thymidine kinase± gene mutation assay, and (c) an in vivo test for chromosomal damage using mammalian hematopoietic cells. These assays were considered the best approach for genotoxic hazard identification and potential carcinogenic risk prediction. However, some limitations of this standard test battery in detecting genotoxicity were found. The current revised guidelines of the Veterinary International Conference on Harmonization and ICH S2 (R1) suggested that it can detect the genetic toxicity of most substances. However, for some special chemicals such as antimicrobial, it was required to supply the bacterial assay with a validated in vitro test for gene mutation in mammalian cells to detect the genetic toxicity (12, 15).

How can we identify and analyze positive genotoxicity results, especially in vitro cytogenetics? Two main factors including cytotoxicity and the highest testing concentration of the tested chemicals have very important effects on the result of genotoxicity. The Organization for Economic Cooperation and Development (OECD) had changed over the years to find the most suitable toxicity required at the highest concentration. In the 1999 revision, it was recommended that at least 50% toxicity should be induced. The ICH S2B suggested that in vitro genotoxicity tests should be conducted up to a top concentration of 10 mM in 1997 (16). In fact, when the dose level exceeds 100 µM, the physiological biological reactions will be disorder and then result in positive findings in in vitro genotoxicity tests. Moreover, a study sponsored by the European Center for the Validation of Alternative Methods indicated that the high testing dose should be reduced because the false-positive results in in vitro genotoxicity occurred at concentration levels from 1 to 10 mM. Recently, the ICH updated the genotoxicity guidelines (Table 1) (11, 17). It reduced the highest dose to 1 mM and supported the in vivo genotoxicity assays.

Summary of the ICH (S2B) and ICH S2 (R1) proposed revision to S2.

ICH (S2B) ICH S2 (R1)
Bacterial mutation (Ames) (positive) Bacterial mutation (Ames) (negative)
Option 1 Option 2
In vitro mammalian cell test (10 mM) In vitro mammalian cell test [1 mM] No requirement
Chromosome aberrations or TK gene mutation test Chromosome aberrations or TK gene mutation test or micronucleus test
In vivo cytogenetic assay In vivo cytogenetic assay In vivo cytogenetic assay
Suggest to be integrated into acute toxicity assays of 28 days

ICH, International Committee on Harmonization of Requirements for Registration Pharmaceuticals for Human Use. It is a summary of the difference between the current ICH (S2B) guideline for testing of pharmaceuticals and the revised guideline of ICH S2 (R1) (15, 18).

Antiparasitics were used in the market for many years, and for a large proportion of them, genotoxicity and carcinotoxicity assays were performed prior to 1980, when the bioassays were not concordant with the present guidelines. Thus, it is necessary to re-evaluate the old data (especially the cytogenetic data) under the current guidelines of ICH S2 (R1) (17).

For pharmaceuticals, whose clinical use is continuous for at least 6 months or intermittent in chronic recurrent conditions, the long-term carcinogenicity studies in rats and mice using lifetime treatment are required (19). This has remained the most frequently chosen testing strategy since proposed by regulatory authorities in 1970s. The objective of carcinogenicity studies is to discover whether a drug has the ability to cause carcinogenicity in animals and whether this tumorigenic potential poses a relevant risk to humans (19, 20). To make an evaluation of carcinogenic risks to humans, the International Agency for Research on Cancer (IARC) in the 1–101 volumes of IARC monographs was published in the years from 1972 to 2011 (21). It examined 940 drugs in various groups: the carcinogenicity studies were sufficient for 107 drugs (11.4%), limited for 59 drugs (6.3%), and inadequate for 266 drugs (28.3%); and the remaining 508 drugs (54.0%) were not classifiable in terms of their carcinogenicity to humans. However, it included only 10 antiparasitics: 2 antiparasitics (Metronidazole and Dichlorvos) were classified as possibly carcinogenic to humans (Group 2B), and 8 antiparasitics (Chloroquine, Chlordimeform, Danex, Deltamethrin, Fenvalerate, Malathion, Permethrin, and Pyrimethamine) were considered non-classifiable in terms of their carcinogenicity to humans (Group 3).

Based on the above mentioned, it is meaningful to verify the extent of antiparasitics having the available results of genotoxicity and carcinogenicity studies. It is also necessary to re-evaluate in vitro genotoxicity results according to the present revised guidance. Due to the bacterial mutagenicity test alone produced misleading positive in predicting the carcinogens, we compared the combinations of bacterial mutagenicity test and other genotoxicity assays (such as cytogeneticity in vivo and in vitro, DNA lesions and mouse bone marrow micronucleus), aiming to work out a novel strategy to predict carcinogenicity.

The 136 antiparasitics that are listed in both the human andveterinary pharmacopeia were authorized by China. Forty-three and 107 antiparasitics were obtained from the human pharmacopeia and veterinary pharmacopeia, respectively. Since some parasites, including helminths, schistosome, and tapeworm, can infect both humans and animals, simultaneously, 14 antiparasitics (Albendazole, Amoscanate, Artesunate, Bithionol, Diethylcarbamazine, Ivermectin, Levamisole, Piperazine, Pyramine, Praziquantel, Mebendazole, Metronidazole, Niclosamide, and Semduramicin Soditium) can be used on both humans and animals.

The methodology of the major carcinogenicity and genotoxicity tests were summarized in Table 2. The collected information of genotoxicity and/or carcinogenicity of antiparasitics was obtained primarily from peer-reviewed journals (e.g., Medline, Toxline, and the Registry of Toxic Effects of Chemical Substances) (22), the US National Toxicology Program, the edition of Physician’s Desk Reference (23–25), the Center for Drug Evaluation and Research of the Food and Drug Administration and some relevant websites, such as http://www.updata.usa.com, http://www.osha.gov, http://www.toxnet.nlm.nih.gov, http://www.ntp.server.niehs.nih.gov, http://www.potency.berkeley.edu, http://www.fda.gov/cder, http://www.scirus.com, and http://www.inchem.org. For some antiparasitics, the genotoxicity and carcinogenicity data are incomplete in terms of the absence of the dose, the indication of an exogenous metabolic system in the genotoxicity assays, and the sex in carcinogenicity assays. In such cases, we presented our data in tables as obtained in these experimental conditions except for special markings. Moreover, regarding the present guidelines, the equivocal results that we found in extensive research were marked as positive in this review.

The methodology of the major carcinogenicity and genotoxicity tests.

Test system Materials Principle of reference
Bacterial mutagenicity The following fi-M Salmonella strains were used for the bacterial reverse mutation assay: TA97a, TA98, TA100, TA102, and TA1535. All strains were checked for maintenance of genetic markers prior to study This test was performed by a plate incorporation procedure as outlined by OECD No.471, 46 Redbook 2000 IV.C.1.a (26), Redbook 2000: IV.C.1.a (27), and Chinese standard guidelines (28)
Mouse lymphoma assay The mouse lymphoma assay using the thymidine kinase (Tk) gene of L5178Y Tk± −3.7.2C mouse lymphoma cell lines was found to be the closest to the in vivo environment among the different in vitro mammalian and bacterial gene-mutation testings The MLA was performed according to FDA toxicological principles for the safety assessment of food ingredients and OECD guidelines for the testing of chemicals. IV.C.1.c Mouse Lymphoma Thymidine Kinase Gene Mutation Assay (29) and Test Guideline 490: In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene (30)
Chromosomal aberration assay The potential of tested compound to induce structural and numerical chromosome aberrations was evaluated in Chinese hamster lung fibroblast cells (V79) Chromosomal aberration assay in vitro according to OECD No.473 (31), Redbook 2000 IV.C.1.b In Vitro Mammalian Chromosomal Aberration Test (32)
Bone marrow erythrocyte micronucleus assay For each treated animal, at least 1,000 polychromatic erythrocytes (PCE) were counted to determine the micronucleus frequencies and record the micronucleus occurrence rate per one thousand PCE, and the proportion of PCE to normochromatic erythrocytes (NCE) was evaluated by counting a total of 1,000 erythrocytes This assay was conducted in accordance with OECD Guideline No.474 (33) and Redbook 2000 IV.C.1.d. Mammalian Erythrocyte Micronucleus Test (27)
HGPRT mutation test Mutations were expressed during a period of 6–7 days, including two subculturing steps. Subsequently, mutant frequencies (mutants/106 cells) and cloning efficiencies were scored This assay was carried out following standard test procedures (34)
Unscheduled DNA synthesis assay Prior to drug treatments, peripheral blood lymphocytes were isolated from healthy individuals. The radioactivity was determined by Beckman Ls3801 liquid scintillation spectrometry This assay was performed according to the OECD guideline number 482 (26, 34)
Long-term carcinogenesis assay in rodent The animal were randomly assigned to four groups based on their body weights, and each group of animal were fed the basal diet mixed with tested compound for a total period of 78 weeks (mice) and 104 weeks (rat) Long-term carcinogenesis assay was conducted according to the guidelines of Ref. (35, 36)
Results Genotoxicity and Carcinogenicity of Antiparatics

For the present analyses, an antiparasitic was regarded as genotoxic when it produced positive or equivocal results in at least one of the standard battery tests, and as a rodent carcinogen when it increased tumor incidence. Table 3 covers the information available on genotoxicity and carcinogenicity findings for each tested antiparasitic. The following genotoxicity assays were used: Ames (bacterial mutagenesis), sex-linked recessive lethal, in vitro cytogenetics (chromosome aberrations), in vivo cytogenetics [chromosome aberrations, micronucleus and sister chromatid exchange (SCE)], unscheduled DNA synthesis in vitro (UDS), MLA (mouselymphoma L5178Y TK± assay), and other types of genotoxicity studies, including DNA fragmentation, mammalian mutagenesis HGPRT, SCE in vitro, DNA strand break analysis in vitro, and the micronucleus assay in vitro. The long-term carcinogenicity test was carried out in mice, rats, and other species.

Genotoxic and carcinogenicity effects of antiparasitics.

