Safety evaluation of a triazine compound nitromezuril by assessing bacterial reverse mutation, sperm abnormalities, micronucleus and chromosomal aberration

1

3 Safety evaluation of a triazine compound nitromezuril by assessing

4 bacterial reverse mutation, sperm abnormalities, micronucleus and

5 chromosomal aberrationQ1

6

7

8 Chenzhong Fei aQ2 , Jie Zhang b, Yang Lin c, Xiaoyang Wang a, Keyu Zhang a, Lifang Zhang a, Wenli Zheng a,

9 Mi Wang a, Tao Li a, Sui Xiao a, Feiqun Xue a,⇑, Chunmei Wang a

10 aKey Laboratory of Veterinary Drug Safety Evaluation and Residues Research, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241,11 PR China12 bChinese Medicine Hospital of Pudong New Area, Shanghai 201200, PR China13 c Institute of Veterinary Feed Control of Zhengzhou, Henan 450003, PR China

1415

1 7a r t i c l e i n f o

18 Article history:19 Received 25 August 201420 Available online xxxx

21 Keywords:22 Coccidiosis23 Anticoccidials24 Nitromezuril25 Bacterial reverse mutation (Ames)26 Sperm abnormalities27 Bone marrow micronucleus28 Chromosome aberration29

3 0

a b s t r a c t

31Nitromezuril (NZL) is a novel triazine compound that exhibits remarkable anticoccidial activity. However,

32mutagenicity and genotoxicity of NZL have not been evaluated to date. This study evaluated the potential

33risks of NZL by testing for bacterial reverse mutation (Ames), mouse sperm abnormality (SA), bone mar-

34row micronucleus (MN) and chromosomal aberration (CA). Mice were orally administered with NZL at

35385, 192 and 96 mg/kg, corresponding to 0.5�, 0.25� and 0.125� the LD50 of NZL, respectively. No sig-

36nificant increases in SA and CA were found in mice treated with NZL for 5 d and 3 d, respectively

37(P > 0.05). NZL at 96–385 mg/kg did not have significant influence on micronucleated polychromatic ery-

38throcyte counts (P > 0.05). These results suggest that NZL is not genotoxic. However, Ames test results

39were positive both with and without the S9 system for Salmonella typhimurium TA98 and TA100, suggest-

40ing that NZL may be mutagenic. The mutagenic effects of NZL were different in in vitro and in vivo assays.

41Further studies should be conducted to confirm the safety of using and developing NZL as a novel antic-

42occidial drug.

43� 2015 Published by Elsevier Inc.

44

45

46

47 1. Introduction

48 Coccidiosis is the most important parasitic diseases affecting

49 the poultry industry. Proper control of coccidiosis depends princi-

50 pally on prophylactic chemotherapy with anticoccidial drugs (Peek

51 and Landman, 2011). However, development of drug resistance

52 poses a tremendous challenge, so identification of alternative

53 drugs is an active area of research. Triazine coccidiostats, including

54 diclazuril and toltrazuril, have been widely used in chickens and

55 turkeys because of their remarkable clinical effects on Eimeria spe-

56 cies. However, they have faced significant problems related to drug

57 resistance in recent years (Conway et al., 2001; Fernandez et al.,

58 2012; Kreiner et al., 2011; Wang et al., 2013).

59 2-(3-Methyl-4-(4-nitrophenoxy)phenyl)-1,2,4-triazine-

60 3,5(2H,4H)-dione, also known as nitromezuril (NZL; CAS:1352755-

61 63-5), is a potential novel anticoccidial agent developed in our

62laboratories (Fei et al., 2013a; Ma et al., 2014). Chemical synthesis

63of this compound has been described in China Patent No. CN

64102285930. The structure of NZL is similar to that of diclazuril

65and toltrazuril, but no cross-resistance was observed. NZL is effec-

66tive against different life cycles of various parasites. A previous

67study reported that the mouse oral LD50 of NZL is 769 mg/kg, with

68a 95% confidence interval of 615–960 mg/kg by the Bliss method

69and an accumulative coefficient of 4.26 (Fei et al., 2012). A 30 d

70subacute toxicity test in mice revealed that NZL has no obvious

71toxic effects (Fei et al., 2013b). A 60 d subchronic toxicity trial in

72chickens demonstrated that NZL exhibits low toxicity and is safe

73for clinical use (Fan et al., 2014). However, mutagenicity and geno-

74toxicity of NZL remain unclear to date. Determining the potential

75risks of NZL is important to establish NZL as a safe drug (Jena

76et al., 2002; Li, 2004; Merino et al., 2010).