Test system Dose or concentration (LED or HID) Result Reference
1. Acriflavine (8048-52-0)
Salmonella typhimurium (none), TA1537, TA1538, TA98 50 μg/plate − (37)
Salmonella typhimurium (rat, liver, S-9, aroclor1254), TA1537, TA1538, TA98 50 μg/plate + (37)
Gene mutation, Aspergillus nidulans + (38)
Chinese hamster ovary (CHO), CHO-K1-BH4 (HGPRT) 0.5–4 µg/l + (39)
Chromosome aberrations in vivo, Mammalian or early embryo − (40)
Forward and reverse gene mutation, host-mediated assay, Salmonella typhimurium# − (41)
Sex-linked recessive lethals and sex-chromosome loss + (42)
Micronucleus test in vivo and in vitro, chromosome aberrations, mammalian polychromatic erythrocytes, mammalian cell culture, non-human + (43)
Mitotic recombination or gene conversion, Saccharomyces cerevisiae NC (44)
Sperm morphology, mouse + (45, 46)
2. Albendazole (54965-21-8)
Bacterial mutation (Ames) − (47)
SCE and micronucleus (MN) on human lymphocytes in vivo 15 mg/kg p.o. in diet for 28 days + (48)
Micronuclei in cultured peripheral blood lymphocytes in vitro and in cultured human lymphocytes 10–100 µg/ml + (49)
Cytogenetics in vitro and in vivo − (47)
Micronucleus assay with CHO-K1 cells in vitro + (50)
Long-term carcinogenesis assay, mice 400 mg/kg/day − (24)
Long-term carcinogenesis assay, rats 20 mg/kg/day − (24)
3. Amitraz (33089-61-1)
Salmonella typhimurium, TA98, TA100, TA97, TA102 0–200 μg/plate − (51)
Genotoxic in the vibrio test 10−3 to 10−5 µg/plate − (52)
DNA damage on hamster cells in vitro, comet assay 3.75 µg/l + (53)
Long-term carcinogenesis assay. rat (oral) 0, 15, 50, 200 mg/l in feed for 104 weeks − (54)
Long-term carcinogenesis assay. mouse (oral) − (54)
4. Amodiaquine (86-42-0)
Salmonella typhimurium, TA100, reverse mutation 0.1–5,000 μg/plate − (55)
Salmonella typhimurium, TA97A, TA102, TA104 0.1–1,000 μg/plate − (56)
Salmonella typhimurium, TA100 (rat, liver S-9, Phenobarbital), reverse mutation 0.1–5,000 μg/plate − (56)
Salmonella typhimurium, TA97A, TA102, TA104 (rat, liver S-9, Phenobarbital), reverse mutation 0.1–1,000 μg/plate − (56)
5. Amoscanate (26328-53-0)
Salmonella typhimurium, TA1537, TA1535, TA100, TA1538, TA98, reverse mutation 0.1–1,000 μg/plate −
Salmonella typhimurium, TA1537, TA1535, TA100, TA1538, TA98 (rat, liver S-9, aroclor 1254 or Phenobarbital), reverse mutation 0.1–1,000 μg/plate −
Salmonella typhimurium, TA100 (rat, liver S-9, aroclor 1254 or Phenobarbital), reverse mutation 20–160 nmol/plate − (57)
6. Amphotericin B (1397-89-3)
Bacterial mutation (Ames) − (47)
Chromosome aberrations, peripheral blood lymphocytes − (58)
Cytogenetics in vitro and in vivo − (47)
MLA − (47)
7. Atovaquone (95233-18-4)
Bacterial mutation (Ames) − (47)
Cytogenetics in vitro and in vivo − (47)
MLA − (47)
Long-term carcinogenesis assay, mice (liver tumors) human AUC × 5 + (24)
Long-term carcinogenesis assay, rats NR − (24)
8. Bithionol (97-18-7)
Salmonella typhimurium (none), TA98, TA100, TA97, TA102, TA100, TA1535, TA1537, TA97 0.1–1,000 μg/plate − (59)
0.1–6.6 µg/plate − (60)
Salmonella typhimurium (rat, liver, S-9, kanechlor 400) TA98, TA100, TA97, TA102 0.1–1,000 μg/plate − (59)
Salmonella typhimurium (Hamster, liver, S-9, Aroclor 1254) TA100, TA1535, TA97, TA98 1–200 μg/plate − (60)
Micronucleus test in vivo, chromosome aberrations, mammalian polychromatic erythrocytes − (61)
9. Bromofenofos (21466-07-9)
Salmonella typhimurium (rat, liver, S-9, kanechlor 400), TA100, TA98, TA1535, TA1537, TA1538; Salmonella typhimurium (none), TA100, TA98, TA1535, TA1537, TA1538 0.005–0.5 mg/plate − (61)
Micronucleus test in vivo, chromosome aberrations, mammalian polychromatic erythrocytes (mouse) − (61)
10. Chlordimeform (6164-98-3)
Salmonella typhimurium, TA1535, TA1537, TA98, TA100 1–7,500 μg/plate − (62)
Salmonella typhimurium, TA98, TA100, TA1535, TA1537, TA1538 1–2,000 μg/plate − (63)
(64)
Recombination assay, Bacillus subtilis (H17 vs. M45) − (65)
(63)
E. coli polA (WP2 uvra), recombination assay, DNA effects (bacterial DNA repair) 10−5 g/ml − (65)
E. coli 1–7,500 μg/plate − (62)
UDS in vitro, DNA effects (Human diploid fibroblasts FL cell) 10−6 to 10−3 g/ml − (66)
+ (66)
Chromosomal aberrations in vitro and in vivo human peripheral lymphocytes MTD − (67)
Chromosomal aberrations in vivo, Chinese hamster cells (CHO), Voles living donor bone marrow cells MTD − (68)
SCE, bone marrow cells in mice, Voles living donor bone marrow cells, Voles fibroblasts 10 mg/kg + (67)
80 mg/kg + (63)
Micronucleus test, mice bone marrow cells in vivo, peripheral lymphocytes 77 mg/kg − (69)
Mitotic recombination or gene conversion, Saccharomyces cerevisiae − (44)
Neoplasms + (70)
Carcinogenicity studies in mouse and rat + (71)
Chromosomal aberrations, mouse bone marrow cells in vivo 100 mg/kg + (55)
11. Chloroquine (54-05-7)
Salmonella typhimurium, TA97, TA1537, reverse mutation 250 µg/plate + (72, 73)
200 µg/l
Salmonella typhimurium, TA1977, TA1535, TA1537, TA1538, reverse mutation 600 µg/l − (74, 75)
10 000 µg/plate
Salmonella typhimurium, TA98, TA100, reverse mutation 0–10,000 µg/plate + (56, 73)
Salmonella typhimurium, TA98, TA100, TA1537, TA1538, reverse mutation 5,000 µg/plate NC (73, 76)
Salmonella typhimurium, TA97A, TA1537, reverse mutation 5,000 µg/plate − (73, 77)
Salmonella typhimurium, TA98, TA100, TA97A, TA100, reverse mutation 50 µg/plate − (73, 78)
10,000 µg/plate
Salmonella typhimurium, TA102, TA104, reverse mutation 5,000 µg/plate − (56, 73)
E. coli WP2 uvra, reverse mutation 5,000 µg/plate NT (72, 73)
E. coli, reverse mutation 300 µg/plate + (78)
Salmonella typhimurium, TA97A, TA100, reverse mutation 20–50 µg/plate + (78)
Salmonella typhimurium, TA97A, TA100 (rat, liver S-9, phenobarbital); Salmonella typhimurium, TA102, TA104; Salmonella typhimurium, TA102, TA104 (rat, liver S-9, phenobarbital), reverse mutation 0.1–10,000 µg/plate − (79)
E. coli polA (W3119 vs. P3478) Rec-assay, DNA effects (bacterial DNA repair) 0.1–10,000 µg/plate + (55)
Chromosome aberrations, mammalian cell culture, non-human, micronucleus test in vitro + (43)
SCE, mouse bone marrow cells in vivo 12.5 mg/kg + (78)
Chromosomal aberrations, mouse bone marrow cells in vivo 100 mg/kg + (55)
12. Closantel (57808-65-8)
Chromosomal aberrations in vivo, bone marrow cells 0, 5, 10, 15, 20 mg/kg + (80)
13. Coumaphos (56-72-4)
Salmonella typhimurium (none), TA98, TA1535, TA1537, TA1538, TA100, TA100, TA98 3.3–3333.3, 3.3–10,000, 0.3–333.3 µg/plate − (81)
Salmonella typhimurium (rat, liver, S-9, aroclor 1254), TA98, TA1535, TA1537, TA1538, TA100, TA100, TA98 3.3–3333.3, 3.3–10,000, 0.3–333.3 µg/plate − (81)
Salmonella typhimurium (none), TA98, TA100, TA1535, TA1537, TA1538 667, 1.000, 3.333, 6.667, 10,000 µg/plate − (82)
E. coli WP2 uvra, (none); E. coli WP2 uvra (rat, liver, S-9, aroclor 1254) 3.3–10,000, 0.3–333.3 µg/plate − (81)
E. coli, mouse, liver, S-9; E. coli, hamster liver, S-9, aroclor 1254 3.3–10,000, 0.3–333.3 µg/plate − (81)
Chromosomal aberrations in vitro, CHO cells (rat, liver, S-9, aroclor 1254) 100, 300, 1,000 µg/l − (83)
Chromosomal aberrations in vitro, CHO cells (none) 99.5, 299, 995 µg/l − (83)
Micronucleus in vivo, polychromatic erythrocytes 480 mg/kg of coumaphos at 98.0% purity + (82)
Carcinogenicity studies, rats 0 (1% peanut oil), 1, 5, 25 mg/l in diet for 24 months − (82)
Carcinogenicity studies, mouse 0, 10, 20 mg/l in diet − (84, 85)
Carcinogenicity studies, rats 0, 10, 20 mg/l in diet − (84, 85)
14. Cyfluthrin (68359-37-5)
Salmonella typhimurium, TA98, TA100 (none); TA98, TA100 (rat liver S9), reverse mutation 1,000–5,000 µg/plate − (86)
Gene mutation, Ames/micronucleus test in cultured human peripheral blood lymphocytes − (86)
Chromosomal aberrations in cultured human peripheral blood lymphocytes; chromosomal aberrations in vivo 1,000, 2,000 mg/ml + (86)
250, 500, 1,000 mg/kg b.w.
SCE, in cultured human peripheral blood lymphocytes 500, 1,000, 2,000 mg/ml − (86)
SCE in blood lymphocytes 500, 1,000, 2,000 µg/l
Micronucleus (MN) formation in cultured human peripheral blood lymphocytes 500, 1,000, 2,000 mg/ml + (86)
DNA damage on the epithelial cells of human nasal mucosa 0.05, 0.1, 0.5, 0.75, 1.0 mg/ml + (87)
DNA damage and comet assay in fish species 5.6 mg/l beta-cyfluthrin for 48 h + (88)
Chromosomal aberrations in vitro 500, 1,000, 2,000 µg/l − (86)
Mouse bone marrow cells in vitro 1,000 µg/l + (86)
15. Cypermethrin (52315-07-8)
Salmonella typhimurium, TA98, TA100, TA1535 − (89)
Micronuclei formation in bone marrow cells in rats; DNA damage in blood cells in rats 25 mg/kg b.w. p.o. for 28 days + (90)
Micronucleus test in mice in vivo NC (91)
Chromosomal aberrations (CAs) on human peripheral lymphocytes; SCE on human peripheral lymphocytes 12.5 + 2.5, 15 + 5, 17.5 + 7.5, 20 + 10 mg/ml + (92)
Micronucleus (MN) tests on human peripheral lymphocytes 12.5 + 2.5, 15 + 5, 17.5 + 7.5 mg/ml + (92)
Excision-repairable DNA damage in ICR mouse hepatocytes − (93)
DNA strand breakage and DNA hypomethylation in ICR mouse hepatocytes + (93)
Chromosomal aberrations on human peripheral lymphocytes 5, 10, 15, 20 mg/ml + (94)
SCE on human peripheral lymphocytes
Micronucleus (MN) tests on human peripheral lymphocytes 5, 10 mg/ml + (94)
Chromosomal aberration (CA) in highly mitotic kidney cells; micronucleus (MN) tests in erythrocytes of a freshwater fish 0.4, 0.8, 1.2 µg/l for 48 and 72 h + (95)
DNA damage in vital organs in mouse 12.5, 25, 50, 100, 200 mg/kg b.w. + (96)
DNA damage using alkaline comet assay 25, 50, 75 mg/kg b.w. for 6–15 days + (97)
Transplacentally genotoxic
Peripheral blood for MN test 20, 30, 40, 50 mg/l + (98)
Excision repairable DNA lesions − (99)
Long-term carcinogenesis assay, rat 75, 1,500 mg/kg b.w. − (100)
Long-term carcinogenesis assay, mouse 240, 1,600 mg/kg b.w. − (100)
16. Danex (52-68-6)
E. coli, WP2 (rat, liver S-9, aroclor 1254) 500–10,000 µg/plate + (101)
E. coli, WP2 UVRA (rat, liver S-9, aroclor 1254)
UDS Human fibroblasis (66)
Salmonella typhimurium, TA100, reverse mutation 1–5,000 µg/plate + (101)
Salmonella typhimurium, TA1535, TA1535 (rat, liver S-9, aroclor 1254), reverse mutation 1.25–5,000 µg/ml − (102)
Salmonella typhimurium, TA104, TA100 (rat, liver S-9, aroclor 1254), reverse mutation 5–25 mg/plate +
Salmonella typhimurium, TA104, TA100, TA1535, TA97, reverse mutation 1–25 mg/plate + (102)
Salmonella typhimurium, TA1535, TA97 (rat, liver S-9, aroclor 1254), Salmonella typhimurium, TA100, TA98, TA104 − (102)
Salmonella typhimurium, TA100, TA98, TA97; Salmonella typhimurium, TA100, TA98, TA104 (rat, liver S-9, aroclor 1254) 0.1–25 mg/plate − (102)
Salmonella typhimurium, TA100, TA98, TA97, TA1535, TA1537 (rat, liver S-9, aroclor 1254), reverse mutation 500–5,000 µg/plate − (102)
Salmonella typhimurium, TA1535, TA1537, reverse mutation 100–10,000 µg/plate − (102)
Salmonella typhimurium, TA98, TA100, reverse mutation 33–10,000 µg/plate − (103)
Chromosomal aberrations, V79 0.4–4,000 mmol − (104)
0.04–0.8 mmol +
Micronucleus in vivo, mouse 100 or 200 mg/kg + (105)
3.13, 6.25, 12.5, 25 mg/kg (106)
UDS human cells − (103)
17. Deltamethrin (52918-63-5)
Salmonella typhimurium, TA98, TA100, TA1535, TA1537, and TA1538 − (70)
Salmonella typhimurium, TA98, TA100 20–600 µg/plate − (107)
Salmonella typhimurium, TA98, TA100, TA1535, TA1537, TA1538 0–5,000 µg/plate − (108)
Chromosomal aberrations, CHO cells in vitro 0, 19, 38, 75, 150 µg/l + (108)
Micronucleus test, mice bone marrow cells in vivo 8.0–90.0 mg/kg + (109)
V79/6-thioguanine, Chinese hamater V79 4–40 µg/l − (107)
Carcinogenesis assay. mouse (dermal) 0, 1, 2,4 mg/kg b.w. for 32 weeks − (110)
Long-term carcinogenesis assay. Rat (intragastric) 0, 3, 6 mg/kg for 120 weeks − (111)
Long-term carcinogenesis assay. Rat (oral) 0, 25, 125, 500, 800 mg/l in feed for 2 years − (108)
Long-term carcinogenesis assay. Mouse (oral) 0, 10, 100, 1,000, 2,000 mg/l in feed for 97 weeks − (108)
Long-term carcinogenesis assay. Mouse (intragastric) 0, 1, 4, 8 mg/kg in diet for 120 weeks − (111)
18. Diaveridine (5355-16-8)
Bacterial umu test, S. typhimurium, TA1535 0.1, 0.3, 1.0, 3.0 µg/l − (112)
Salmonella typhimurium, TA100, TA98, TA97, TA102 0.5, 1.0, 2.5, 5.0, 10, 25 µg/plate − (112)
E. coli, WP2 uvra/pkm101 0.5, 1.0, 2.5, 5.0, 10, 25 µg/plate − (112)
Chromosome aberration in cultured Chinese hamster CHL cells 12.5, 25, 50, 100 µg/l + (112)
Micronucleus test in rodent bone marrow, mice and rats 500, 1,000, 1,500, 2,000 mg/kg b.w. − (112)
Comet assay in five mouse organs in vivo 1,000, 1,500, 2,000 mg/kg b.w. + (112)
Salmonella typhimurium, TA98 (rat, liver, S9) − (113)
Salmonella typhimurium, TA98 (Hamster, liver, S9), TA100 (rat, liver, S9) reverse mutation − (113)
Salmonella typhimurium, TA100 (Hamster, liver, S9) + (113)
Salmonella typhimurium, TA97, TA98, TA100, TA102 (rat, liver, S9) reverse mutation 0.1–3.0 µg/l − (112)
Salmonella typhimurium, TA1535, TA1535 (rat, liver, S9) 10 µg/l − (112)
Chromosomal aberrations 100 µg/l,48 h + (112)
Mouse bone marrow cells in vivo, rat − (112)
Comet assay (liver, kidney, lung, spleen) + (112)
Comet assay (bone marrow) − (112)
19. Diazinon (333-41-5)
Salmonella typhimurium, TA1535, TA1536, TA1537, TA1538 carcinogenicity studies in vivo (114)
Salmonella typhimurium (TA98, TA100, TA1535, TA1537, and TA1538), reverse mutation − (115, 116)
Salmonella typhimurium, TA98, TA97, TA102, TA1535, TA1537, TA100 reverse mutation 20–80 mg/l, 100–10,000 µg/plate − (117, 60)
E. coli WP2 uvra, tryptophan reverse gene mutation − (101)
E. coli (rat, liver S-9, aroclor 1254), mouse, Hamster 0.3–333.3, 1–100, 10–10,000 µg/plate − (81)
MNs (micronuclei) in rat lymphocytes 150 mg/kg b.w. + (118)
SCE, non-human CHO cells in vitro + (119)
SCE, human Laz-007 B lymphoid cells in vitro + (120)
DNA effects (bacterial DNA repair), Bacillus subtilis (H17 vs. M45), recombination assay, NC (79)
DNA damage in human blood lymphocytes in vitro 750 µg/l + (121)
UDS in vitro, DNA effects human diploid fibroblasts − (66)
Mitotic recombination or gene conversion, Saccharomyces cerevisiae − (44)
Long-term carcinogenesis assay. mice 0, 100, 200 mg/l in diet − (122, 123)
Long-term carcinogenesis assay. rats 0, 400, 800 mg/l in diet (84, 85)
20. Dichlorvos(DDVP) (62-73-7)
Salmonella typhimurium, TA100 500–1,000 µg/plate + (124)
100–6,666 µg/plate + (125)
0.5–500 µg/plate ++ (126)
100–5,000 µg/plate + (60)
100–1,000 µg/plate + (127)
Salmonella typhimurium, TA98 100–6,666 µg/plate + (125)
100–5,000 µg/plate + (60)
Salmonella typhimurium (TA98, TA100, TA1535, TA1537, TA1538), histidine reverse gene mutation + (115, 116)
Salmonella typhimurium, forward and reverse gene mutation, mitotic recombination and gene conversion, DNA effects, host-mediated assay − (128)
SCE in vitro, human lymphocytes − (129)
SCE in vitro, non-human With dose response + (130)
SCE in vitro, human, human lymphocytes NC
E. coli (rat, liver S-9, aroclor 1254) 22.6 µg/l + (101, 131)
E. coli polA (W3119 vs. P3478), Recombination assay, DNA effects (bacterial DNA repair) + (79)
E. coli WP2 uvra, tryptophan reverse gene mutation + (101)
E. coli 5 mg/ml + (132)
+ (101)
+ (133)
Chromosome aberrations, mammalian polychromatic erythrocytes NC (134)
Chromosomal aberrations in vitro, CHO cells 16, 50, 100, 160 µg/l + (135)
50, 160, 500, 1,600 µg/l +
500, 750, 1,000 µg/l +
Chromosome aberrations, Allium cepa With dose response + (136)
Chromosome aberrations, non-human bone marrow in vivo − (40)
Chromosome aberration, mammalian germ cells in vivo − (137)
Chinese hamster V79 1.25–5 µg/l − (104)
CHO, CHO-k1-bh4 (HGPRT)/6-thioguanine 50–150 µg/l + (138)
Mitotic recombination or gene conversion, Saccharomyces cerevisiae + (44)
Mouse lymphoma, L5178Y (TK+/TK−) 0–0.33 µg/l, 0–0.12 µg/l, 0–0.24 µg/ml + (130)
Micronucleus in vivo, erythrocytes − (130)
Mouse lymphoma, L5178Y (TK+/TK−) 6.25–200 µg/l + (125)
UDS human cells 6.5–650 mg/ml + (104)
UDS rat hepatocytes 0.005–1.25 mg/ml − (131)
UDS mouse forestomach epithelium 1–100 mg/kg − (139)
Sex-linked recessive lethal gene mutation, Drosophila melanogaster − (140)
Sperm morphology, mouse NC (45, 46)
Dominant lethal test, rodents With dose response NC (137)
Recombination assay, spot test, DNA effects, Bacillus subtilis (H17 vs. M45) + (79)
Carcinogenicity studies in vivo, non-human NC (141)
Carcinogenicity studies, mouse 0, 317, 635 mg/l in diet − (142)
Carcinogenicity studies, rat 0, 150, 318, 326, 635 mg/l in diet − (142)
Long-term carcinogenesis assay. Rat 0, 4, 8 mg/kg in corn oil for 105 weeks + (125)
Long-term carcinogenesis assay. Mouse 0, 10, 20 mg/kg in corn oil for 105 weeks + (125)
Long-term carcinogenesis assay. Rat 0, 0.1 mg in 0.2 ml water for 111 weeks − (143)
Long-term carcinogenesis assay. Mouse 0, 10, 20 mg/kg in corn oil for 104 weeks + (144)
Long-term carcinogenesis assay. Rat 0, 4, 8 mg/kg in corn oil for 104 weeks + (125)
21. Dimetridazole (551-92-8)
Salmonella typhimurium, TA98, TA100, TA1535, TA1537, TA1538 + (145)
Salmonella typhimurium, TA100 + (146)
Salmonella typhimurium, TA97, TA98, TA100, TA102 50–200 µg/plate + (147)
Comet assay in human lymphocytes 354.3 mg/ml + (148)
22. Fenbendazole (43210-67-9)
Salmonella typhimurium (none), TA100, TA97, TA98, TA102 (rat, liver, S-9, aroclor 1254), TA100, TA97, TA98, TA102. 5–1,000 µg/plate − (149)
Chromosomal damage in Chinese hamster lung (CHL) cells 0.78 mg/ml + (150)
Cytotoxicity to 10T1/2 cells 0.04–1.60 mg/ml + (150)
Morphological transformation in mouse embryo fibroblasts 0.08–0.4 mg/ml + (150)
23. Fenchlorphos (299-84-3)
SCE, human somatic cells in vitro + (120)
24. Fenthion (55-38-9)
Ames reverse gene mutation 0.1–20 µg/plate − (151)
Bacillus subtilis (H17 vs. M45) 20 µg/plate − (132)
E. coli polA (W3119 vs. P3478), recombination assay, DNA effects(bacterial DNA repair) NC (79)
E. coli WP2 uvra, tryptophan reverse gene mutation − (101)
SCE, non-human V79 cells in vitro + (152)
SCE, human somatic cells in vitro NC (120)
Mitotic recombination or gene conversion, Saccharomyces cerevisiae − (44)
Bacillus subtilis (H17 vs. M45), recombination assay, spot test, DNA effects (bacterial DNA repair) NC (79)
Drosophila melanogaster, sex-linked recessive lethal mutation NC (140)
UDS, human diploid fibroblasts in vitro − (66)
UDS, thymidine incorporation, rat hepatocytes 0, 5.0, 7.5, 10.0, 15.0, 30.0 µg/l + (153)
Chromosomal aberrations, CHO cells in vitro 0, 0.02, 0.04, 0.08, 0.15 µg/l − (153)
SCE in vivo and UDS in vitro + (154)
Chromosomal aberrations, human peripheral lymphocytes in vitro 0.5, 1.5, 2.5, 5.0 µg/ml + (151)
Long-term carcinogenesis assay. Mice 0, 0.1, 1, 5, 25 mg/l in diet for 2 years (153)
Long-term carcinogenesis assay. Rats 0, 5, 20, 100 mg/l in diet for 2 years − (153)
Long-term carcinogenesis assay. B6C3F1 male mice 10 mg/l in diet for 103 weeks + (155)
Long-term carcinogenesis assay. B6C3F1 female mice 10 mg/l in diet for 103 weeks − (155)
Long-term carcinogenesis assay. F341 rat 200 mg/l in diet for 103 weeks − (155)
25. Fenvalerate (51630-58-1)
Salmonella typhimurium, TA104 100–3,500 µg/plate − (156)
TA100 500–4,000 µg/plate −
TA97 100–4,000 µg/plate −
TA100 500–4,000 µg/plate −
TA98 100–3,000 µg/plate −
Micronuclei in bone marrow in mice in vivo 10, 20 mg/kg by i.p. + (157)
Peripheral blood for MN test 25, 50, 75, 100 mg/l + (98)
Chinese hamster V79 gene mutation 4–40 µg/l − (107)
Excision repairable DNA lesions − (99)
Chromosomal aberrations, Chinese hamster ovary (CHO-K1) in vitro 10, 25, 50, 100,150 µg/l + (158)
Long-term carcinogenesis assay. Rat (oral) 0, 1, 5, 25, 250 mg/l in diet for 2 years − (159)
Long-term carcinogenesis assay. Rat (oral) 1, 5, 25, 250, 1,000 mg/l in diet for 2 years − (160)
Long-term carcinogenesis assay. Mouse (oral) 0, 10, 50, 250, 1,250 mg/l in the diet for 2 years − (161)
Long-term carcinogenesis assay. Mouse (intragastric) 0, 40, 80 mg/kg in arachis oil for 120 weeks (111)
26. Fipronil (120068-37-3)
Salmonella typhimurium, TA98, TA100, TA1535, TA1537 0–0.5 mg/plate of 90.6% fipronil + (162)
Chromosomal aberrations, human lymphocytes in vitro 0, 4.69, 9.38, 18.75, 37.5, 75, 150, 300 μg/l + (162)
Sister chromatid exchanges (SCEs); DNA damage, comet assay in vitro; micronuclei (MN) in human peripheral blood lymphocytes 0.7,0.3 µg/l + (163)
Comet assay with gillsin, the fish Rhamdia Quelen; nuclear morphological alterations 0.05, 0.10, 0.23 µg/l − (164)
Micronucleus test in the Piscine 0.10, 0.23 µg/l + (164)
Chinese hamster V79 cells, HGPRT mutations 0, 0.8, 4, 20, 100, 500 µg/l + (162)
Bone marrow polychromatic erythrocytes, mouse micronucleus in vivo 0, 1, 5, 25 mg/kg b.w. + (162)
Long-term carcinogenesis assay. Rat (oral) 0, 0.5, 1.5, 30, 300 mg/l of 95.4% fipronil in diet for 104 weeks + (162)
Long-term carcinogenesis assay. Mouse (oral) 0, 0.1, 0.5, 10, 30 mg/l of 95.4% fipronil in diet for 78 weeks + (162)
27. Flubendazole (31430-15-6)
Salmonella typhimurium (none), TA100, TA98; Salmonella typhimurium (rat, liver, S-9, aroclor 1254), TA98, TA100 0.01–10 µg/plate − (165)
28. Furapromide (1951-56-0)
Chromosomal aberrations, V79 cells + (166)
Salmonella typhimurium, TA98, reverse mutation Neurospora crassa, forward gene mutation + (167)
(168)
Chromosomal aberrations, V79, HPRT 10–120 µmol + (166)
Saccharomyces cerevisiae, mitotic recombination or gene conversion 7–567 µmol + (44)
Salmonella typhimurium (TA98, TA100, TA1535, TA1537, and TA1538) + (115, 116, 169)
29. Furapyrimidone (75888-03-8)
Salmonella typhimurium, TA98, TA100; Salmonella typhimurium, TA98, TA100 (S-9), reverse mutation 0.01–10 µg/plate + (170)
30. Imidacloprid (138261-41-3)
Salmonella typhimurium, TA100 (rat, Liver, S-9) 25–10 µg/plate − (171)
Salmonella typhimurium, TA98 (rat, Liver, with or without S-9) 25–100 µg/plate + (171)
Salmonella typhimurium, TA97, TA98, TA100, TA102 (S9) 40, 200, 1,000, 5,000 µg/plate − (172)
Micronuclei test in mouse bone marrow 23, 45, 90 mg/kg b.w. − (172)
Chromosome aberration in primary spermatocytes testicle 38, 75, 150 mg/kg b.w. − (172)
Micronucleus (MN) test in vivo, amphibian 165 mg/kg b.w. + (173)
Comet assay in vivo, amphibian 0.05, 0.1, 0.2, 0.5 mg/kg b.w.
Bone marrow polychromatic erythrocytes in rats 100, 200, 300 mg/kg b.w. + (174)
Micronucleus in vitro, Human peripheral blood lymphocytes (rat, liver, S9) 0.2, 2, 20 µg/l + (175)
Micronuclei test in human peripheral lymphocytes SCE test in human peripheral lymphocytes 0.1, 0.5 mg/l + (176)
Comet assay, DNA damage, SCGE 0.05, 0.1, 0.2, 0.5 mg/l + (176)
Micronucleus (MN) formation in human lymphocytes in vitro 50 µg/l + (174)
SCE induction in human lymphocytes Combination with metalaxyl at 100, 200 μg/l + (174)
SCE induction in human lymphocytes 0.1, 1, 5, 10, 50, 100 µg/l − (174)
Micronucleus in the rat bone marrow 200, 300, 400 mg/kg b.w. + (174)
DNA damage, Comet assay, SCGE + (177)
Micronucleus (MN) tests on Hypsiboas pulchellus tadpoles 25 mg/l for 96 h + (178)
DNA single-strand breaks on Hypsiboas pulchellus tadpoles 37.5 mg/l for 96 h + (178)
Nuclear abnormalities 12.5–37.5 mg/l − (178)
Chromosome abnormality on sperm deformity of the earthworm 0.2 mg/kg dry soil + (179)
DNA damage in human peripheral blood lymphocytes exposed in vitro + (180)
Long-term carcinogenesis assay. Rat (male) 0, 100, 300, 900, 1,800 mg/l + (181)
Long-term carcinogenesis assay. Mice 0, 100, 330, 1,000, 2,000 mg/l − (181)
31. Ivermectin (70288-86-7)
Carcinogenicity studies, rats 0, 2 mg/l in diet for 1 year − (182)
32. Lindane (58-89-9)
Salmonella typhimurium, Serratia marcescens, forward and reverse gene mutation, host-mediated assay NC (128)
MN-forming activity in MCF-7 and PC-3 cells 10−12, 2 × 10−12, 10−11, 2 × 10−11, 5 × 10−11 g/ml + (183)
Chromosomal aberrations in human peripheral lymphocytes in vitro + (184)
Micronucleus (MN) formation in bone marrow in vivo + (185)
Sex-linked recessive lethal gene mutation, Drosophila melanogaster NC (140)
Chromosome aberrations, Allium cepa + (136)
Chromosome aberrations, Hordeum vulgare (barley) + (186)
Chromosome aberrations, Vicia faba + (187)
Chromosome aberrations, Tradescantia species + (187)
Mitotic recombination or gene conversion, Saccharomyces cerevisiae + (44)
DNA damage and the risk for cancer on human tonsillar 0.5, 0.75, 1.0 mg/ml + (188)
Carcinogenicity studies in mouse 12.5, 25 and 50 ppm for 80 weeks − (189)
Carcinogenicity studies in vivo, non-human + (141)
Long-term carcinogenesis assay. AVy/AVy, AVy/a, A/a mouse 160 mg/kg/day + (190)
Long-term carcinogenesis assay. Rats 0, 0.05, 0.45, 4.5, 18.7 mg/kg/day (male)0, 0.06, 0.57, 5.6, 23.1 mg/kg/day (female) − (54)
33. Malathion (121-75-5)
Salmonella typhimurium, TA98, TA100, TA97A, TA102, TA1535, TA1537, reverse mutation 33–1,650, 80–400 mg/l − (191, 117)
E. coli WP2 uvra, tryptophan reverse gene mutation − (101)
Recombination assay, spot test, DNA effect (bacterial DNA repair) NR (79)
SCE, non-human, V79 cells in vitro NR + (119)
SCE, human somatic cells in vitro NR + (120)
Chromosome aberrations NR + (186)
Micronuclei in bone marrow in vivo (mice) 2.5, 5, 10 mg/kg i.p. or p.o. + (157)
Chromosomal aberrations, mouse (injection) 400 mg/kg b.w. + (192)
Chromosomal aberrations, mouse (oral) bone marrow cells in vivo 240 mg/l for 4 or 8 weeks, 120 mg/l for 8 weeks
Chromosomal aberrations, CHO cells in vivo 25, 50, 76 µg/l − (193)
Chromosomal aberrations (rat, liver S-9, aroclor1254), CHO cells in vivo 303, 352,402 µg/l + (193)
Drosophila melanogaster, sex-linked recessive lethal mutation − (140)
UDS, human diploid fibroblasts in vitro − (66)
Histidine reverse gene mutation, Ames assay − (146)
Mitotic recombination or gene conversion − (44)
Micronucleus test, chromosome aberrations − (194)
Micronucleus test, mice(oral) bone marrow cells in vivo 120, 240 mg/l in diet for 2 weeks + (192)
Micronucleus test, mice (injection) bone marrow cells in vivo 200, 300 mg/kg b.w. (192)
Micronucleus test, human peripheral lymphocytes in vivo 20, 50, 75, 100 µg/l + (195)
Micronucleus test, rat peripheral blood lymphocytes in vivo 0, 25, 50, 100, 150 mg/kg b.w. − (196)
Micronucleus test, rat peripheral blood polychromatic and normochromatic erythrocytes in vivo 150 mg/kg b.w. + (196)
Long-term carcinogenesis assay. Rats 0, 2,000, 4,000 mg/l in diet − (84, 85)
Long-term carcinogenesis assay. Mice 0, 8,000, 16,000 mg/l in diet − (122, 123)
Carcinogenicity studies in vivo − (141)
34. Mebendazole (31431-39-7)
Salmonella typhimurium (rat, liver, S-9, aroclor 1254), TA100, TA98 0.5–5, 0.5–5 µg/plate − (165)
0.01–10 µg/plate  + +
Salmonella typhimurium (none), TA100, TA98 0.01–10 µg/plate − (165)
Forward and reverse gene mutation, body fluid assay, Salmonella typhimurium, host-mediated assay + (197)
Genotoxicity in a diploid mitotic recombination or gene mutation; genotoxicity in a haploid yeast reversion assay; gene conversion assay (strain D5 of Saccharomyces cerevisiae) − (198)
35. Mefloquine (53230-10-7)
Bacterial mutation (Ames) − (47)
Cytogenetics in vivo − (47)
Long-term carcinogenesis assay, mice 30 mg/kg/day − (75, 199)
36. Metronidazole (443-48-1)
Salmonella typhimurium, forward and reversegene mutation, host-mediated assay + (197)
Salmonella typhimurium, forward and reverse gene mutation, body fluid assay + (200)
Salmonella typhimurium, TA98, TA100, TA1535, TA1537, and TA1538 + (201)
Salmonella typhimurium, TA100 25–1,000 µg/plate + (202)
300 µg/plate + (203)
50–200 µg/plate + (147)
1–66 µg/plate + (204)
50–12,800 µg/plate + (205)
Salmonella typhimurium, TA97, TA100, TA102, TA98 50–200 µg/plate + (147)
Salmonella typhimurium, TA1538, TA1537, TA100, TA98, TA1535 − (206)
E. coli, none 0.01–0.5 mg/ml + (101)
25–1,000 µg/l + (207)
E. coli (rat, liver, S-9, Aroclor 1254) 25–1,000 µg/l, 25–500 µg/l − (207)
E. coli WP2 uvra, Tryptophan reverse gene mutation With dose response + (101)
Comet assay in human lymphocytes 292.1 mg/ml + (148)
Chromosome aberration (CA) in vivo 10, 20, 40 mg/kg b.w. + (208)
Micronucleus (MN) in the bone marrow cells of Balb/c mice in vivo 10, 20, 40 mg/kg b.w. + (208)
SCE in vivo, non-human NC (209)
SCE in vitro, human lymphocytes With dose response + (210)
Micronucleus in vivo, bone marrow polychromatic lymphocytes 23, 70, 160 mg/kg b.w. + (211)
Mitotic recombination or gene conversion, Saccharomyces cerevisiae − (44)
Micronucleus test in vivo, chromosome aberrations, mammalian polychromatic erythrocytes − (43)
Chromosomal aberrations in vitro, human lymphocytes 0.1, 1, 10, 50 µg/l + (119)
Forward gene mutation, Neurospora crassa + (168)
Aneuploidy, chromosome aberrations, Neurospora crassa − (168)
Neurospora crassa, human NC (45, 46)
Sex-linked recessive lethal gene mutation, Drosophila melanogaster NC (140)
Carcinogenicity studies in vivo, non-human + (141)
UDS and cytogenetics in vitro + (47)
Carcinogenicity studies, mouse + (212)
Carcinogenicity studies, rat + (212)
Tumor promotion studies, mouse (213)
37. Niclosamide (50-65-7)
Salmonella typhimurium (none), TA1978, UTH8413, TA1538, TA98; Salmonella typhimurium (rat, liver, S-9, aroclor 1254), TA1978, UTH8413, TA1538, TA98 1–50 µg/plate − (74)
Salmonella typhimurium (rat, liver, S-9, aroclor 1254), TA98 (NR), YG1020, YG1021, YG1024 0.5–15 µg/plate − (74)
0.5–20 µg/plate + (214)
SCE in vitro, Human lymphocytes + (215)
38. Nitroscanate (19881-18-6)
Salmonella typhimurium (none), TA98, TA98(NR), TA98(1,8-Dnp6), TA100, TA100(NR), YG1024, YG1021, TA98, TA98(1,8-Dnp6), TA100 1–160 µg/plate + (216)
20–160 µg/plate −
20–320 µg/plate +
10–80 µg/plate +
10–80 µg/plate −
0–9 µg/plate +
0–40 µg/plate +
10–320 µg/plate +
Salmonella typhimurium (rat, liver, S-9, aroclor 1254), TA98, TA98(NR), TA100, TA100(NR), TA98, TA98(1,8-Dnp6), TA100 10–160 µg/plate + (216)
10–160 µg/plate −
10–80 µg/plate +
10–160 µg/plate − (216)
10–320 µg/plate +
39. Nitroxinil (1689-89-0)
Salmonella typhimurium (rat, liver, S-9, kanechlor 400), TA100, TA98, TA1535, TA1537, TA1538; Salmonella typhimurium (none), TA100, TA98, TA1535, TA1537, TA1538 0.05–5 mg/plate − (61)
Chromosomal aberrations in vivo, mouse bone marrow cells 0, 10, 20, 30, 40 mg/kg once + (80)
Salmonella typhimurium (none), TA1537 0–1,000 µg/plate − (56)
Micronucleus test in vivo, chromosome aberrations, mammalian polychromatic erythrocytes − (61)
40. Oxfendazole (53716-50-0)
Chromosomal aberrations in vivo, spermatocytes and bone marrow cells 1,000 µg/kg + (217)
41. Pentamidine (100-33-4)
Salmonella typhimurium, TA98, TA100, reverse mutation; Salmonella typhimurium, TA98, TA100 (rat, liver S-9, Phenobarbital), reverse mutation 0.01–1 µmol/plate − (218)
42. Permethrin (52645-53-1)
Salmonella typhimurium, TA98, TA100 100–3,000 µg/plate − (107)
Salmonella typhimurium, TA98, TA100 5–1,000 µg/plate − (219)
Salmonella typhimurium, TA98, TA100 1–20 mg/plate − (220)
Salmonella typhimurium, TA98, TA100, TA97A 39–2,730 mg/l − (191)
Salmonella typhimurium, TA1535, TA1537, TA98, TA100, E. coli 1–7,500 µg/plate − (62)
Chinese hamster V79, rat hepatocytes 4–40 µg/l − (107)
UDS in vitro, DNA effects, human diploid fibroblasts − (66)
Mitotic recombination or gene conversion, Saccharomyces cerevisiae − (44)
43. Piperazine (110-85-0)
Salmonella typhimurium (none), TA100, TA1535, TA1537, TA98, TA100; Salmonella typhimurium (rat, liver, S-9, aroclor 1254), TA100, TA1535, TA1537, TA98; Salmonella typhimurium (hamster, liver, S-9, aroclor 1254), TA100, TA1535, TA1537, TA98 33–2,167 µg/plate − (221)
Salmonella typhimurium (rat, liver, S-9, aroclor 1254), TA100, TA1535, TA1537, TA98
Salmonella typhimurium (hamster, liver, S-9, aroclor 1254), TA100, TA1535, TA1537, TA98
Salmonella typhimurium (rat, liver, S-9, PCB), TA100, TA98 (222)
44. Praziquantel (55268-74-1)
Salmonella typhimurium (TA98, TA100, TA1535, TA1537, TA1538) − (223)
Salmonella typhimurium (none), TA1537, TA1535, TA100, TA1538, TA98; Salmonella typhimurium (rat, liver, S-9, kanechlor 400), TA1537, TA1535, TA100, TA1538, TA98 0–1,000 µg/plate − (56)
Salmonella typhimurium, forward and reverse gene mutation, host-mediated assay + (197)
Forward and reverse gene mutation, body fluid assay, Salmonella typhimurium NC (224)
Forward gene mutation, Schizo saccharomyces pombe − (225)
Sex-linked recessive lethal gene mutation, Drosophila melanogaster − (140)
Mitotic recombination or gene conversion, Saccharomyces cerevisiae − (44)
Dominant lethal test, rodents − (226)
Carcinogenicity studies, Hamster 0, 300 mg/kg in corn oil for 40 weeks − (227)
45. Pyrimethamine (58-14-10)
Bacterial mutation (Ames) − (47)
Micronucleus test (MN) bone marrow in mice in vivo 40 mg/kg b.w. + (228)
The transplacental MN test in mice in vivo 40 mg/kg b.w. − (228)
Cytogenetics in vitro + (47)
DNA damage on ICR mice (oral) 50 mg/kg b.w. + (229)
Embryonic and maternal genotoxicity 50 mg/kg b.w. + (229)
Cytogenetics in vivo + (47)
DNA damage, SCGE, Comet assay in mice and rats 50, 120 mg/kg b.w., respectively + (230)
MLA + (47)
Micronucleus assay in vitro, cultured human lymphocytes − (231)
Long-term carcinogenesis assay, B6C3F1 mice (female) 1,000 mg/l in diet − (75, 122, 123)
Long-term carcinogenesis assay, mice (lung tumors) 25 mg/kg i.p. + (75, 122, 123)
Long-term carcinogenesis assay, F344 rats 400 mg/l in diet − (75, 122, 123)
46. Quinine (130-95-0)
Salmonella typhimurium, TA98, TA100, reverse mutation, Salmonella typhimurium, TA98, TA100 (rat, liver S-9) 20–50 µg/plate − (78)
47. RH-5849 (112225-87-3)
Salmonella typhimurium, TA98, TA100, TA97A, TA102, TA100 reverse mutation 5, 50, 500, 5,000 µg/plate − (232)
Micronuclei test in mouse bone marrow in vivo 42, 84, 168 mg/kg b.w. − (232)
Chromosome aberration, primary spermatocytes of testis 50, 100, 200 mg/kg/d for 5days − (232)
Micronuclei test in human peripheral lymphocytes SCE test in human peripheral lymphocytes 25, 100 mg/l + (176)
Comet assay, DNA damage, SCGE 5, 25, 50, 100 mg/l + (176)
Chromosome abnormality on sperm deformity of the earthworm 100 mg/kg dry soil + (179)
Micronucleus(MN) test in human lymphocytes in vitro, Micronucleus(MN) test in rat bone marrow in vivo 50 mg/ml + (174)
300 mg/kg b.w.
SCE in human lymphocytes 100, 200 mg/ml + (174)
DNA strand breaks and DNA damage + (177)
Micronucleus(MN) test in mouse 23, 45, 90 mg/kg b.w. − (172)
Chromosome aberration Primary spermatocytes of testis 38, 75, 150 mg/kg b.w. − (172)
48. Tetramethrin (7696-12-0)
Salmonella typhimurium, TA98; Salmonella typhimurium, TA98 (rat, liver S-9, polychlorinated biphenyl) 0.1–1 mg/plate + (220)
Salmonella typhimurium, TA100; Salmonella typhimurium, TA100 (rat, liver S-9, polychlorinated biphenyl) 0.1–1 mg/plate +
Salmonella typhimurium, TA98, TA100; Salmonella typhimurium, TA98, TA100 (S9) 5–1,000 µg/plate − (219)
49. Thiophanate (23564-05-8)
Salmonella typhimurium (none), TA100, TA98, TA1535, TA1537, TA97 33–10,000 µg/plate − (204)
Salmonella typhimurium [hamster, liver, S-9, aroclor 1254 (10% or 30%)], TA100, TA1535, TA97, TA98, TA100; Salmonella typhimurium [liver, S-9, aroclor 1254 (10 or 30%)], TA100, TA1535, TA97, TA98, TA1537 100–10,000 µg/plate − (204)
Chromosome aberrations, aneuploidy, Aspergillus nidulans + (233)
Chromosome aberrations in vivo, mammalian germ cells − (40)
50. Tiabendazole (148-79-8)
Salmonella Typhimurium (none), TA100, TA98; Salmonella typhimurium (hamster, liver, S-9, aroclor 1254, 30%), TA100, TA98; Salmonella typhimurium (rat, liver, S-9, aroclor 1254, 30%), TA100, TA98 100–10,000 µg/plate − (60)
Salmonella typhimurium (hamster, liver, S-9, aroclor 1254, 10%), TA98 100–10,000 µg/plate + (60)
Salmonella typhimurium (none), TA98, TA100, TA97, TA104, E. coli, WP2S/PKM101 50–400 µg/l + (234)
Micronucleus test in vivo, chromosome aberrations, mammalian polychromatic erythrocytes + (235)
Mitotic recombination, Aspergillus nidulans NC (233)
Chromosome aberrations, Aspergillus nidulans, aneuploidy + (233)
Micronucleus (none) in vitro, V79 cells 0.5–700 µg/l + (236)
Micronucleus (none) in vitro, human lymphoblastoid wtk1 cells 0, 50, 100, 200 µg/l + (234)
Carcinogenicity studies, mouse 0, 0.8, 1.2, 1.6% in diet for 44 weeks − (237)
0, 0.031, 0.125, 0.5% in diet for 78 weeks
Long-term carcinogenesis assay, rats 0, 0.05, 0.1, 0.2, 0.4% in diet for 104 weeks − (238)
51. Tinidazole (19387-91-8)
Salmonella typhimurium, TA100, reverse mutation 10–100 µg/plate + (239)
Salmonella typhimurium, TA100 (rat, liver S-9, aroclor 1254), reverse mutation 10–100 µg/plate − (239)
Salmonella typhimurium, TA98, reverse mutation 10–100 µg/plate + (239)
Salmonella typhimurium, TA98 (rat, liver S-9, aroclor 1254), reverse mutation 10–800 µg/plate + (205)
Salmonella typhimurium, UTH8414, reverse mutation 50–12,800 nmol/plate +
Salmonella typhimurium, TA98, TA100, reverse mutation 50–3,200 nmol/plate + +
Salmonella typhimurium, TA100, reverse mutation
Salmonella typhimurium, TA100(1,8-DNP6),YG1029, TA100 (NR), reverse mutation
Salmonella typhimurium, TA100 (NR), TA100 (rat, liver S-9, aroclor 1254), reverse mutation
52. Triclabendazole (68786-66-3)
Chromosomal aberrations in vitro, river buffalo lymphocytes 25, 50, 100 µg/l + (239)
Micronucleus in vitro, river buffalo Lymphocytes, micronucleus formation in lymphocyte cultures of the river buffalo 25, 50, 100 µg/l + (239)
SCEs in lymphocyte cultures of the river buffalo 25, 50, 100 µg/ml