77Drugs for registration must be thoroughly tested for safety

78because of the nominal association between mutagenicity and car-

79cinogenesis. In recent years, many mutagenicity tests with varying

80degrees of usefulness and extrapolation to humans have been

81developed. Scientists and regulatory agencies have found that no

http://dx.doi.org/10.1016/j.yrtph.2015.01.011

0273-2300/� 2015 Published by Elsevier Inc.

⇑ Corresponding author at: No. 518, Ziyue Road, Minhang District, Shanghai

200241, PR China.

E-mail address: [email protected] (F. Xue).

Regulatory Toxicology and Pharmacology xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Regulatory Toxicology and Pharmacology

journal homepage: www.elsevier .com/locate /yr tph

YRTPH 3223 No. of Pages 6, Model 5G

29 January 2015

Please cite this article in press as: Fei, C., et al. Safety evaluation of a triazine compound nitromezuril by assessing bacterial reverse mutation, sperm abnor-

malities, micronucleus and chromosomal aberrationQ1 . Regul. Toxicol. Pharmacol. (2015), http://dx.doi.org/10.1016/j.yrtph.2015.01.011

82 single mutagenicity test can detect all types of potential human

83 mutagens with 100% accuracy or prediction (Hwang and Kim,

84 2012; Kamath and Rao, 2013). Therefore, most regulatory agencies

85 such as the Food and Drug Administration and the Environmental

86 Protection Agency require a battery of mutagenicity tests, includ-

87 ing sperm abnormalities (SA), bacterial reverse mutation (Ames),

88 bone marrow micronucleus (MN) and chromosomal aberration

89 (CA) tests (Kamath and Rao, 2013; Mitchell and Skibinski, 2012).

90 Comprehensive assessment of mutagenicity and genotoxicity of

91 the novel triazine drug NZL is necessary to understand the risks of

92 potential genotoxicity and its safety for clinical use. In this study,

93 we conducted the Ames test and the mouse SA, MN and CA tests

94 in accordance with the guidelines of veterinary safety evaluation

95 to evaluate the mutagenicity and genotoxicity of NZL and thereby

96 determine its safety for use in dietary supplements.

97 2. Materials and methods

98 2.1. Chemicals and animals

99 NZL (>98%) was synthesized by the Shanghai Veterinary

100 Research Institute of the Chinese Academy of Agricultural Sciences.

101 Sodium carboxy-methyl cellulose (CMC, >98%) was purchased

102 from Aladdin Chemical Co. (Shanghai, China). Cyclophosphamide

103 (CPA, >98%) was obtained from Aesar Chemical Co. (Shanghai, Chi-

104 na), while nicotinamide-adenine dinucleotide phosphate (NADP)

105 was bought from Merck (Rahway, NJ, USA). Sodium azide (SA), D-

106 biotin, L-histidine, ampicillin, tetracycline were obtained from San-

107 gon Biotech Co. (Shanghai, China). 2-Amino-anthracene (2AA), 2-

108 amino-fluorene, fenaminosulf, glucose-6-phosphate (G6P),

109 dimethyl sulphoxide (DMSO) and colchicines were obtained from

110 Sigma–Aldrich (St. Louis, MO, USA). Citric acid monohydrate,

111 NaOH, KCl, NaCl, and other chemicals were purchased from Sino-

112 pharm Chemical Reagent Co. Ltd. (Shanghai, China).

113 Kunming mice weighing 25–30 g were purchased from the

114 Shanghai Laboratory Animal Centre of Chinese Academy of

115 Sciences (License No. SCXK (Hu) 2007-0005). The mice were quar-

116 antined in a pathogen-free, well-ventilated room to enable them to

117 acclimatize to their environment. The animals were housed in

118 wire-topped polypropylene cages with rice husk bedding and

119 maintained under standard laboratory conditions at 28 ± 5 �C,

120 60 ± 5% relative humidity and 12 h photoperiod. The animals were

121 given food in pellet form and water ad libitum.