The name of each drug is followed by the CAS number. For each type of assay: “+,” positive response; “−,” negative response; NR, not reported; NT, not tested; p.o., oral; i.p., intraperitoneal; UDS, DNA repair synthesis; MLA, gene mutation, mouse lymphoma L5178Y cells, TK locus; HGPRT, gene mutation, hgprt locus; SCE, sister chromatid exchange; MN, micronucleus; Trans., cell transformation;HID, highest ineffective dose; LED, lowest effective dose.

Pharmaceuticals without retrievable data: Amicarbalide, Abamectin, Acetarsone, Amprolium, Arecoline Hydrobromide, Artemether, Artemisinine, Artesunate, Avermectin, Azamethiphos, Amprolium Hydrochloride, Bunamidine, Carbarsone, Chiniofon, Clopidol, Clorsulon, Closantel Sodium, Cyromazine, Destomycin A, Diamphenethide, Diclazuril, Diethylcarbamazine, Diethylcarbamazine, Dihydroartemisinin, Diiodohydroxyquinoline, Diloxanide, Diminazene, Dinitolmide, Dithiazanine Iodide, Doramectin, Emetine, Epsiprantel, Ethopabate, Febantel, Fexinidazole, Fluvalinate, Hainanmycin, Halofuginone, Haloxon, Hetolin, Hexachloroparaxylene, Hydroxychloroquine, Hygromycin B, Imidocarb, Dipropionate, Isometamidium, Levamisole, Lumefantrine, Maduramicin, Malaridine, Metrifonate, Milbemycin Oxime, Monensin Sodium, Morantel, Moxidectin, Naftalofos, Naphthalophos, Nicarbazin, Nitazoxanide, Nitroquine, Oxantel, Oxibendazole, Oxinothiophos, Phanquinone, Phoxim, Piperanitrozole, Piperaquine, Primaquine, Propetamphos, Pyramine, Pyrantel, Quinapyramine, Rafoxanide, Resorantel, Robenidine, Salinomycin, Secnidazole, Semduramicin, Sodium stibogluconate, Sulfaquinoxaline, Sulfur Sublimat, Tetramisole, Thiacetarsamide, and Toltrazuril.

Table 4 summarizes the total number of antiparasitics and the following are included: the number of antiparasitics with at least one genotoxicity or carcinogenicity test result and with data required by the present guidelines; the number of antiparasitics only tested for genotoxicity or carcinogenicity. It also presents the antiparasitics with results in in vitro data required by present guidelines; the number of antiparasitics that have at least one result in long-term carcinogenesis assays in rats or mice; and the number of antiparasitics in genotoxicity assays (bacterial mutagenicity, in vitro tests for gene mutation and for chromosomal damage, in vivo cytogenetic tests, and other types of genotoxicity assays). Of 136 antiparasitics examined, 52 (38.2%) had at least one genotoxicity or carcinogenicity test result, and 32 (23.5%) were tested only for either genotoxicity or carcinogenicity. Among 20 antiparasitics with results available for both genotoxicity and carcinogenicity, 16 had all the results required by the present guidelines for testing of pharmaceuticals: 8 of them—Albendazole, Coumaphos, Cypermethrin, Deltamethrin, Diazinon, Fenvalerate, Malathion and Tiabendazole—tested positive in genotoxicity assays but gave at least one negative result in carcinogenesis assays; 8 antiparasitics (Chlordimeform, Dichlorvos, Fenthion, Fipronil, Lindane, Metronidazole, Pyrimethamine, and Imidacloprid) gave positive responses in both genotoxicity and carcinogenicity. The remaining four with both genotoxicity and carcinogenicity data were not in agreement with the current guidelines: Amitraz and Praziquantel gave positive responses in genotoxicity but were non-carcinogenic; Atovaquone tested negative in genotoxicity but positive in mouse carcinogenicity; and Mefloquine produced negative responses in both genotoxicity and carcinogenicity.

Overview of genotoxicity and carcinogenicity testing of antiparasitics.

Antiparasitics with at least one genotoxicity or carcinogenicity tests results (Table 3) 52 (38.2%)a
Antiparasitics without retrievable genotoxicity or carcinogenicity data 84 (61.8%)
Antiparasitics with all genotoxicity and carcinogenicity data required by present guidelines (Table 3: 2, 10, 13, 15, 17, 19, 20, 24–26, 30, 32, 33, 36, 45, 50)b 16 (11.8%)
Antiparasitics tested not according to present guidelines 36 (26.5%)
Antiparasitics with least one genotoxicity and carcinogenicity test results (Table 3: 2, 3, 7, 10, 13, 15, 17, 19, 20, 24–26, 30, 32, 33, 35, 36, 44, 45, 50) 20 (14.7%)
Antiparasitics tested only for genotoxicity (Table 3: 1, 4–6, 8, 9, 11, 12, 14, 16, 18, 21–23, 27–29, 34, 37–43, 46–49, 51, 52) 31 (22.8%)
Antiparasitics tested only for carcinogenicity (Table 3: 31) 1 (0.7%)
Antiparasitics with at least one results in tests for bacterial mutagenicity (Table 3: 1–11, 13–22, 24–30, 32–39, 41–51) 47 (34.6%)
Antiparasitics with at least one results in tests for gene mutation in mammalian cells (Table 3: 1, 6, 7, 10, 17, 19, 20, 24–26, 28, 32–34, 36, 44, 45, 50) 18 (13.2%)
Antiparasitics with at least one results in in vitro tests for SCE, chromosomal aberrations, aneuploidy, or micronucleus in animal or human cells (Table 3: 6, 7, 9, 10, 13–26, 28, 30, 32, 33, 36, 37, 42, 45, 47, 49, 50, 52) 33 (24.3%)
Antiparasitics with results in in vitro data required by present guidelines (Table 3: 1–3, 13, 17–19, 24–26, 28, 30, 42, 50, 52) 15 (11.0%)
Antiparasitics with at least one results in in vivo tests for SCE, chromosomal aberrations, or micronucleus in animal or human cells (Table 3: 6–20, 24–26, 30, 32, 33, 35, 36, 39, 40, 45, 47, 49, 50) 31 (22.8%)
Antiparasitics which underwent testing for DNA damage or DNA repair synthesis (Table 3: 3, 10, 11, 14–16, 18–21, 24, 25, 30, 32, 33, 36, 42, 45, 47) 19 (14.0%)
Antiparasitics which underwent testing in other types of genotoxicity assays (Table 3: 1, 10, 15, 19, 20, 22, 24, 26, 28, 30, 32–34, 36, 42, 44, 45) 17 (12.5%)
Antiparasitics examined for genotoxicity in human cells (Table 3: 2, 6, 14–16, 19–21,23, 24, 26, 30, 32, 33, 36, 37, 42, 45, 47, 50) 20 (14.7%)
Antiparasitics tested for carcinogenicity in mice (Table 3: 2, 3, 7, 10, 13, 15, 17, 19, 20, 24–26, 30, 32, 33, 35, 36, 45, 50) 19 (14.0%)
Antiparasitics tested for carcinogenicity in rats (Table 3: 2, 3, 7, 10, 13, 15, 17, 19, 20, 24–26, 30–33, 35, 36, 45, 50) 20 (14.7%)
Antiparasitics tested for carcinogenicity in both mice and rats (Table 3: 2, 3, 7, 10, 13, 15, 17, 19, 20, 24–26, 30, 32, 33, 35, 36, 45, 50) 19 (14.0%)
Antiparasitics tested for carcinogenicity in other species (Table 3: 44) 1 (0.7%)

aValues in parentheses indicate the percentage of the 136 antiparasitics considered.

bNumber and percentage in parentheses are those of antiparasitics of Table 3.

Additional 32 antiparasitics were only tested in either genotoxicity or carcinogenicity. Only one (Ivermectin) had retrievable results in carcinogenicity. As for the rest, 31 antiparasitics had the data of genotoxicity. Twenty-one antiparasitics (Acriflavine, Closantel, Chloroquine, Cyfluthrin, Danex, Diaveridine, Dimetridazole, Fenbendazole, Fenchlorphos, Furapyrimidone, Furapromide, Mebendazole, Nitroscanate, Nitroxinil, Niclosamide, Oxfendazole, RH-5849, Tetramethrin, Thiophanate, Tinidazole, and Triclabendazole) gave positive responses in at least one genotoxicity assay; 10 antiparasitics (Amodiaquine, Amoscanate, Amphotericin B, Bithionol, Bromofenofos, Flubendazole, Pentamidine, Permethrin, Piperazine, and Quinine) were found to be negative in all the considered genotoxicity assays. With regard to the different types of genotoxicity assays: there were 47 antiparasitics with at least one result in tests for bacterial mutagenicity; 18 antiparasitics with at least one result in tests for gene mutation in mammalian cells; 33 antiparasitics in in vitro tests for SCE, chromosomal aberrations, aneuploidy, or micronucleus in animal or human cells; 15 antiparasitics with results in in vitro data required by present guidelines; 31 antiparasitics in in vivo tests for SCE, chromosomal aberrations, or micronucleus in animal or human cells; 19 antiparasitics in DNA damage or DNA repair synthesis; 17 antiparasitics in other types of genotoxicity assays; and 20 antiparasitics examined for genotoxicity in human cells. With respect to carcinogenesis assays, 19 and 20 antiparasitics were tested for carcinogenicity in mice and rats, respectively. Among the antiparasitics with both the genotoxicity and carcinogenicity data, 19 antiparasitics tested for carcinogenicity in both mice and rats and only 1 in hamsters.

Table 5 provides the number of antiparasitics tested for each type of assay, including the genotoxicity and carcinogenicity studies. The results are indicated as positive, negative and discordant. When carcinogenicity testing is considered, 57.9% of antiparasitics were tested negative in mice, and 73.7% in rats. Five antiparasitics (nos. 7, 10, 26, 32, and 36) and three antiparasitics (nos. 10, 26, and 36) were carcinogenic in mice and rats, respectively. The percentage of concordant results in carcinogenicity assays between mice and rats is 85.7% (12 out of 14) and only 2 (nos. 7 and 32) antiparasitics have discordant results: no. 32 tested positive in mice and negative in rats, while no. 7 produced the opposite result. The occurrence of discordant results between mice and rats may be the differences in species (e.g., metabolic enzymes). Ten antiparasitics were in IARC of 2B and 3 ground classifications of carcinogens: Chloroquine, Danex, and Permethrin do not have available carcinogenicity data; Deltamethrin, Fenvalerate, and Malathion tested negative in rodents while positive results were given by Chlordimeform and Metronidazole. Dichlorvos (DDVP) and Pyrimethamine have discordant results of carcinogenicity in mice and rats. To interpret the tumor findings in a carcinogenicity study and provide a perspective on the relevance of rodents to human, the mechanism and some investigations in tumor profile (trans-species, trans-sex, and multisite versus single species, single sex, and single site) were suggested by the guidelines (15).

Summary per assays type of antiparasitics with positive, negative, and discordant results.

Bacterial mutagenitity Positive 4 (8.5%) (Table 3: 21, 26, 28, 29)
Negative 29 (61.7%) (Table 3: 2–10, 13–15, 17, 19, 22, 24, 25, 27, 32, 33, 35, 39, 41–47, 49)
Discordant 14 (29.8%) (Table 3: 1, 11, 16, 18, 20, 30, 34, 36–38, 44, 48, 50, 51)
Gene mutation in cultured mammalian cells Positive 7 (38.9%) (Table 3: 1, 20, 26, 28, 32, 45, 50)
Negative 11 (61.1%) (Table 3: 6, 7, 10, 19, 24, 25, 33, 34, 36, 42, 44)
Discordant 0
In vitro cytogenetics Positive 18 (54.5%) (Table 3: 1, 15, 16, 18, 19, 21, 22, 23, 26, 28, 32, 33, 36, 37, 45, 47, 50, 52)
Negative 7 (21.2%) (Table 3: 3, 6, 7, 9, 13, 34, 42)
Discordant 8 (24.2%) (Table 3: 2, 10, 14, 17, 20, 24, 25, 30)
In vivo cytogenetics Positive 13 (41.9%) (Table 3: 11–15, 17, 19, 24–26, 32, 40, 50)
Negative 8 (25.8%) (Table 3: 6–9, 18, 20, 35, 49)
Discordant 10 (32.3%) (Table 3: 1, 2, 10, 16, 30, 33, 36, 39, 45, 47)
DNA lesions (in vitro and in vivo) Positive 9 (47.4%) (Table 3: 3, 14, 16, 21, 30, 32, 36, 45, 47)
Negative 3 (15.8%) (Table 3: 25, 33, 42)
Discordant 7 (36.8%) (Table 3: 10, 15, 18–20, 24, 26)
Carcinogenesis in mice Positive 5 (26.3%) (Table 3: 7, 10, 26, 32, 36)
Negative 11 (57.9%) (Table 3: 2, 3, 13, 15, 17, 19, 22, 25, 30, 33, 35)
Discordant 3 (15.8%) (Table 3: 20, 24, 45)
Carcinogenesis in rats Positive 3 (15.8%) (Table 3: 10, 26, 36)
Negative 14 (73.7%) (Table 3: 2, 3, 7, 13, 15, 17, 24, 25, 31–33, 35, 45, 50)
Discordant 2 (10.5%) (Table 3: 20, 30)
Carcinogenesis in mice and rats Discordant 2 (14.3%) (Table 3: 7, 32)
Carcinogenesis in mice and rats Concordant 12 (85.7%) (Table 3: 2, 3, 10, 13, 15, 17, 25, 26, 33, 35, 36, 50)

The antiparasitic was considered as positive when it gave only positive results and as negative when it gave only negative or inconclusive results. Discordant indicates the number of antiparasitics, of which the results of genotoxicity assays were both positive and negative or inconclusive and he results of carcinogenicity assays performed in the same species were carcinogenic to mice or rats but not to rats or mice. In parentheses is the number of drugs in Table 3.

 Re-Evaluation of <italic>In Vitro</italic> Genotoxicity Results

Table 6 presents the incidence of misleading positive effects in in vitro cytogenicity when using the reduction in a top dose of 1 mM. Of 33 antiparasitics with at least one result in in vitro tests for SCE, chromosomal aberrations, or micronucleus in animal or human cells, 25 (75.8%) antiparasitics had at least one retrievable dose in in vitro cytogenicity assays, while 8 (24.2%) antiparasitics had no available dose. Under the current in vitro genotoxicity testing guidelines for dose limits, 10 (nos. 10, 14, 15, 16, 20, 21, 22, 32, 36, and 47) antiparasitics were identified as genotoxins at dose levels more than 1 mM. The re-evaluation results indicated the misleading positive response in the previous reports. Fifteen (nos. 1, 2, 3, 13, 17, 18, 19, 24, 25, 26, 28, 30, 42, 50, and 52) antiparasitics had in vitro genotoxicity results consistent with ICH S2 (R1).

Re-evaluate the in vitro cytogenetic results according to the ICH S2 (R1).

Test system (in vitro cytogenetic assays) Dose or concentration (LED or HID) Result Conversion unit (mM) ICH S2 (R1), 1 mM Concordant
1. Acriflavine (1) (259.70)
CHO, CHO-K1-BH4 (HGPRT) 0.5–4 µg/l + 1.54 × 10−5 Y
2. Albendazole (2) (265.33)
MN, peripheral blood lymphocytes 10–100 µg/ml + 0.377 Y
MN, human lymphocytes 10–100 µg/ml + 0.377 Y
3. Amitraz (3) (293.23)
DNA damage on hamster cells, comet assay 3.75 µg/l + 1.28 × 10−5 Y
4. Chlordimeform (10) (196.68)
DNA effects (human diploid fibroblasts FL cell) 10−6 to 10−3 g/ml + 5.08 N
5. Coumaphos (13) (362.78)
CA in vitro, CHO cells (rat, liver, S-9, aroclor 1254) 100, 300, 1,000 µg/l − 2.76 × 10−3 Y
CA in vitro, CHO cells (none) 99.5, 299, 995 µg/l − 2.7 × 10−3 Y
6. Cyfluthrin (14) (434.29)
CA, human peripheral blood lymphocytes 1,000, 2,000 mg/ml + 4.61 × 103 N
SCE, human peripheral blood lymphocytes 500, 1,000, 2,000 mg/ml − 4.61 × 103 N
MN, human peripheral blood lymphocytes 500, 1,000, 2,000 mg/ml + 4.61 × 103 N
DNA damage, epithelial cells of human nasal mucosa 0.05, 0.1, 0.5, 0.75, 1.0 mg/ml + 2.303 N
DNA damage and comet assay in fish 5.6 mg/l beta-cyfluthrin for 48 h + 1.29 × 10−2 Y
CA in vitro 500, 1,000, 2,000 µg/l − 4.61 × 10−3 Y
SCE in blood lymphocytes 500, 1,000, 2,000 µg/l − 4.61 × 10−3 Y
Mouse bone marrow cells in vitro 1,000 µg/l + 2.30 × 10−3 Y
7. Cypermethrin (15) (416.32)
CAs, human peripheral lymphocytes 5, 10, 15, 20 mg/ml + 48.0 N
SCE, human peripheral lymphocytes 5, 10, 15, 20 mg/ml + 48.0 N
MN, human peripheral lymphocytes 5, 10 mg/ml + 24.0 N
CA in highly mitotic kidney cells 0.4, 0.8,1.2 µg/l for 48 and 72 h + 2.88 × 10−6 Y
MN, erythrocytes of a freshwater fish 0.4, 0.8,1.2 µg/l for 48 and 72 h + 2.88 × 10−6 Y
Peripheral blood for MN test 20, 30, 40, 50 mg/l + 0.120 Y
8. Danex (16) (257.45)
UDS human cells 0.4–4,000 mmol + 4.0 × 103 N
CA, V79 cell 0.04–0.8 mmol − 8.0 × 102 N
9. Deltamethrin (17) (505.20)
CA, CHO cells in vitro 0, 19, 38, 75, 150 µg/l + 2.97 × 10−4 Y
V79/6-thioguanine, Chinese hamater V79 4–40 µg/l − 7.92 × 10−5 Y
10. Diaveridine (18) (260.29)
CA in cultured CHL cells 12.5, 25, 50, 100 µg/l + 3.84 × 10−4 Y
CA 100 µg/l,48 h + 3.84 × 10−4 Y
11. Diazinon (19) (304.35)
DNA damage, human blood lymphocytes 750 µg/l + 2.46 × 10−3 Y
12. Dichlorvos(DDVP) (20) (220.98)
CA in vitro, CHO cells 16, 50, 100, 160 µg/l + 7.24 × 10−4 Y
50, 160, 500, 1,600 µg/l + 7.24 × 10−3 Y
500, 750, 1,000 µg/l + 4.53 × 10−3 Y
CA, V79 1.25–5 µg/l − 2.26 × 10−5 Y
CHO, CHO-k1-bh4 (HGPRT)/6-thioguanine 50–150 µg/l + 6.79 × 10−4 Y
Mouse lymphoma, L5178Y (TK+/TK−) 0–0.33 µg/l, 0–0.12 µg/l, + 1.49 × 10−6 Y
0–0.24 µg/ml + 1.09 × 10−3 Y
Mouse lymphoma, L5178Y (TK+/TK−) 6.25–200 µg/l + 9.05 × 10−4 Y
UDS human cells 6.5–650 mg/ml + 2.94 × 103 N
UDS rat hepatocytes 0.005–1.25 mg/ml − 5.66 N
13. Dimetridazole (21) (141.12)
Comet assay, human lymphocytes 354.3 mg/ml + 2.51 × 103 N
14. Fenbendazole (22) (299.34)
Chromosomal damage in CHL cells 0.78 mg/ml + 2.61 N
Cytotoxicity to 10T1/2 cells 0.04–1.60 mg/ml + 5.35 N
Morphological transformation in mouse embryo fibroblasts 0.08–0.4 mg/ml + 1.34 N
15. Fenthion (24) (278.33)
UDS, thymidine incorporation, rat hepatocytes 0, 5.0, 7.5, 10.0, 15.0, 30.0 µg/l + 1.08 × 10−4 Y
CA, CHO cells in vitro 0, 0.02, 0.04, 0.08, 0.15 µg/l − 5.39 × 10−7 Y
CA, human peripheral lymphocytes 0.5, 1.5, 2.5, 5.0 µg/ml + 1.80 × 10−2 Y
16. Fenvalerate (25) (419.90)
Peripheral blood for MN test 25, 50, 75, 100 mg/l + 0.238 Y
Chinese hamster V79 gene mutation 4–40 µg/l − 9.53 × 10−5 Y
CA, CHO-K1, in vitro 10, 25, 50, 100,150 µg/l + 3.57 × 10−4 Y
CA, CHO-K1, in vitro 5, 10, 25, 50 µg/l + 1.19 × 10−4 Y
17. Fipronil (26) (437.20)
CA, human lymphocytes in vitro 0, 4.69, 9.38, 18.75, 37.5, 75, 150, 300 µg/l + 6.86 × 10−4 Y
SCEs, DNA damage, comet assay 0.3,0.7 µg/l + 1.60 × 10−6 Y
MN, human peripheral blood lymphocytes 0.3, 0.7 µg/l + 1.60 × 10−6 Y
Comet assay with gillsin, the fish Rhamdia quelen 0.05, 0.10, 0.23 µg/l − 5.26 × 10−7 Y
Nuclear morphological alterations 0.05, 0.10, 0.23 µg/l − 5.26 × 10−7 Y
CA, V79 cells, HGPRT mutations 0, 0.8, 4, 20, 100, 500 µg/l + 1.14 × 10−3 Y
18. Furapromide (28) (224.22)
CA, V79 cell 10–120 µmol + 1.20 × 10−4 Y
19. Imidacloprid (30) (255.70)
MN, human peripheral blood lymphocytes 0.2, 2, 20 µg/l + 7.82 × 10−5 Y
MN, human peripheral lymphocytes 0.1, 0.5 mg/l + 1.96 × 10−3 Y
SCE, human peripheral lymphocytes 0.1, 0.5 mg/l + 1.96 × 10−3 Y
Comet assay, DNA damage, SCGE 0.05, 0.1, 0.2, 0.5 mg/l + 1.96 × 10−3 Y
MN, Human lymphocytes in vitro 50 µg/l + 1.96 × 10−4 Y
SCE in human lymphocytes Combination with metalaxyl at 100, 200 µg/l + 7.82 × 10−4 Y
SCE induction in human lymphocytes 0.1, 1, 5, 10, 50, 100 µg/l − 3.91 × 10−4 Y
20. Lindane (32) (290.82)
Comet-forming activity in MCF-7 cells 10−4 g/ml + 3.44 N
DNA damage and the risk for cancer on human tonsillar 0.5, 0.75, 1.0 mg/ml + 34.4 N
21. Metronidazole (36) (171.16)
Comet assay in human lymphocytes 292.1 mg/ml + 1.71 × 103 N
CA in vitro, human lymphocytes 0.1, 1, 10, 50 µg/l + 2.92 × 10−4 Y
22. Permethrin (42) (391.28)
Chinese hamster V79, rat hepatocytes 4–40 µg/l − 1.02 × 10−4 Y
23. RH-5849 (47) (296.40)
MN, human peripheral lymphocytes 25, 100 mg/l + 0.337 Y
SCE, human peripheral lymphocytes 25, 100 mg/l + 0.337 Y
Comet assay, DNA damage, SCGE 5, 25, 50, 100 mg/l + 0.337 Y
MN, human lymphocytes in vitro 50 mg/ml + 1.69 × 102 N
SCE, human lymphocytes 100, 200 mg/ml + 6.75 × 102 N
24. Tiabendazole (50) (210.19)
MN (none) in vitro, V79 cells 0.5–700 µg/l + 3.33 × 10−3 Y
MN, human lymphoblastoid wtk1 cells 0, 50, 100, 200 µg/l + 9.52 × 10−4 Y
25. Triclabendazole (52) (359.66)
CA in vitro, river buffalo lymphocytes 25, 50, 100 µg/l + 2.78 × 10−4 Y
MN in vitro, river buffalo lymphocytes 25, 50, 100 µg/l + 2.78 × 10−4 Y
CA in lymphocyte 25, 50, 100 µg/l + 2.78 × 10−4 Y
SCEs in lymphocyte 25, 50, 100 µg/ml + 0.278 Y
MN in lymphocyte 25, 50, 100 µg/ml + 0.278 Y

The name of each antiparasitic is followed by the number in the Table 1 and molecular weight. For each type of assay: “+,” positive response; “−,” negative response; “Y,” consistent with results of the current guideline of ICH S2 (R1); “N,” discordant with results of the current guideline of ICH S2 (R1); UDS, DNA repair synthesis; MN, micronucleus; MLA, gene mutation, mouse lymphoma L5178Y cells, TK locus; HGPRT, gene mutation, hgprt locus; SCE, sister chromatid exchange; Trans., cell transformation; HID, highest ineffective dose; LED, lowest effective dose; CHO, Chinese hamster ovary; CHL, Chinese hamster lung. Pharmaceuticals with in vitro cytogenetic results but without the retrievable dose: Amphotericin B, Atovaquone, Bromofenofos, Fenchlorphos, Malathion, Niclosamide, Pyrimethamine, Thiophanate.

 Correlation between the Genotoxicity Assays

Table 7 provides the correlation among the different types of genotoxicity assays of antiparasitics, the numbers and percentages of antiparasitics that tested concordant and discordant between each other. On the whole, the degree of coincident correlation was higher than the discordant results, which ranged from 84.6% between bacterial mutagenicity and gene mutation in mammalian cells to 55.6% between gene mutation in mammalian cells and in vivo cytogenetics. When bacterial mutagenicity was compared with the following assays: gene mutation in mammalian cells, in vitro cytogenetics, in vivo cytogenetics and DNA lesions, 13 (nos. 3, 14, 15, 17, 19, 22, 24, 25, 32, 33, 45, 47, and 49) antiparasitics gave negative results in bacterial mutagenicity. Among these antiparasitics, there were 2 (nos. 32 and 45), 8 (nos. 15, 22, 24, 25, 32, 33, 47, and 49), 7 (nos. 14, 15, 17, 19, 24, 25, and 32) and 5 (nos. 3, 14, 32, 45, and 47) antiparasitics that tested positive in gene mutation in mammalian cells, in vitro cytogenetics, in vivo cytogenetics and DNA lesions, respectively.

Correlation between the results of genotoxicity assays of antiparasitics.

Couples of assays considered No. of drugs with
Concordant results Discordant results
Bacterial mutagenicity—gene mutation in mammalian cells 11 (84.6%) (6, 7, 10, 19, 20, 24–26, 28, 33, 42) 2 (16.7%) (32, 45)
Bacterial mutagenicity—in vitro cytogenetics 12 (60.0%) (3, 6, 7, 10, 13, 16, 20, 26, 28, 30, 36, 42) 8 (40.0%) (15, 19, 22, 25, 32, 33, 47, 49)
Bacterial mutagenicity—in vivo cytogenetics 11 (57.9%) (1, 6–9, 16, 26, 30, 35, 36, 49) 8 (42.1%) (13–15, 17, 19, 24, 25, 32)
Bacterial mutagenicity—DNA lesions 7 (58.3%) (16, 18, 20, 21, 25, 33, 42) 5 (41.7%) (3, 14, 32, 45, 47)
Gene mutation in mammalian cells—in vitro cytogenetics 9 (75.0%) (1, 6, 7, 10, 26, 28, 32, 42, 50) 3 (25.0%) (19, 25, 33)
Gene mutation in mammalian cells—in vivo cytogenetics 5 (55.6%) (6, 7, 26, 32, 50) 4 (44.4%) (19, 20, 24, 25)
Gene mutation in mammalian cells—DNA lesions 5 (83.3%) (25, 32, 33, 42, 45) 1 (16.7%) (36)
in vitro cytogenetics—in vivo cytogenetics 13 (81.2%) (2, 6, 7, 15, 16, 19, 25, 26, 30, 32, 36, 45, 50) 3 (18.8%) (13, 18, 49)
DNA lesions—in vitro cytogenetics 6 (66.7%) (16, 20, 24, 32, 42, 47) 3 (33.3%) (3, 25, 33)
DNA lesions—in vivo cytogenetics 4 (80.0%) (10, 14, 16, 32) 1 (20.0%) (25)

In these comparisons, the drug gave only positive result (s) or only negative or inconclusive result (s) in the considered assays. In parentheses are indicated the number and corresponding percentages, as well as the numbers of Table 3.

The highly consistent correlation between bacterial mutagenicity and gene mutation in mammalian cells indicated that the same genetic end point tests might have the high consistency. The discordance (nos. 32 and 45) may be due to the xenobiotic metabolism in the liver and other organs between the bacteria and animals. With the comparison between in vitro cytogenetics and in vivo cytogenetics, 2 (nos. 18 and 49) antiparasitics gave positive responses in in vitro cytogenetics while no. 13 gave negative. These results were inconsistent with that in in vivo cytogenetics. With regard to the discordant results between DNA lesions and in vitro cytogenetics of the three (nos. 3, 19 and 33) antiparasitics, two (nos. 19 and 33) antiparasitics tested negative and no. 3 yield positive in DNA lesions, respectively. These results were opposite to that in in vitro cytogenetics.

 A Novel Strategy for Predicting Carcinogenicity Based on the Genotoxicity Assays

Antiparasitics with both genotoxicity and carcinogenicity data are reported in Table 8 to analyze the correlation between the results of the various types of genotoxicity and carcinogenicity. The results are marked positive or negative or inconclusive. It is obvious that the concordant and discordant results occurred in all the 15 pairs of assays considered. When carcinogenicity in mice or rats was considered, the percentage of discordant results ranged from 71.4% between in vivo cytogenetics and carcinogenicity in both mice and rats to 10.0% between bacterial mutagenicity and carcinogenicity in both mice and rats. The rank order of the consistency between genotoxicity and carcinogenicity was bacterial mutagenicity > DNA lesions > in vitro cytogenetics > gene mutation in mammalian cells > in vivo cytogenetics.

Correlation between the multiple genotoxicity and carcinogenicity in mice and rats assays of antiparasitics.

Couples of assays considered No. of antiparasitics with
Concordant results Discordant results
Bacterial mutagenicity—carcinogenicity in mice 11 (78.6%) (2, 3, 13, 15, 17, 19, 20, 25, 26, 33, 35) 3 (21.4%) (7, 10, 32)
Bacterial mutagenicity—carcinogenicity in rats 15 (93.75%) (2, 3, 7, 13, 15, 17, 20, 24–26, 30, 32, 33, 35, 45) 1 (6.25%) (10)
Bacterial mutagenicity—carcinogenicity in both mice and rats 9 (90.0%) (2, 3, 13, 15, 17, 20, 25, 33, 35) 1 (10.0%) (10)
Gene mutation in mammalian cells—carcinogenicity in mice 5 (55.6%) (19, 25, 26, 32, 33) 4 (44.4%) (7, 10, 36, 50)
Gene mutation in mammalian cells—carcinogenicity in rats 5 (50.0%) (7, 24, 25, 26, 33) 5 (50.0%) (10, 32, 36, 45, 50)
Gene mutation in mammalian cells—carcinogenicity in both mice and rats 3 (50.0%) (25, 26, 33) 3 (50.0%) (10, 36, 50)
In vitro cytogenetics—carcinogenicity in mice 7 (53.8%) (3, 13, 20, 24, 26, 32, 45) 6 (46.2%) (7, 15, 19, 25, 33, 50)
In vitro cytogenetics—carcinogenicity in rats 7 (58.3%) (3, 7, 13, 19, 20, 26, 30) 5 (41.7%) (15, 25, 32, 33, 50)
In vitro cytogenetics—carcinogenicity in both mice and rats 4 (50.0%) (3, 13, 20, 26) 4 (50.0%) (15, 25, 33, 50)
In vivo cytogenetics—carcinogenicity in mice 4 (36.4%) (26, 32, 35, 45) 7 (63.6%) (7, 13, 15, 17, 19, 25, 50)
In vivo cytogenetics—carcinogenicity in rats 5 (41.7%) (7, 19, 26, 30, 35) 7 (58.3%) (13, 15, 17, 24, 25, 32, 50)
In vivo cytogenetics—carcinogenicity in both mice and rats 2 (28.6%) (26, 35) 5 (71.4%) (13, 15, 17, 25, 50)
DNA lesions—carcinogenicity in mice 6 (75.0%) (20, 24, 25, 32, 33, 36) 2 (25.0%) (3, 30)
DNA lesions—carcinogenicity in rats 4 (57.1%) (20, 25, 33, 36) 3 (42.9%) (3, 32, 45)
DNA lesions—carcinogenicity in both mice and rats 4 (80.0%) (20, 25, 33, 36) 1 (20.0%) (3)

In these comparisons, the antiparasitics gave only positive results or only negative or inconclusive results in genotoxicity assay and tested positive in at least one sex of mice or rats or gave negative or inconclusive results in both species in carcinogenicity assays. The following indicated the number and corresponding percentages, as well as the numbers of drugs of Table 3.

Table 9 showed 2 types and 10 combinations of gene-tox assays based on bacterial mutagenicity to indicate the predictivity for rodent carcinogenicity. The sequence of the predictivity was (Ames–DNA lesions) = (Ames–DNA lesions–in vitro) = (Ames–DNA lesions–gene mutation in mammalian cells) = (Ames–In vivo–DNA) > (Ames–in vitro) = (Ames–in vivo) > (Ames–gene mutation in mammalian cells) > (Ames–in vivo–in vitro) > (Ames–gene mutation in mammalian cells–in vivo) = (Ames–gene mutation in mammalian cells–in vitro).

Predictivity of multiple combinations with Ames for rodent carcinogenicity assays of antiparasitics.

Couples of assays considered No. of antiparasitics with concordant results Carcinogenicity
Concordant results Discordant results Without results
Ames–Gene 11 (6, 7, 10, 19, 20, 24–26, 28, 33, 42) 5 (62.5%) (19, 20, 25, 26, 33) 3 (37.5%) (7, 10, 24) 3 (6, 28, 42)
Ames–In vitro 16 (1, 3, 6, 7, 10, 13,16, 18, 20, 26, 28, 30, 36, 37, 42, 50) 6 (66.7%) (3, 13, 20, 26, 30, 36) 3 (33.3%) (7, 10, 50) 7 (1, 6, 16, 18, 28, 37, 42)
Ames–In vivo 13 (1, 6–9, 11, 16, 26, 30, 35, 36, 49, 50) 4 (66.7%) (26, 30, 35, 36) 2 (33.3%) (7, 50) 7 (1, 6, 8, 9, 11, 16, 49)
Ames–DNA 10 (16, 18, 20, 21, 25, 26, 30, 33, 36, 42) 6 (100.0%) (20, 25, 26, 30, 33, 36) 0 4 (16, 18, 21, 42)
Ames–Gene–In vitro 7 (6, 7, 10, 20, 26, 28, 42) 2 (50.0%) (20, 26) 2 (50.0%) (7, 10) 3 (6, 28, 42)
Ames–Gene–In vivo 3 (6, 7, 26) 1 (50.0%) (26) 1 (50.0%) (6) 1 (7)
Ames–Gene–DNA 5 (20, 25, 26, 33, 42) 4 (100.0%) (20, 25, 26, 33) 0 1 (42)
Ames–In vitro–In vivo 8 (1, 6, 7, 16, 26, 30, 36, 50) 3 (60.0%) (26, 30, 36) 2 (40.0%) (7, 50) 3 (1, 6, 16)
Ames–In vitro–DNA 7 (16, 18, 20, 26, 30, 36, 42) 4 (100.0%) (20, 26, 30, 36) 0 3 (16, 18, 42)
Ames–In vivo–DNA 1 (26) 1 (100.0%) (26) 0 0

Ames, bacterial mutagenicity; Gene, gene mutation in mammalian cells; In vitro, in vitro cytogenetics; In vivo, in vivo cytogenetics; DNA, DNA lesions. In these comparisons, all the combinations took the Ames as center. The antiparasitics gave only positive results or only negative or inconclusive results in genotoxicity assay, and tested positive in at least one sex of mice or rats or gave negative or inconclusive results in both species in carcinogenicity assays. The following indicated the number and corresponding percentages, as well as the numbers of antiparasitics of Table 3.

Table 10 presents the number and the percentage of antiparasitics that were classified as non-genotoxic non-carcinogens, genotoxic non-carcinogens, non-genotoxic carcinogens, and genotoxic carcinogens according to the genotoxicity assays considered. An antiparasitic was regarded as genotoxic when a positive response was given in at least one genotoxicity assay, and carcinogenic when it was tested positive in at least one rodent sex. Of the 20 antiparasitics with retrievable results of both genotoxicity and carcinogenicity, Malathion, Diazinon, Deltamethrin, Fenvalerate, Coumaphos, Tiabendazole, Albendazole, Cypermethrin, Amitraz and Praziquantel might be classified as genotoxic non-carcinogens; Fenthion, Lindane, Chlordimeform, Fipronil, Dichlorvos, Metronidazole, Pyrimethamine, and Imidacloprid can be classified as genotoxic carcinogens; Mefloquine was considered a non-genotoxic non-carcinogen, while the non-genotoxic carcinogens only contained Atovaquone, which tested negative in bacterial mutagenicity, in vitro and in vivo cytogenetic assays, but was found to induce liver tumors in mice in a long-term carcinogenesis assay (75, 122, 123).

Correlation between the results of genotoxicity and carcinogenicity assays of antiparasitics.

Assay type No. of non-genotoxic non-carcinogens No. of genotoxic non-carcinogens No. of non-genotoxic carcinogens No. of genotoxic carcinogens
Ames 8 (42.1%) (2, 3, 13, 17, 19, 25, 33, 35) 1 (5.3%) (50) 6 (31.6%) (7, 10, 19, 24, 32, 45) 4 (21.1%) (20, 26, 30, 36)
Gene 2 (16.7%) (25, 33) 1 (8.3%) (50) 5 (41.7%) (7, 10, 19, 24, 36) 4 (33.3%) (20, 26, 32, 45)
In vitro 2 (11.1%) (3, 13) 6 (33.3%) (2, 15, 17, 25, 33, 50) 2 (11.1%) (7, 10) 8 (44.4%) (19, 20, 24, 26, 30, 32, 36, 45)
In vivo 1 (5.6%) (35) 7 (38.9%) (2, 13, 15, 17, 25, 33, 50) 2 (11.1%) (1, 20) 8 (44.4%) (10, 19, 24, 26, 30, 32, 36, 45)
DNA lesions 2 (15.4%) (25, 33) 2 (15.4%) (3, 15) 0 9 (69.2%) (10, 19, 20, 24, 26, 30, 32, 36, 45)

Ames, bacterial mutagenicity; Gene, gene mutation in mammalian cells; In vitro, in vitro cytogenetics; In vivo, in vivo cytogenetics. The data show the number of antiparasitics that classified as non-carcinogens and carcinogens, which were examined in each genotoxicity assay and the result was negative (non-genotoxic) and positive (genotoxic) in the same assay. In this analysis, the antiparasitics that did not increase tumor incidence in mice and/or rats of both sexes were considered as non-carcinogens, and that increased tumor incidence in at least one sex of mice or rats were considered as carcinogens. An antiparasitic was considered non-genotoxic when it gave a single negative result, and genotoxic when it gave a single positive or concordant positive result in the indicated genotoxicity assay. The following indicated the number and corresponding percentages, as well as the numbers of antiparasitics of Table 3.

The bacterial mutagenicity has the highest specificity but the lowest sensitivity (Table 8), while DNA lesions (in vitro and/or in vivo) have the highest sensitivity and a lower specificity. A test with a low specificity induced a high proportion of misleading positive results. Therefore, the combination of bacterial mutagenicity and DNA lesions has high accuracy in relation to rodent cancer, which is consistent with the above analysis results. A proportion of 5.3% of antiparasitics gave positive in bacterial mutagenicity and was classified as non-carcinogens. There were 31.6% of antiparasitics that were regarded as carcinogenic while gave a negative result in bacterial mutagenicity.

 Discussion

The economic importance of parasitic infections in livestock and humans has long been recognized. Meanwhile, the most important advances in antiparasitics have come from the animal health area. Although many antiparasitics have been developed and applied to control parasitism in humans and animals, genotoxicity and carcinogenicity studies have not been conducted on a large proportion of them. Since a relationship between exposure to genotoxic compounds and carcinogenesis has been established, genotoxicity tests have been proposed for all medicinal products for human use except for some compounds (e.g., anticancer) that can interact with DNA (11). Therefore, this review was to assess the extent of antiparasitics that have been tested for genotoxic and carcinogenic activity. In addition, the ability of various types of genotoxicity assays was summarized to discriminate rodent carcinogens, which benefit to analyze the relative predictivity of carcinogenicity in rodents and humans. Furthermore, it is necessary to re-evaluate in vitro genotoxicity according the present revised guidelines.

With regard to the genotoxicity assays, compared to the positive and discordant results, the incidence of negative responses is 61.7, 61.1, 21.2, 25.8, and 15.8% for bacterial mutagenicity, gene mutation in cultured mammalian cells, in vitro cytogenetics, in vivo cytogenetics, and DNA lesions (in vitro and in vivo), respectively. It was observed that the incidence of negative responses was higher than the positive and discordant results in bacterial mutagenicity and gene mutation in cultured mammalian cells. Kasper et al. (240) reviewed the advantages and limitations of the standard genotoxicity tests in predicting the ability and the mode of action for carcinogens, which demonstrated that a totally negative response in all the standard genotoxicity assays was sufficient to prove the non-genetic toxicity of the chemicals, while the presence of a positive response in some genotoxicity assays, particularly in Ames and in vitro genotoxicity studies, did not afford support for the genetic definition of the chemicals. There have been a number of experiences in the literature regarding the high correlation among the various types of genotoxicity assays with respect to carcinogens (241, 242), which suggested that a chemical that tested positive in Salmonella tended to yield positive responses in any other in vitro genotoxicity studies, for instance, chromosome aberrations (CA), SCEs, and mutations in mouse lymphoma cells (MLA) (243).

A high percentage of antiparasitics tested positive in the following assays: in vitro cytogenetics, in vivo cytogenetics, and DNA lesions (in vitro and in vivo). It is worth noting that the proportion of positive responses in in vitro cytogenetics is higher than in other types of assays. The in vitro cytogenetics seems to be more sensitive to genetic substance. However, the in vitro assays always lead to a number of false-positive results in genotoxicity and the carcinogenicity in rodents (244, 245). It was learned from the literature that the massive positive results only occurred at high levels of concentration. Recent surveys for in vitro cytogenetics were taken from compilations such as that of Müller et al. (246), Kirkland and Müller (247), Müller and Kasper (248), and Hilliard et al. (249). The conclusion was that the highest testing concentrations might lead to an increase in the emergence of misleading, toxicity-related positive results. In cytotoxicity and chromosome aberrations in vitro, Galloway (250) found that the positive response in genetic toxicology was caused by the cytotoxicity rather than the true drug or DNA interactions. Parry et al. (251) examined 24 carcinogens that gave positive results in in vitro genotoxicity at 1–10 mM, yet almost half of them were not mechanistically genotoxic carcinogens or had carcinogenic effects only in excessive doses. In the present review, we re-evaluate the in vitro genotoxicty according to current ICH S2 (R1) guidance. We find that the percentage of antiparasitics in agreement with the current ICH S2 (R1) guidance for in vitro genotoxicity data acceptance was 15 (45.5%). Thus, it is essential to re-evaluate in vitro genotoxicty that conducted prior to the update guideline of ICH S2 (R1) to provide a comprehensive assessment of the genotoxic effects.

Misleading positive results were found not only in in vitro but also in in vivo genotoxic assays. Increasing experience suggested that the occurrence of a positive response in rats and mice micronucleus tests was not the consequence of intrinsic genotoxicity but drug-related disturbances in the physiology (252), such as lysosomal damage, ATP depletion or impairment of mitochondrial function and the release of DNA endonucleases. However, at the time of writing, there has still been no amendment to the guidelines requirements of in vivo genotoxicity for dose limitations and toxicity to avoid irrelevant physiological responses. Furthermore, there is no consensus as to the highest testing concentration in in vitro genotoxicity assays. The method for the detection of toxicity has greatly changed in recent years, and the limitations of dose and toxicity in genotoxicity testing in OECD and ICH should be adjusted to adapt to the new changes. The standard genotoxicity system also needs to identify the cytotoxicity and genotoxicity clearly.

There are many explanations that could account for the existence of different results in the various types of genetic tests. The differences are the following: the detection of the genetic end point; the xenobiotic metabolism between bacterial mutagenicity and mammalian cells; the effective dose between in vitro and in vivo, especially the in vivo decomposition; the relative sensitivities of various genotoxicity assays to genetic damage; the metabolic activation pathway and metabolizing enzymes among species. In vivo activity, which is designed to study the mechanisms of mutagenicity in the potential target organs of rodents, is the best method to confirm the differences in cytogenetics between in vivo and in vitro. Except for the irrelevant biological reaction at high doses, it is also accepted that the metabolic activation process and metabolites could induce genetic toxicity. Some evidence suggested that the genetic toxicity of compounds may be prototypes or metabolites. For the drugs that are theoretically nitrosatable in the presence of amine, the interaction resulted in the formation of genotoxic–carcinogenic N-nitroso compounds (253). However, the current standard of genotoxicity assays cannot distinguish whether the positive results are derived from the drugs or their metabolites directly.

In Table 7, the percentage of concordant results between bacterial mutagenicity and carcinogenicity in both mice and rats is 90.0%, which is higher than any other correlation pairs. The same conclusion was drawn by Snyder and Green (19) in a review of the genotoxicity of marketed pharmaceuticals. Data from 467 marketed drugs were collected and no combination of gene-tox assays provided a higher predictivity of rodent carcinogenesis than the bacterial mutagenicity test itself (19). In two studies conducted by Zeiger, one identified 172 chemicals that gave negative or equivocal results in 2-year rodent assays, yet 38 (22.1%) chemicals produced positive results in Salmonella (243). Another found that among 158 drugs that tested negative in carcinogenicity assays, 33 (21%) were Salmonella mutagens (254). However, a chemical that tested negative in Salmonella testing cannot be regarded as a non-carcinogenicity because the percentage of rodent carcinogens that are not mutagenic is about 50% (254). It was also reported that the predictivity for rodent carcinogenicity of bacterial mutagenicity ranged from approximately 77 to 98% (254, 255). The remaining 2–23% was classified as non-carcinogen with positive result in bacterial mutagenicity, which demonstrated the flaw and insufficiency on the prediction carcinogenicity of bacterial mutagenicity.

Therefore, it requires efforts to overcome the deficiencies of bacterial mutagenicity and improve the predictivity for carcinogenicity. We try to find which genotoxicity assay(s) considered could enhance the prediction of bacterial mutagenicity to rodent carcinogenicity. Our approach has many differences and improvement compared to Snyder and Green (19), who examined only five combinations of gene-tox assays, such as Ames–in vitro cytogenetics, Ames–in vivo cytogenetics, In vitro cytogenetics–in vivo cytogenetics, MLA–in vivo cytogenetics, and MLA–in vitro cytogenetics (19). These combinations have no DNA lesions tests and no taking bacterial mutagenicity as center. A review suggested that DNA lesion alone could contribute to the prediction of carcinogenicity in mice (255). In the present article, as shown in Table 8, DNA lesion testing can significantly increase the predictivity of Ames from 90 to 100%, suggesting that the combination of DNA lesions and bacterial mutagenicity obtained higher prediction of carcinogenicity.

There are three types of DNA lesions: (a) the formation of DNA adducts; (b) DNA repair synthesis (UDS); and (c) the induction of DNA strand breaks and cross-links. An analysis of correlations between the induction of DNA lesions and carcinogenic activity was conducted in 2010 (256). It noted that the carcinogenic activity of some drugs can be correctly predicted by DNA lesion assays, yet neglected in the standard 3-test battery. Thus, DNA lesion assays were considered the best supplement for the standard 3-test battery. The occurrence of the highest predictivity in a combination of bacterial mutagenicity and DNA lesions in our review suggested a close relationship between genotoxicity and carcinogenic activity. The bacterial mutagenicity test was often used to measure the ability of a drug to cause mutations rather than a definitive test of the carcinogens. The in vivo DNA lesion tests can detect the chemicals that reach the appropriate target with an effective dose to convert into a permanent mutation by reacting with DNA. In a few cases, the mutation escaped monitoring to survive and subsequently, carcinogenicity was generated through a loss of restriction of cell division. The in vivo DNA lesions can identify this “survived mutation.” Thus, the combination of bacterial mutagenicity and DNA lesions showed a higher and more accurate predictivity of carcinogenicity.

The correlation between the results of genotoxicity and carcinogenicity assays of antiparasitics was indicated in Table 9. Among the antiparasitics that were classified as genotoxic carcinogens, 69.2% tested positive in in vitro and/or in vivo DNA lesions exhibiting a greater sensitivity to carcinogens than any other types of genotoxicity assays. Eight out of 19 (42.1%) antiparasitics gave negative results in bacterial mutagenicity and were identified as non-carcinogens. Sensitivity and specificity are commonly used to describe the capability of in vitro genotoxicity assays (257). Sensitivity is defined as the percentage of genotoxic carcinogens that produced positive results in the considered test, and specificity is regarded as the ratio of non-carcinogens that gave negative responses. The ability of a battery of three in vitro genotoxicity tests to discriminate between rodent carcinogens and non-carcinogens was made by Kirkland et al. to increase the specificity of a valid test (258). The conclusion was that the “profile” of the genotoxicity results, such as the concentration, the level of toxicity and magnitude of response, provided a body of evidence to predict the carcinogenic results (259).

The rodent bioassays were useful and relevant for predicting risks of human cancers (260). The epigenetic changes with a loss of restriction of cell division (261) and the DNA oxidative stress damage were likely to produce cancer. Trosko and Upham found that the changes in gene expression caused by cell communication systems play a key role in the imbalance of cell proliferation, differentiation, and apoptosis, eventually promoting the tumor process (262). A large number of rodent tumor findings were found not relevant for humans (262) recently. It is worth noting that traditional carcinogenicity studies are largely not predictive of human cancer risk, therefore the well-suited approaches were proposed, for instance, the genetically modified animal models (15), and in vitro carcinogenicity screening assays based on gene expression profiling (16, 263). From the perspective of prospects, a more useful and accurate method to predict the carcinogenicity in humans is very urgent.

Herein, 136 antiparasitics were collected from both human and veterinary pharmacopeia. Due to the design of toxicity and the highest concentration in in vitro genetic toxicity tests have changed enormously in current guidelines, the reliably of old data were evaluated and as low as 45.5%. For a larger proportion of antiparasitics, whose genotoxicity and/or carcinogenicity results were not retrievable, the retesting based on revised guidelines should be done to make a safety assessment of human health. The combination of DNA lesions and bacterial mutagenicity is more accurate for predicting carcinogenicity than bacterial mutagenicity alone or together with any other genotoxicity testing. Development of this method for predicting carcinogens should be applied to reduce the misleading hazard alerts of the new and effective drugs.

 Author Contributions

ZY conceived the idea. XW analyzed and discussed data. QL analyzed and discussed data and wrote the article. ZL performed and revised the experiments. AI and FZ revised the article. All the authors discussed the results and contributed to the final manuscript.

 Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding. This work was supported by the National Key Research and Development Program of China (2017YFD0501405 and 2017YFD0501401), Natural Science Foundation of China (grant nos. 31272614 and 31502115), grants from 2017 National Risk Assessment of Quality and Safety of Livestock and Poultry Products (GJFP2017008), and Research on the detection standard of veterinary drug residue (2662015PY021).

 References Pimentel D Greiner A Bashore T. Economic and Environmental Costs of Pesticide Use. Environmental Toxicology: Current Developments. UK: Gordon and Breach Science Publishers (1998). p. 121–87. Fraumeni JF Jr Miller RW. Drug-induced cancer. J Natl Cancer Inst (1972) 48:1267–70. Hoover R Fraumeni JF Jr. Drugs in clinical use which cause cancer. J Clin Pharmacol (1975) 15:16–23.10.1002/j.1552-4604.1975.tb01423.x Barthel E. Increased risk of lung cancer in pesticide-exposed male agricultural workers. J Toxicol Environ Health (1981) 8:1027–40.10.1080/15287398109530135 Ji BT Silverman DT Stewart PA Blair A Swanson GM Baris D Occupational exposure to pesticides and pancreatic cancer. Am J Ind Med (2001) 39:92–9.10.1002/1097-0274(200101)39:1<92::AID-AJIM9>3.0.CO;2-P11148019 Viel JF Challier B. Bladder cancer among French farmers: does exposure to pesticides in vineyards play a part? Occup Environ Med (1995) 52:587–92.10.1136/oem.52.9.5877550798 Brown LM Blair A Gibson R Everett GD Cantor KP Schuman LM Pesticide exposures and other agricultural risk factors for leukemia among men in lowa and Minnesota. Cancer Res (1990) 50:6585–91. Blair A Zahm SH Pearce NE Heinemann EF Fraumeni JF. Clues to cancer etiology from studies of farmers. Scand J Work Environ Health (1992) 18:209–15.10.5271/sjweh.15781411362 Blair A Zahm SH. Agricultural exposures and cancer. Environ Health Perspect (1995) 103:205–8.10.2307/3432311 Meinert R Schuz J Kaletsch U Kaatsch P Michaelis J. Leukemia and non-Hodgkin’s lymphoma in childhood and exposure to pesticides: results of a register-based case-control study in Germany. Am J Epidemiol (2000) 151:639–46.10.1093/oxfordjournals.aje.a010256 (ICH) S2B. Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. (2012). VICH. Veterinary International Conference on Harmonization. Safety Studies for Veterinary Drug Residues in Human Food: Genotoxicity Studies. (2000). FDA. Toxicological principles for the safety assessment of food ingredients. IV.C.1.a. Bacterial Reverse Mutation Test. (2000). (ICH) S2 (R1). Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use. (2001). (ICH) S2 (R1). Guidance for Industry Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human use. (2012). Vinken M Doktorova T Ellinger-Ziegelbauer H Ahr HJ Lock E Carmichael P The carcinogenomics project: critical selection of model compounds for the development of omics-based in vitro carcinogenicity screening assays. Mutat Res (2008) 659:202–10.10.1016/j.mrrev.2008.04.00618514569 (ICH) S2 (R1). Revised Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use. (2010). ICH. (2011) Snyder RD Green JW. A review of the genotoxicity of marketed pharmaceuticals. Mutat Res (2001) 488:151–69.10.1016/S1383-5742(01)00055-211344042 (ICH) S1A. The Need for Carcinogenicity Studies of Pharmaceuticals. CPMP/ICH/140/95. (1996). IARC. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Lyon: IARC (1972). p. 1–90. (ICH) S1B. Carcinogenicity: Testing for Carcinogenicity of Pharmaceuticals, CPMP/ICH/299/95. (1998). HHS. Registry of Toxic Effects of Chemical Substances, 1985–1986. Washington: US Department of Health and Human Sciences (1988). Physicians’ Desk Reference. (1999). 59th ed, Thomson PDR, Montvale, NJ. Physicians’ Desk Reference. (2005). 59th ed, Thomson PDR, Montvale, NJ. OECD. OECD Guidelines for the Testing of Chemicals. Test Guideline 471. Bacterial Reverse Mutation Test. (1997). FDA. Toxicological principles for the safety assessment of food ingredients. IV.C.1.d. Mammalian Erythrocyte Micronucleus Test. (2000). Chinese. Toxicology Assessment Procedure and Method for Food Safety. GB15193.13-2003. (2003). FDA. Toxicological principles for the safety assessment of food ingredients. IV.C.1.c. In Vitro Mouse Lymphoma TK ± Gene Mutation Assay. (2000). OECD. OECD Guidelines for the Testing of Chemicals. Test No. 490: In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene. OECD – Organisation for Economic Cooperation and Development (2015). OECD. OECD Guidelines for the Testing of Chemicals. Test Guideline 473. In Vitro Mammalian Chromosomal Aberration Test. OECD – Organisation for Economic Cooperation and Development (2014). FDA. Toxicological principles for the safety assessment of food ingredients. IV.C.1.b. In Vitro Mammalian Chromosomal Aberration Test. (2000). OECD. OECD Guidelines for the Testing of Chemicals. Test Guideline 474. Mammalian Erythrocyte Micronucleus Test. OECD – Organisation for Economic Cooperation and Development (2014). OECD. OECD Guidelines for the Testing of Chemicals. Test Guideline 476. In Vitro Mammalian Cell Gene Mutation Test. OECD – Organisation for Economic Cooperation and Development (1997). GBI. Chronic Toxicity and Carcinogencity Study. Toxicological Sciences (2003). p. 109–13. OECD. Guideline for the Testing of Chemicals. Guideline 453:Combined. Paris: Chronic Toxicity\Carcinogenicity Studies (2009). Brown JP Brown RJ. Mutagenesis by 9,10-anthraquinone derivatives and related compounds in Salmonella typhimurium. Mutat Res (1976) 40:203–24.10.1016/0165-1218(76)90046-X Scott BR Dorn GL Kaefer E Stafford R. Aspergillus nidulans: systems and results of tests for induction of mitotic segregation and mutation, 2. haploid assay systems and overall response of all systems: a report of the US EPA Gene–Tox Program. Mutat Res (1982) 98:49–94.10.1016/0165-1110(82)90003-3 Oberly TJ Rexroat MA Bewsey BJ Richardson KK Michaelis KC. An evaluation of the CHO/hgprt mutation assay involving suspension cultures and soft agar cloning: results for 33 chemicals. Environ Mol Mutagen (1990) 16:260–71.10.1002/em.28501604082253605 Preston RJ Au W Bender MA Brewen JG Carrano AV Heddle JA. Mammalian in vivo and in vitro cytogenetic assays: a report of the U.S. EPA’s Gene–Tox Program. Mutat Res (1981) 87(2):143–88.10.1016/0165-1110(81)90030-0 Propping P Röhrborn G Buselmaier W. Comparative investigations on the chemical induction of point mutations and dominant lethal mutations in mice. Mol Gen Genet (1972) 117:197–209. Alba P Ferrs MD Xamena N Creus A Marcos R. Genotoxicity of acridine orange and acriflavine in Drosophila melanogaste. Mutat Res (1983) 121:199–203.10.1016/0165-7992(83)90204-X Heddle JA Hite M Kirkhart B Mavournin K Mac Gregor JT Newell GW The induction of micronuclei as a measure of genotoxicity, a report of the U.S. Environmental Protection Agency Gene–Tox Program. Mutat Res (1983) 123:61–118.10.1016/0165-1110(83)90047-7 Zimmermann FK Von Borstel RC Von Halle ES Parry JM Siebert D Zetterberg G Testing of chemicals for genetic activity with Saccharomyces cerevisiae: a report of the U.S. Environmental Protection Agency Gene–Tox Program. Mutat Res (1984) 133:199–244.10.1016/0165-1110(84)90017-4 Wyrobek AJ Gordon LA Burkhart JG. An evaluation of human sperm as indicators of chemically induced alterations of spermatogenic function: a report of the U.S. Environmental Protection Agency Gene–Tox Program. Mutat Res (1983) 115:73–148.10.1016/0165-1110(83)90015-5 Wyrobek AJ Gordon LA Burkhart JG Francis MW Kapp RW Jr Letz G An evaluation of the mouse sperm morphology test and other sperm tests in nonhuman mammals, a report of the US Environmental Protection Agency Gene–Tox Program. Mutat Res (1983) 115:1–72.10.1016/0165-1110(83)90014-3 Physicians’ Desk Reference. (2000). 59th ed, Thomson PDR, Montvale, NJ. Oztas S Salman AB Tatar A Yigiter M Yazgi H Ertek M Genotoxic effect of albendazole in pediatric patients with hepatic hydatid disease. Int J Infect Dis (2007) 11:446–9.10.1016/j.ijid.2007.01.00217331781 Ramírez T Benítez-Bribiesca L Ostrosky-Wegman P Herrera LA. In vitro effects of albendazole and its metabolites on the cell proliferation kinetics and micronuclei frequency of stimulated human lymphocytes. Arch Med Res (2001) 32:119–22.10.1016/S0188-4409(01)00259-411343808 Ermler S Scholze M Kortenkamp A. Seven benzimidazole pesticides combined at sub-threshold levels induce micronuclei in vitro. Mutagenesis (2013) 28:417–26.10.1093/mutage/get01923547264 Tudek B Gajewska J Szczypka M Rahden-Staron I Szymczyk T. Screening for genotoxic activity of amitraz with short-term bacterial assays. Mutat Res (1988) 204:585–91.10.1016/0165-1218(88)90061-43280992 Osano O Admiraal W Klamer HJ Pastor D Bleeker EA. Comparative toxic and genotoxic effects of chloroacetanilides, formamidines and their degradation products on Vibrio fischeri and Chironomus riparius. Environ Pollut (2002) 119:195–202.10.1016/S0269-7491(01)00334-712152826 Padula G Ponzinibbio MV Picco S Seoane A. Assessment of the adverse effects of the acaricide amitraz: in vitro evaluation of genotoxicity. Toxicol Mech Methods (2012) 22:657–61.10.3109/15376516.2012.66668322394339 CEPAD. California Environmental Protection Agency Department. (1998). Chatterjee T Mukopadhyay A Khan KA Giri K. Comparative mutagenic and genotoxic effects of three antimalarial drugs, chloroquine, primaquine and amodiaquine. Mutagenesis (1998) 13:619–24.10.1093/mutage/13.6.6199862194 Shubber EK Jacobson–Kram D Williams JR. Comparison of the Ames assay and the induction of sister chromatid exchanges: results with ten pharmaceuticals and five selected agents. Cell Biol Toxicol (1986) 2:379–99.10.1007/BF001218533267457 Gupta RL Kaur IP Juneja TR. Mutagenicity of the antischistosomal agent amoscanate and its hydrolytic and related parasubstituted 4-nitrodiphenylamine products. Med Sci Res (1992) 20:365–8. Egito LC Medeiros SR Medeiros MG Price JC Egito ES. Evaluation of the relationship of the molecular aggregation state of amphotericin B in medium to its genotoxic potential. J Pharm Sci (2004) 93:1557–65.10.1002/jps.2003815124213 Mourot D Mourot A. Mutagenicity of bithionol sulfoxide and its metabolites in the salmonella/mammalian microsome test. Mutat Res (1987) 188:53–5.10.1016/0165-1218(87)90114-53553922 Zeiger E Anderson B Haworth S Lawlor T Mortelmans K. Salmonella mutagenicity tests: IV. Results from the testing of 300 chemicals. Environ Mol Mutagen (1988) 11:1–157.10.1002/em.28501106023277844 Yoshimura H Nakamura M Koeda T. Lack of mutagenicity of fasciolicides. Vet Res Commun (1985) 9:147–52.10.1007/BF022151384002614 Simmon VF Riccio ES Robinson DE Mitchell AD. In Vitro Microbiological Mutagenicity and Unscheduled DNA Synthesis Studies of Fifteen Pesticides. (1985). EPA/600/1–85/006, ntis pb. 85–193761. IARC. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans (Chlordimeform). (Vol. 30). (1983). p. 61–72. Chen XR. China Acad J College Med (1980) 9:181–5. Shirasu Y. Origins of Human Cancer. (1997). p. 267. Mitchell AD Casciano DA Meltz ML Robinson DE San RC Williams GM Unscheduled DNA synthesis tests: a report of the U.S. Environmental Protection Agency Gene–Tox Program. Mutat Res (1983) 123:363–410.10.1016/0165-1110(83)90029-5 Zhao JM. China Pub Health Occup Dis J (1984) 2:46. Lang R. The mammalian spot test and its use for testing of mutagenic and carcinogenic potential: experience with the pesticide chlordimeform, its principal metabolites and the drug lisuride hydrogen maleate. Mutat Res (1984) 135:219–24.10.1016/0165-1218(84)90125-36708963 Li F. A research of chlorphenamidine on labour hygiene and the mutation effect on human. China Acad J Electron Publish House (1985). Leslie C Reidy GF Murray M Stacey NH. Induction of xenobiotic biotransformation by the insecticide chlordimeform, a metabolite 4-chloro-o-toluidine and a structurally related chemical o-toluidine. Biochem Pharmacol (1988) 37:2529–35.10.1016/0006-2952(88)90242-0 Weisbuurger EK Russfield AB Homburger F Weisburger JH Boger Van E Dongen CG Testing of twenty-one environmental aromatic amines or derivatives for long-term toxicity or carcinogenicity. Environ Pathol Toxicol (1978) 2:325–56. Thomas SM Silburn KA MacPhee DG. Frameshift mutagenesis by chloroquine in Escherichia coli and Salmonella typhimurium. Mutat Res (1987) 192:233–7.10.1016/0165-7992(87)90062-52446128 U.S. National Library of Medicine. Espinosa-Aguirre JJ Ramírez Santos J Cortinas de Nava C. Influence of the UVR repair system on the mutagenicity of antiparasitic drugs. Mutat Res (1989) 222:161–6.10.1016/0165-1218(89)90132-82646533 Available from: http://www.toxnet.nlm.nih.gov Kadotani S Arisawa M Maruyama HB. Mutagenicity examination of several non-steroidal anti-inflammatory drugs in bacterial systems. Mutat Res (1984) 138:133–6.10.1016/0165-1218(84)90035-1 O’Donovan MR. The comparative responses of Salmonella typhimurium TA1537 and TA97A to a range of reference mutagens and novel compounds. Mutagenesis (1990) 5:267–74.10.1093/mutage/5.3.2672200947 Sideropoulos AS Specht SM. Evaluation of microbial testing methods for the mutagenicity of quinoline and its derivatives. Current Microbiol (1984) 11:59–65.10.1007/BF01567705 Leifer Z Kada T Mandel M Zeiger E Stafford R Rosenkranz HS. Evaluation of tests using DNA repair-deficient bacteria for predicting genotoxicity and carcinogenicity: a report of the U.S. EPA’s Gene–Tox Program. Mutat Res (1981) 87:211–97.10.1016/0165-1110(81)90015-4 Donya SM Souria M Hassan EE. Clastogenetic effects of the fasciolicides closantel and nitroxynil on mice somatic and germ cells. Cytologia (2007) 72:29–36.10.1508/cytologia.72.29 Dunkel VC Zeiger E Brusick D McCoy E McGregor D Mortelmans K Reproducibility of microbial mutagenicity assays: II. Testing of carcinogens and noncarcinogens in Salmonella typhimurium and Escherichia coli. Environ Mol Mutagen (1985) 7:1–248.10.1002/em.28600709023905369 CEPAD. California Environmental Protection Agency Department. (1992). Loveday KS Lugo MH Resnick MA Anderson BE Zeiger E. Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells in vitro: II. Results with 20 chemicals. Environ Mol Mutagen (1989) 13:60–94.10.1002/em.28501301082642806 NCINTP. Carcinogenesis Technical Report Series. National Cancer Institute/National Toxicology Program; US Department of Health and Human Services. National Toxicology Program Technical Report Series 192. (1979). NCINTP. Carcinogenesis Technical Report Series. National Cancer Institute/National Toxicology Program; US Department of Health and Human Services. National Toxicology Program Technical Report Series 96. (1979). Ila HB Topaktas M Rencuzogullari E Kayraldiz A Donbak L Daglioglu YK. Genotoxic potential of cyfluthrin. Mutat Res (2008) 656:49–54.10.1016/j.mrgentox.2008.07.00518692594 Tisch M Faulde MK Maier H. Genotoxic effects of pentachlorophenol, lindane, transfluthrin, cyfluthrin, and natural pyrethrum on human mucosal cells of the inferior and middle nasal conchae. Am J Rhinol (2005) 19:141–51.15921213 Marinowic DR Mergener M Pollo TA Maluf SW Silva LB. In vivo genotoxicity of the pyrethroid pesticide beta-cyfluthrin using the comet assay in the fish Bryconamericus iheringii. Z Naturforsch C (2012) 67:308–11.10.1515/znc-2012-5-61022888536 Isidori M Caterino E Criscuolo E Fatigati V Liguori G Parrella A. Antimutagenic and antigenotoxic effects of vegetable matrices on the activity of pesticides. Food Addit Contam Part A Chem Anal Control Expo Risk Assess (2009) 26:1049–62.10.1080/0265203090289619419680980 Sankar P Telang AG Manimaran A. Curcumin protects against cypermethrin-induced genotoxicity in rats. Environ Toxicol Pharmacol (2010) 30:289–91.10.1016/j.etap.2010.07.005 Bhunya SP Pati PC. Genotoxic effects of a synthetic pyrethroid insecticide, cypermethrin, in mice in vivo. Toxicol Lett (1988) 41:223–30.10.1016/0378-4274(88)90058-63376150 Kocaman AY Topaktaş M. Genotoxic effects of a particular mixture of acetamiprid and alpha-cypermethrin on chromosome aberration, sister chromatid exchange, and micronucleus formation in human peripheral blood lymphocytes. Environ Toxicol (2010) 25:157–68. Cui Y Guo J Xu B Chen Z. Genotoxicity of chlorpyrifos and cypermethrin to ICR mouse hepatocytes. Toxicol Mech Methods (2011) 21:70–4.10.3109/15376516.2010.52919221047179 Kocaman AY Topakta M. The in vitro genotoxic effects of a commercial formulation of alpha-cypermethrin in human peripheral blood lymphocytes. Environ Mol Mutagen (2009) 50:27–36.10.1002/em.20434 Ansari RA Rahman S Kaur M Anjum S Raisuddin S. In vivo cytogenetic and oxidative stress-inducing effects of cypermethrin in freshwater fish, Channa punctata Bloch. Ecotoxicol Environ Saf (2011) 74:150–6.10.1016/j.ecoenv.2010.08.036 Patel S Pandey AK Bajpayee M Parmar D Dhawan A. Cypermethrin-induced DNA damage in organs and tissues of the mouse: evidence from the comet assay. Mutat Res (2006) 607:176–83.10.1016/j.mrgentox.2006.04.010 Murkunde YV Sathya TN Subashini N Murthy PB. Transplacental genotoxicity evaluation of cypermethrin using alkaline comet assay. Hum Exp Toxicol (2012) 31:185–92.10.1177/096032711141209121659343 Kasimoglu C Uysal H. Mutagenic biomonitoring of pirethroid insecticides in human lymphocyte cultures: use of micronuclei as biomarkers and recovery by Rosa canina extracts of mutagenic effects. Pharm Biol (2014) 21:1–5. Surrallés J Xamena N Creus A Marcos R. The suitability of the micronucleus assay in human lymphocytes as a new biomarker of excision repair. Mutat Res (1995) 342:43–59.10.1016/0165-1218(95)90089-67885393 JMPR. The WHO/FAO. (2006). Brusick DJ Simmon VF Rosenkranz HS Ray VA Stafford RS. An evaluation of the Escherichia coli WP2 and WP2 UVRA reverse mutation assay. Mutat Res (1980) 76:169–90.10.1016/0165-1110(80)90009-37005668 Barrueco C Herrera A de la Pena E. Mutagenic evaluation of trichlorfon using different assay methods with Salmonella typhimurium. Mutagenesis (1991) 6:71–6.10.1093/mutage/6.1.712038275 Zeiger E Anderson B Haworth S Lawlor T Mortelmans K Speck W. Salmonella mutagenicity tests: III. Results from the testing of 255 chemicals. Environ Mol Mutagen (1987) 9:1–110.10.1002/em.28600906023552650 Aquilina G Benigni R Bignami M Calcagnile A Dogliotti E Falcone E Genotoxic activity of dichlorvos, trichlorfon and dichloroacetaldehyde. Pestic Sci (1984) 15:439–42.10.1002/ps.2780150504 Tian Y Ishikawa H Yamauchi T. Analysis of cytogenetic and developmental effects on preimplantation mouse embryos from dams treated with organophosphate insecticide trichlorfon. Huanjing yu zhiye yixue (2004) 21:391–3. Igarashi M Setoguchi M Takada S Itoh S Furuhama K. Optimum conditions for detecting hepatic micronuclei caused by numerical chromosome aberration inducers in mice. Mutat Res (2007) 632:89–98.10.1016/j.mrgentox.2007.04.01217561434 Pluijmen M Drevon C Montesano R Malaveille C Hautefeuille A Bartsch H. Lack of mutagenicity of synthetic pyrethroids in Salmonella typhimurium strains and in V-79 Chinese hamster cells. Mutat Res (1984) 137:7–15.10.1016/0165-1218(84)90106-X CEPAD. California Environmental Protection Agency Department. (2000). Grisolia CK. A comparison between mouse and fish micronucleus test using cyclophosphamide, mitomycin C and various pesticides. Mutat Res (2002) 518:145–50.10.1016/S1383-5718(02)00086-412113765 Shukla Y Arora A Singh A. Tumorigenic studies on deltamethrin in swiss albino mice. Toxicology (2001) 163:1–9.10.1016/S0300-483X(00)00416-9 Cabral JR Galendo D Laval M Lyandrat N. Carcinogenicity studies with deltamethrin in mice and rats. Cancer Lett (1990) 49:147–52.10.1016/0304-3835(90)90151-M2306708 Ono T Sekiya T Takahashi Y Sasaki YF Izumiyama F Nishidate E The genotoxicity of diaveridine and trimethoprim. Environ Toxicol Pharmacol (1997) 3:297–306.10.1016/S1382-6689(97)00026-421781790 Yoshimura H. Mutagenicity of the coccidiostat diaveridine in the Salmonella mammalian microsome assay. Mutat Res (1991) 261:149–52.10.1016/0165-1218(91)90061-P Marshall TC Dorough HW Swim HE. Screening of pesticides for mutagenic potential using Salmonella typhimurium mutants. Jagric Food Chem (1976) 24:560–3.10.1021/jf60205a013 Kier LD Brusick DJ Auletta AE The Salmonella typhimurium/mammalian microsome mutagenicity assay; an report of the US Environmental Protection Agency Gene–Tox Program. Mutat Res (1986) 168:67–238. Kier LD Brusick DJ Auletta AE Von Halle ES Brown MM Simmon VF The Salmonella typhimurium/mammalian microsomal assay, a report of the U.S. Environmental Protection Agency Gene–Tox Program. Mutat Res (1986) 168:69–240.10.1016/0165-1110(86)90002-3 Wong PK Wai CC Liong E. Comparative study on mutagenicities of organophosphorus insecticides in salmonella. Chemosphere (1989) 18:2413–22. Shadboorestan A Shokrzadeh M Ahangar N Abdollahi M Omidi M Hosseini Payam SS. The chemoprotective effects of L-carnitine against genotoxicity induced by diazinon in rat blood lymphocyte. Toxicol Ind Health (2013). Nishio A Uyeki EM. Induction of sister chromatid exchanges in Chinese hamster ovary cells by organophosphate insecticides and their oxygen analogs. Toxicol Environ Health (1981) 8:939–46.10.1080/152873981095301287338954 Sobti RC Krishan A Pfaffenberger CD. Cytokinetic and cytogenetic effects of some agricultural chemicals on human lymphoid cells in vitro: organophosphates. Mutat Res (1982) 102:89–102.10.1016/0165-1218(82)90149-56981766 Shokrzadeh M Ahmadi A Ramezaninejhad S Shadboorestan A. Hesperidin, a citrus bioflavonoid, ameliorates genotoxicity-induced by diazinon in human blood lymphocytes. Drug Res (2014) 65:57–60.10.1055/s-0033-1363998 NCINTP. Carcinogenesis Technical Report Series. National Cancer Institute/National Toxicology Program; US Department of Health and Human Services. National Toxicology Program Technical Report Series. 24. (1978). NCINTP. Carcinogenesis Technical Report Series. National Cancer Institute/National Toxicology Program; US Department of Health and Human Services. National Toxicology Program Technical Report Series 77. (1978). Choi EJ Kim YK Roh IK. Genetic toxicity of pesticides used in Korea on salmonella typhimurium and saccharomyces cerevisiae. Environ Mutagens Carcinog (1985) 5:11–8. NCINTP. Carcinogenesis Technical Report Series. National cancer institute/national toxicology program, US Department of Health and Human Services. National Toxicology Program Technical Report Series 342. (1989). Breau AP Mitchell WM Swinson J Field L. Mutagenic and cell transformation activities of representative phosphorothioate esters in vitro. J Toxicol Environ Health (1985) 16:403–13.10.1080/152873985095307504087308 Moriya M Ohta T Watanabe K Miyazawa T Kato K Shirasu Y. Further mutagenicity studies on pesticides in bacterial reversion assay systems. Mutat Res (1982) 116:185–216.10.1016/0165-1218(83)90059-9 Buselmaier W Roehrborn G Propping P. Mutagenicity investigations with pesticides in the host-mediated assay and the dominant lethal test in mice. Biol Zentralbl (1972) 91:311–25. Latt SA. Sister-chromatid exchanges: a report of the gene–tox program. Mutat Res (1981) 87:17–62.10.1016/0165-1110(81)90003-8 CEPAD. California Environmental Protection Agency Department. (2005). Selden JR Dolbeare F Clair JH Miller JE McGettigan K DiJohn JA Validation of a flow cytometric in vitro DNA repair (UDS) assay in rat hepatocytes. Mutat Res (1994) 315(2):147–67.10.1016/0921-8777(94)90015-97520997 Shirasu Y Moriya M Kato K Furuhashi A Kada T. Mutagenicity screening of pesticides in the microbial system. Mutat Res (1976) 40:19–30.10.1016/0165-1218(76)90018-5814455 Green MH Muriel WJ Bridges BA. Use of a simplified fluctuation test to detect low levels of mutagens. Mutat Res (1976) 38:33–42.10.1016/0165-1161(76)90077-7768758 Paik SG Lee SY. Genetic effects of pesticides in the mammalian cells: 1. Induction of micronucleus. Tongmul Hakhoe Chi (1977) 20:19–28. Anderson BE Zeiger E Shelby MD Resnick MA Gulati DK Ivett JL Chromosome aberration and sister chromatid exchange test results with 42 chemicals. Environ Mol Mutagen (1990) 16:55–137.10.1002/em.28501605052091924 Grant WF. Chromosome aberration assays in allium, a report of the U.S. Environmental Protection Agency Gene–Tox Program. Mutat Res (1982) 99:273–91.10.1016/0165-1110(82)90046-X Dean BJ Thorpe E. Studies with dichlorvos vapor in dominant lethal mutation tests on mice. Arch Toxikol (1970) 30:51–9.10.1007/BF00605273 Oshiro Y Piper CE Balwierz PS Soelter SG. Chinese hamster ovary cell assays for mutation and chromosome damage: data from non-carcinogens. J Appl Toxicol (1991) 11:167–77.10.1002/jat.25501103041918790 Benford DJ Price SC Lawrence JN Grasso P Bremmer JN. Investigations of the genotoxicity and cell proliferative activity of dichlorvos in mouse forestomach. Toxicology (1994) 92:203–15.10.1016/0300-483X(94)90178-37940561 Lee WR Abrahamson S Valencia R Von Halle ES Wuerglre FE Zimmering S. The sex-linked recessive lethal test for mutagenesis in drosophila melanogaster: a report of the U.S. Environmental Protection Agency Gene–Tox Program. Mutat Res (1983) 123:183–279.10.1016/0165-1110(83)90025-8 Nesnow S Argus M Bergman H Chemical carcinogens: a review and analysis of the literature of selected chemicals and the establishment of the gene–tox carcinogen data base: a report of the U.S. Environmental Protection Agency Gene–Tox Program. Mutat Res (1987) 185:1–195.10.1016/0165-1110(87)90017-0 NCINTP. Carcinogenesis Technical Report Series. National Cancer Institute/National Toxicology Program; US Department of Health and Human Services. National Toxicology Program Technical Report Series 10. (1977). Horn KH Teichmann B Schramm T Nischan P. Investigation of dichlorvos (DDVP), part II. Testing of dichlorvos for carcinogenic activity in rats. Arch Geschwulstforsch (1988) 58:1–10. Chan PC Huff J Haseman JK Alison R Prejean JD Carcinogenesis studies of dichlorvos in Fischer rats and B6C3F1 mice. J Cancer Res (1991) 82:157–64.1900819 Rosenkranz HS Jr Speck WT Stambaugh JE. Mutagenicity of metronidazole: structure-activity relationships. Mutat Res (1976) 38:203–6.10.1016/0165-1161(76)90191-6 McCann J Choi E Yamasaki E Ames BN. Detection of carcinogens as mutagens in the salmonella/microsome test: assay of 300 chemicals. Proc Natl Acad Sci U S A (1975) 2:5135–9.10.1073/pnas.72.12.5135 Vanelle P Dmeo MP Maldonado J Nouguier R Crozet MP Laget M Genotoxicity in oxazolidine derivatives: influence of the nitro group. Eur J Med Chem (1990) 25:241–50.10.1016/0223-5234(90)90207-J Ré JL De Méo MP Laget M Guiraud H Castegnaro M Vanelle P Evaluation of the genotoxic activity of metronidazole and dimetridazole in human lymphocytes by the comet assay. Mutat Res (1997) 375:147–55.10.1016/S0027-5107(97)00010-99202725 Przybojewska B Barański B Spiechowicz E Dziubałtowska E Szymczak W. Potential carcinogenicity assessment ofalpha-aescin and phenbendasol. Acta Pol Pharmal (1994) 51:89–93.7976438 Huang N Cerepnalkoski L Nwankwo JO Dews M Landolph JR. Induction of chromosomal aberrations, cytotoxicity, and morphological transformation in mammalian cells by the antiparasitic drug flubendazole and the antineoplastic drug harringtonine. Fundam Appl Toxicol (1994) 22:304–13.10.1006/faat.1994.10348005380 Rani MV Rao MS. In vitro effect of fenthion on human lymphocytes. Bull Environ Contam Toxicol (1991) 47:316–20.10.1007/BF01688658 Chen HH Sirianni SR Huang CC. Sister chromatid exchanges in Chinese hamster cells treated with seventeen organophosphorus compounds in the presence of a metabolic activation system. Environ Mutagen (1982) 4:621–4.10.1002/em.2860040513 CEPAD. California Environmental Protection Agency Department. (1997). Bai CL Qiao CB Zhang WD Chen YL Qu SX. A study of the pesticide fenthion: toxicity, mutagenicity, and influence on tissue enzymes. Biomed Environ Sci (1990) 3:262–75.2147549 National Cancer Institute USA. Technical. Rep Ser pb–293 832 (1997). p. 3–34. Herrera A Laborda E. Mutagenic activity in synthetic pyrethroids in Salmonella typhimurium. Mutagenesis (1988) 3:509–14.10.1093/mutage/3.6.5093070291 Giri A Giri S Sharma GD. Malathion and fenvalerate induce micronuclei in mouse bone marrow cells. Environ Mol Mutagen (2011) 52:607–13.10.1002/em.2064921538555 Caballo C Herrera A Barrueco C Santa-María A Sanz F de la Peña E. Analysis of cytogenetic damage induced in CHO cells by the pyrethroid insecticide fenvalerate. Teratog Carcinog Mutagen (1992) 12:243–9.10.1002/tcm.1770120602 CEPAD. California Environmental Protection Agency Department. (2001). Parker CM Patterson DR Van Gelder GA Gordon EB Valerio MG Hall WC. Chronic toxicity and carcinogenicity evaluation of fenvalerate in rats. Toxicol Environ Health (1984) 13:83–97.10.1080/152873984095304836716513 Parker CM Mccullough CB Gellatly JBM Johnston CD. Toxicologic and carcinogenic evaluation of fenvalerate in the B6C3F1 mouse. Fundam Appl Toxicol (1983) 3:114–20.10.1016/S0272-0590(83)80066-96873529 CEPAD. California Environmental Protection Agency Department. (2011). Çelik A Ekinci SY Güler G Yildirim S. In vitro genotoxicity of fipronil sister chromatid exchange, cytokinesis block micronucleus test, and comet assay. DNA Cell Biol (2014) 33:148–54.10.1089/dna.2013.215824460388 Ghisi NC Ramsdorf WA Ferraro MV de Almeida MI Ribeiro CA Cestari MM. Evaluation of genotoxicity in Rhamdia quelen (Pisces, Siluriformes) after sub-chronic contamination with fipronil. Environ Monit Assess (2011) 180:589–99.10.1007/s10661-010-1807-7 Sinsheimer JE Giri AK Osorio S Wise DS Townsend LB. Comparative mutagenicity and genotoxicity of the antiparasitic drugs, mebendazole, flubendazole and flubendazole oxime. Prog Clin Biol Res (1990) 340:225–34. Bradley MO Bhuyan B Francis MC Langenbach R Peterson A Huberman E. Mutagenesis by chemical agents in V79 Chinese hamster cells: a review and analysis of the literature, a report of the Gene–Tox Program. Mutat Res (1981) 87:81–142.10.1016/0165-1110(81)90029-4 Sturdik E Rosenberg M Stibranyi L Balaz S Chreno O Ebringer L Structure-mutagenicity relationships of 5-nitro-2-furylethylenes in Salmonella typhimurium TA98. Chem Biol Interact (1985) 53:145–53.10.1016/S0009-2797(85)80092-2 Brockman HE Serres FJ Ong TM De Marini DM Katz AJ Griffiths AJ Mutation tests in Neurospora crassa, a report of the U.S. Environmental Protection Agency Gene–Tox Program. Mutat Res (1984) 133:87–134.10.1016/0165-1110(84)90004-6 Emicback/26452. Mutat Res (1977):5613–20. Ni YC Shao BR. Comparison of the mutagenicity of 4 nitrofurans in Salmnella/microsome system. Zhongguo Yao Li Xue Bao (1985) 6:134–7. Karabay NU Oguz MG. Cytogenetic and genotoxic effects of the insecticides, imidacloprid and methamidophos. Genet Mol Res (2005) 4:653–62.16475109 Jia Q Guo M Xie L Liu YZ Sai LL. A experimental mutagenesis study on active compound of imidacloprid. Chin J Ind Med (2008) 21(2):114–5. Feng S Kong Z Wang X Zhao L Peng P. Acute toxicity and genotoxicity of two novel pesticides on amphibian, Rana N. Hallowell. Chemosphere (2004) 56:457–63.10.1016/j.chemosphere.2004.02.01015212911 Demsia G Vlastos D Goumenou M Matthopoulos DP. Assessment of the genotoxicity of imidacloprid and metalaxyl in cultured human lymphocytes and rat bone-marrow. Mutat Res (2007) 634:32–9.10.1016/j.mrgentox.2007.05.018 Costa C Silvari V Melchini A Catania S Heffron JJ Trovato A Genotoxicity of imidacloprid in relation to metabolic activation and composition of the commercial product. Mutat Res (2009) 672:40–4.10.1016/j.mrgentox.2008.09.01818977458 Feng S Kong Z Wang X Peng P Zeng EY. Assessing the genotoxicity of imidacloprid and RH-5849 in human peripheral blood lymphocytes in vitro with comet assay and cytogenetic tests. Ecotoxicol Environ Saf (2005) 61:239–46.10.1016/j.ecoenv.2004.10.00515883095 Lin A Zhu Y Tong Y. Evaluation of genotoxicity of combined soil pollution by cadmium and imidacloprid. Sci China C Life Sci (2005) 48:7–13.10.1007/BF0288979616089324 Pérez–Iglesias JM Ruiz de Arcaute C Nikoloff N Dury L Soloneski S Natale GS The genotoxic effects of the imidacloprid-based insecticide formulation Glacoxan Imida on Montevideo tree frog Hypsiboas pulchellus tadpoles (Anura, Hylidae). Ecotoxicol Environ Saf (2014) 104:120–6.10.1016/j.ecoenv.2014.03.00224675439 Kong ZM Zang Y Shen JP Luo Y Zhong Y. Effect of two new pesticides on sperm morphology of earthworms (Eisenia foetida). Carcinog Teratog Mutagen (1999) 11(1). Calderón-Segura ME Gómez-Arroyo S Villalobos-Pietrini R Martínez-Valenzuela C Carbajal-López Y Calderón-Ezquerro Mdel C Evaluation of genotoxic and cytotoxic effects in human peripheral blood lymphocytes exposed in vitro to neonicotinoid insecticides news. J Toxicol (2012) 2012:612647.10.1155/2012/61264722545045 CEPAD. California Environmental Protection Agency Department. (2004). O’Connor PJ MacNaught F Butler WH Cooper DP Margison GP Povey AC. Increased pathology incidence in the forestomach of rats maintained on a diet containing ivermectin and given a single dose of N-methyl-N1-nitro-N-nitrosoguanidine. J Environ Pathol Toxicol Oncol (2001) 20:223–7.10.1615/JEnvironPatholToxicolOncol.v20.i3.6011797831 Kalantzi OI Hewitt R Ford KJ Cooper L Alcock RE Thomas GO Low dose induction of micronuclei by lindane. Carcinogenesis (2004) 25:613–22.10.1093/carcin/bgh04814688026 Rupa DS Reddy PP Reddi OS. Genotoxic effect of benzene hexachloride in cultured human lymphocytes. Hum Genet (1989) 83:271–3.10.1007/BF002851702477324 Bhunya SP Jena GB. Genotoxic potential of the organochlorine insecticide lindane (g–BHC): an in vivo study in chicks. Mutat Res (1992) 272:175–81.10.1016/0165-1161(92)90045-N Constantin MJ Nilan RA. Chromosome aberration assays in barley (Hordeum vulgare): a report of the U.S. Environmental Protection Agency Gene–Tox Program. Mutat Res (1982) 99:11–36.10.1016/0165-1110(82)90029-X Ma TH. Tradescantia cytogenetic tests (root-tip mitosis, pollen mitosis, pollen mother-cell meiosis); a report of the U.S. Environmental Protection Agency Gene–Tox Program. Mutat Res (1982) 99:293–302.10.1016/0165-1110(82)90047-1 Tisch M Bergenthal S Maier H. Genotoxic effect of PCP and lindane on human epithelial tonsil cells. HNO (2002) 50:920–7.10.1007/s00106-001-0621-5 Weisse I Herbst M. Carcinogenicity study of lindane in the mouse. Toxicology (1977) 7:233–8.10.1016/0300-483X(77)90069-5 Wolff GL Suber RL. Hepatic glutathione S-transferase activity in mice: effects of Avy/-genotype, obesity, lindane treatment, and sex. Proc Soc Exp Biol Med (1986) 181:535–41.10.3181/00379727-181-42289 Pednekar MD Gandhi SR Netrawali MS. Evaluation of mutagenic activities of endosulfan, phosalone, malathion, and permethrin, before and after metabolic activation in the ames salmonella test. Bull Environ Contam Toxicol (1987) 38:925–33.10.1007/BF01609074 Amer SM Fahmy MA. Genotoxicity studies on the organophosphorus insecticide “malathion” in mouse bone-marrow. Bull Natl Res Cent (2004) 29:93–108. Galloway SM Armstrong MJ Reuben C Colman S Brown B Cannon C Chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells: evaluations of 108 chemicals. Environ Mol Mutagen (1987) 10:1–175.10.1002/em.28501005023319609 Dulout FN Olivero OA Guradze Von H Pastori MC. Cytogenetic effect of malathion assessed by the micronucleus test. Mutat Res (1982) 105:413–6.10.1016/0165-7992(82)90186-5 Titenko–Holland N Windham G Kolachana P Reinisch F Parvatham S Osorio AM Genotoxicity of malathion in human lymphocytes assessed using the micronucleus assay in vitro and in vivo: a study of malathion-exposed workers. Mutat Res (1997) 388:85–95.10.1016/S1383-5718(96)00140-49025795 Réus GZ Valvassori SS Nuernberg H Comim CM Stringari RB Padilha PT DNA damage after acute and chronic treatment with malathion in rats. J Agric Food Chem (2008) 56:7560–5.10.1021/jf800910q18616275 Batzinger RP Ou SY Bueding E. Antimutagenic effects of 2(3)-tert-butyl-4-hydroxyanisole and of antimicrobial agents. Cancer Res (1978) 38:4478–85.363261 Hennig UG Galindo-Prince OC Cortinas de Nava C Savage EA Von Borstel RC. A comparison of the genetic activity of pyrvinium pamoate with that of several other anthelmintic drugs in Saccharomyces cerevisiae. Mutat Res (1987) 187:79–89.10.1016/0165-1218(87)90120-03543669 CDER. Center for Drug Evaluation and Research (CDER). Available from: http://www.fda.gov Connor TH Stoeckel M Evrard J Legator MS. The contribution of metronidazole and two metabolites to the mutagenic activity detected in urine of treated humans and mice. Cancer Res (1977) 37:629–33.318924 Nagasawa HT Muldoon WP Shirota FN. Nitramino acids. Synthesis and biological evaluation of 1-nitroproline, 1-nitropipecolic acid, and N-nitrosarcosine. J Med Chem (1977) 20:1588–91.10.1021/jm00222a010 Chacko M Nagabhushan M Sarkar S Bhide SV. Studies on the possible mutagenic action of metronidazole. Indian J Exp Boil (1987) 25:240–3. Ehlhardt WJ Beaulieu BB Jr Goldman P. Mammalian cell toxicity and bacterial mutagenicity of nitrosoimidazoles. Biochem Pharmacol (1988) 37:2603–6.10.1016/0006-2952(88)90252-33390219 Zeiger E Anderson B Haworth S Lawlor T Mortelmans K. Salmonella mutagenicity tests: V. Results from the testing of 311 chemicals. Environ Mol Mutagen (1992) 19:2–141.10.1002/em.28501906031541260 Gupta RL Vats V Juneja TR. Activation of tinidazole, an antiprotozoal drug to a mutagen by mammalian liver S9. Mutat Res (1996) 370:195–201.10.1016/S0165-1218(96)00076-68917666 Dubini F Riviera L Scaltrito MM Bellotti MG. Susceptibility of Helicobacter pylori and pharmacokinetic properties in mice of new 5-nitroimidazole derivatives devoid of mutagenic activity in the Ames test. J Chemother (1993) 5:168–73.10.1080/1120009X.1993.117392288371126 Grzybowska E Szala S. Mutagenic activity of two nitroimidazole derivatives in the WP2 repair test. Bull Pol Acad Sci (1985) 33:73–8. Das Roy L Giri S Singh S Giri A. Effects of radiation and vitamin C treatment on metronidazole genotoxicity in mice. Mutat Res (2013) 753:65–71.10.1016/j.mrgentox.2013.02.00123416157 Neal SB Probst GS. Chemically-induced sister-chromatid exchange in vivo in bone marrow of Chinese hamsters, An evaluation of 24 compounds. Mutat Res (1983) 113:33–43. Wulf HC. Sister chromatid exchanges in lymphocytes treated with metronidazole, preliminary communication. Dan Med Bull (1980) 27:38–40. Mudry MD Carballo M Labal de Vinuesa M Gonzalez Cid M Larripa I. Mutagenic bioassay of certain pharmacological drugs: III. Metronidazole (MTZ). Mutat Res (1994) 305:127–32.10.1016/0027-5107(94)90230-57510021 IARC. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Lyon: IARC (1977). 113 p. Kelly BE Meikle WD Moore DE. Enhancement of UV-induced skin carcinogenesis by azathioprine: role of photochemical sensitization. Photochem Photobiol (1989) 49:59–65.10.1111/j.1751-1097.1989.tb04078.x Espinosa-Aguirre JJ Reyes RE Cortinas de Nava C. Mutagenic activity of 2-chloro-4-nitroaniline and 5-chlorosalicylic acid in Salmonella typhimurium: two possible metabolites of niclosamide. Mutat Res (1991) 264:139–45.10.1016/0165-7992(91)90131-M1944396 Ostrosky-Wegman P García G Montero R Pérez Romero B Alvarez Chacón R Cortinas de Nava C. Susceptibility to genotoxic effects of niclosamide in human peripheral lymphocytes exposed in vitro and in vivo. Mutat Res (1986) 173:81–7.10.1016/0165-7992(86)90015-13941681 Gupta RL Kaur IP Juneja TR. Genotoxicity of potential metabolites of nitroscanate-an antischistosomal drug. Mutat Res (1995) 335:235–43.10.1016/0165-1161(95)90045-4 E-Makawy A Radwan HA Ghaly IS El-Raouf AA. Genotoxical, teratological and biochemical effects of anthelmintic drug oxfendazole maximum residue limit (MRL) in male and female mice. Reprod Nutr Dev (2006) 46:139–56.10.1051/rnd:2006007 Stauffert I Paulini H Steinmann U Sippel H Estler CJ. Investigations on mutagenicity and genotoxicity of pentamidine and some related trypanocidal diamidines. Mutat Res (1990) 245:93–8.10.1016/0165-7992(90)90006-62215556 Mori Y Hasegawa K Utsunomiya A Setsuda S Goto S Onodera S Ames mutagenicity of pyrethroid pesticides (tetramethrin, permethrin, empenthrin, and allethrin) and 1,1’-oxybis(2,3,3,3-tetrachloropropane) as a synergist. Kankyo hen’igen Kenkyu (1998) 20:29–33. Nishi K Tatsuo N Nishioka H. DNA damaging capacity and mutagenicity of pyrethroid compounds in E. coli DNA repair and salmonella/microsome tests. Sci Eng Rev Doshisha Univ (1985) 26:93–9. Haworth S. Salmonella mutagenicity test results for 250 chemicals. Environ Mutagen (1983) 5:1–142.10.1002/em.2860050703 Kahashi K Ono H Yoshida T Tuboi S. Integration of porin into the outer mitochondrial membrane through the common import machinery located in contact sites. Biochem Mol Biol Int (1993) 29:429–34.8485460 Obermeier J Frohberg H. Mutagenicity studies with praziquantel, a new anthelmintic drug: tissue-, host-, and urine-mediated mutagenicity assays. Arch Toxicol (1977) 38:149–61.10.1007/BF00293649 Suling WJ Struck RF Woolley CW Shannon WM. Comparative disposition of phosphoramide mustard and other cyclophosphamide metabolites in the mouse using the Salmonella/mutagenesis assay. Cancer Treat Rep (1978) 62:1321–8. Loprieno N Barale R Von Halle ES Von Borstel RC. Testing of chemicals for mutagenic activity with Schizosaccharomyces pombe. Mutat Res (1983) 115:215–23.10.1016/0165-1110(83)90004-06343850 Machemer L Lorke D. Mutagenicity studies with praziquantel, a new anthelmintic drug, in mammalian systems. Arch Toxicol (1978) 39:187–97.10.1007/BF00368227580366 Thamavit W Moore MA Ruchirawat S Ito N. Repeated exposure to Opisthorchis viverrini and treatment with the antihelminthic Praziquantel lacks carcinogenic potential. Carcinogenesis (1992) 13:309–11.10.1093/carcin/13.2.3091740024 Vijayalaxmi KK Vishalakshi M. Evaluation of the genotoxic effects of pyrimethamine, an antimalarial drug in the in vivo mouse. Teratog Carcinog Mutagen (2000) 20:65–71.10.1002/(SICI)1520-6866(2000)20:2<65::AID-TCM2>3.0.CO;2-K10679750 Tsuda S Kosaka Y Matsusaka N Sasaki YF. Detection of pyrimethamine-induced DNA damage in mouse embryo and maternal organs by the modified alkaline single cell gel electrophoresis assay. Mutat Res (1998) 415:69–77.10.1016/S1383-5718(98)00057-6 Sasaki YF Izumiyama F Nishidate E Ohta T Ono T Matsusaka N Simple detection of in vivo genotoxicity of pyrimethamine in rodents by the modified alkaline single-cell gel electrophoresis assay. Mutat Res (1997) 392:251–9.10.1016/S1383-5718(97)00079-X9294025 Migliore L Nieri M. Evaluation of twelve potential aneuploidogenic chemicals by the in vitro human lymphocyte micronucleus assay. Toxicol In Vitro (1991) 5:325–36.10.1016/0887-2333(91)90009-320732033 Ji L Shanguan XL Cen JJ Xu YZ Liu YL Xia YE. A mutagenesis, acute and subchronic study of RH-5849. China Acad J Electron Publish House (2005) 36(11). Kafer E Scott BR Dorn GL Stafford R. Aspergillus nidulans: systems and results of tests for chemical induction of mitotic segregation and mutation, I. Diploid and duplication assay systems, a report of the US EPA Gene–Tox Program. Mutat Res (1982) 98:1–48.10.1016/0165-1110(82)90002-1 Watanabe–Akanuma M Ohta T Sasaki YF. A novel genotoxic aspect of thiabendazole as a photomutagen in bacteria and cultured human cells. Toxicol Lett (2005) 158:213–9.10.1016/j.toxlet.2005.03.01315905048 Mudry de Pargament MD Labal de Vinuesa M Larripa I. Mutagenic bioassay of certain pharmacological drugs. I. Thiabendazole (TBZ). Mutat Res (1987) 188:1–6.10.1016/0165-1218(87)90107-83574333 Von der Hude W Kalweit S Engelhardt G McKiernan S In vitro micronucleus assay with Chinese hamster V79 cells – results of a collaborative study with in situ exposure to 26 chemical substances. Mutat Res (2000) 468:137–63.10.1016/S1383-5718(00)00045-0 Tada Y Fujitani T Yano N Yuzawa K Nagasawa A Yoneyam M. Thiabendazole induces urinary tract toxicity in male ICR mice. Toxicology (2001) 162(1):1–10.10.1016/S0300-483X(00)00460-111311453 Fujii T Mikuriya H Sasaki M. Chronic oral toxicity and carcinogenicity study of thiabendazole in rats. Food Chem Toxicol (1991) 29:771–5.10.1016/0278-6915(91)90186-B1761257 Ahmed S Othman OE. Clastogenic effects of the fasciolicide drug fasinex on river buffalo lymphocyte cultures in vitro. Mutat Res (2003) 541:115–21.10.1016/S1383-5718(03)00185-214568300 Kasper P Uno Y Mauthe R Asano N Douglas G Matthews E Follow-up testing of rodent carcinogens not positive in the standard genotoxicity testing battery: IWGT workgroup report. Mutat Res (2007) 627:106.10.1016/j.mrgentox.2006.10.007 Benigni R. Analysis of the National Toxicology Program data on in vitro genetic toxicity tests using multivariate statistical methods. Mutagenesis (1989) 4:412–9.10.1093/mutage/4.6.4122695754 Zeiger E Haseman JK Shelby MD Margolin BH Tennant RW. Evaluation of four in vitro genetic toxicity tests for predicting rodent carcinogenicity: confirmation of earlier results with 41 additional chemicals. Environ Mol Mutagen (1990) 16:1–14.10.1002/em.28501605022091921 Zeiger E. Mutagens that are not carcinogens: faulty theory or faulty tests? Mutat Res (2001) 492:29–38.10.1016/S1383-5718(01)00153-X11377241 Kirkland DJ Hayashi M Jacobson-Kram D Kasper P MacGregor JT Muller L Summary of major conclusions from the 4th IWGT, San Francisco. Mutat Res (2005) 627:5–9.10.1016/j.mrgentox.2006.08.009 Thybaud V Aardema M Clements J Dearfield K Galloway S Hayashi M Strategy for genotoxicity testing: hazard identification and risk assessment in relation to in vitro testing. Mutat Res (2007) 627:41–58.10.1016/j.mrgentox.2006.10.00317126066 Müller L Kikuchi Y Probst G Schechtman L Shimada H Sofuni T ICH-harmonised guidances on genotoxicity testing of pharmaceuticals: evolution, reasoning and impact. Mutat Res (1999) 436:195–225.10.1016/S1383-5742(99)00004-6 Kirkland DJ Müller L. Interpretation of the biological relevance of genotoxicity test results: the importance of thresholds. Mutat Res (2000) 464:137–47.10.1016/S1383-5718(99)00175-810633186 Müller L Kasper P. Human biological relevance and the use of threshold-arguments in regulatory genotoxicity assessment: experience with pharmaceuticals. Mutat Res (2000) 464:19–34.10.1016/S1383-5718(99)00163-110633174 Hilliard CA Armstrong MJ Bradt CI Hill RB Greenwood SK Galloway SM. Chromosome aberrations in vitro related to cytotoxicity of non-mutagenic chemicals and metabolic poisons. Environ Mol Mutagen (1998) 31:316–26.10.1002/(SICI)1098-2280(1998)31:4<316::AID-EM3>3.0.CO;2-G Galloway S. Cytotoxicity and chromosome aberrations in vitro: experience in industry and the case for an upper limit on toxicity in the aberration assay. Environ Mol Mutagen (2000) 35:191–201.10.1002/(SICI)1098-2280(2000)35:3<191::AID-EM6>3.0.CO;2-410737954 Parry JM Parry E Phrakonkha P Corvi R. Analysis of published data for top concentration considerations in mammalian cell genotoxicity testing. Mutagenesis (2010) 25:531–8.10.1093/mutage/geq04620720196 Tweats DJ Blakey D Heflich RH Jacobs A Jacobsen SD Morita T Report of the IWGT working group on strategies and interpretation of regulatory in vivo tests. I. Increases in micronucleated bone marrow cells in rodents that do not indicate genotoxic hazards. Mutat Res (2007) 627:78–91.10.1016/j.mrgentox.2006.10.00517116417 Brambilla G Martelli A. Genotoxic and carcinogenic risk to humans of drugnitrite interaction products. Mutat Res (2007) 635:17–52.10.1016/j.mrrev.2006.09.003 Zeiger E. Identification of rodent carcinogens and non-carcinogens using genetic toxicity tests: premises, promises, and performance. Regul Toxicol Pharmacol (1998) 28:85–95.10.1006/rtph.1998.1234 Brambilla G Mattioli F Martelli A. Genotoxic and carcinogenic effects of antipsychotics and antidepressants. Toxicology (2009) 261:77–88.10.1016/j.tox.2009.04.05619410629 Brambilla G Mattioli F Robbiano L Martelli A. Genotoxicity and carcinogenicity testing of pharmaceuticals: correlations between induction of DNA lesions and carcinogenic activity. Mutat Res (2010) 705:20–39.10.1016/j.mrrev.2010.02.00420206292 Cooper JA Saracci R Cole P. Describing the validity of carcinogen screening tests. Br J Cancer (1979) 39:87–9.10.1038/bjc.1979.10 Brambilla G Martelli A. Update on genotoxicity and carcinogenicity testing of 472 marketed pharmaceuticals. Mutat Res (2009) 681:209–29.10.1016/j.mrrev.2008.09.00218845271 Kirkland D Aardema M Muller L Hayashi M. Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens II. Further analysis of mammalian cell results, relative predictivity and tumour profiles. Mutat Res (2006) 608:29–42.10.1016/j.mrgentox.2006.04.01716769241 Trosko JE Upham BL. The emperor wears no clothes in the field of carcinogen risk assessment: ignored concepts in cancer risk assessment. Mutagenesis (2005) 20:81–92.10.1093/mutage/gei01715784692 LeBarona MJ Rasoulpoura RJ Klapacza J Ellis–Hutchings RG Hollnagel HM Gollapudi BB. Epigenetics and chemical safety assessment. Mutat Res (2010) 705:83–95.10.1016/j.mrrev.2010.04.00320399890 Friedrich A Olejniczak K. Evaluation of carcinogenicity studies of medicinal products for human use authorised via the European Centralised Procedure (1995–2009). Regul Toxicol Pharmacol (2011) 60:225–48.10.1016/j.yrtph.2011.04.001 Bercu JP Jolly RA Flagella KM Baker TK Romero P Stevens JL. Toxicogenomics and cancer risk assessment: a framework for key event analysis and dose-response assessment for nongenotoxic carcinogens. Regul Toxicol Pharmacol (2010) 58:369–81.10.1016/j.yrtph.2010.08.00220801182