122 2.2. Selection of dose

123 The NZL concentrations used in the Ames test were based on

124 bacterial toxicity estimated in a pre-trial and were as follows:

125 1000, 200, 40 and 8 lg/plate. The doses selected for in vivo muta-

126 genic and genotoxic assays were set to 385, 192 and 96 mg/kg b.w.,

127 corresponding to 0.5�, 0.25� and 0.125� the LD50 of NZL, respec-

128 tively based on previous toxicity tests and recommended guideli-

129 nes for toxicity studies of drugs (Kamath and Rao, 2013). Test

130 substances of three concentrations were prepared by serial dilu-

131 tions using 0.5% CMC. The suspension volumes by oral gavages

132 were approximately 0.2 mL/10 g weight.

133 2.3. Ames test

134 Four strains of Salmonella typhimurium TA97, TA98, TA100 and

135 TA1535 were kindly given by Dr. Jin Ma, National Shanghai Center

136 for New Drug Safety Evaluation Research Center. Genotypes of the-

137 se strains were confirmed using the method described by the state

138 standard for Ames (GB 15193.4-2003, PR China). Positive mutage-

139 nesis controls were seen in legends of Table 1.

140NZL was assayed via the Ames test by standard plate-incorpora-

141tion assay according to OECD Guideline 471 (1997) and the state

142standard for Ames (GB 15193.4-2003, PR China). The preliminary

143Ames test was performed both with and without the S9 activation

144system. The metabolic activation system (S9 mix) was freshly pre-

145pared before each test using an Aroclor-1254-induced rat liver

146fraction (S-9, MolTox™, Boone, NC, USA). Concentrations of NZL

147used in this study were 1000, 200, 40 and 8 lg/plate. All of them

148were diluted in DMSO. Experiments were performed in triplicate.

149All strains were tested for spontaneous revertant colonies using

150DMSO as negative (solvent) control. The mutagenic index (MI)

151was calculated for each dose as the average number of revertants

152per plate divided by the average number of revertants per plate

153of the negative (solvent) control. A sample was considered positive

154when MI was above 2 for at least one of the test doses, and when

155the response was dose-dependent (Biso et al., 2010).

1562.4. SA test

157The SA test was carried out according to the veterinary guideli-

158nes on SA test in mice and the state standard for sperm abnor-

159malities (GB15193.7-2003, PR China). Fifty male mice were

160divided into five groups, with 10 mice in each group, using a

161restricted randomization procedure. Mice in groups 1–3 were

162treated once daily with 0.5�, 0.25� and 0.125� the LD50 of NZL

163through oral gavages for five consecutive days. Mice in group 4 (-

164positive control) were administered by gavages with 50 mg/kg b.w.

165CPA, and mice in group 5 (negative control) received 0.5 mL of nor-

166mal 0.5% CMC.

167At 35 d after the first oral administration, the mice from each

168dosage group and the controls were sacrificed by cervical disloca-

169tion and their epididymis Q3were surgically removed. Sperm smears

170were prepared from the epididymis as reported by Otubanjo and

171Mosuro (2001) and described in the guidelines. Slides were ran-

172domly coded prior to microscopic analysis, and 1000 sperm cells

173from each mouse were assessed for morphological abnormalities

174of the sperm head according to the criteria of Wyrobek and

175Bruce (1975).

1762.5. Bone marrow MN test

177The MN test was performed according to the veterinary guide-

178lines on MN test in mice and the state standard for bone marrow

179cell micronucleus (GB15193.5-2003, PR China). One hundred mice

180were divided into five groups, with 10 male and 10 female mice in

181each group, using a restricted randomization procedure. Mice in

182groups 1–3 were treated twice with 0.5�, 0.25� and 0.125� the

183LD50 of NZL by oral gavages at 24 h intervals. Mice in group 4 (-

184positive control) were administered with 50 mg/kg b.w. CPA, and

185mice in group 5 (negative control) received 0.5 mL of normal

1860.5% CMC.

187At 30 h after the first oral administration, the mice were sacri-

188ficed by cervical dislocation. The femur bones of each mouse were

189separated and cleaned of surrounding muscle tissue. Two ends of

190the femur were cut until a small opening became visible.

191Approximately 0.3 mL of bovine albumin was injected into one

192opening via syringe. The bone marrow was flushed into a clean,

193dry glass slide, mixed well and smeared onto the slides. Subse-

194quent processing was performed as described by Hwang and Kim

195(2012). Micronucleus slides were randomly coded prior to micro-

196scopic analysis, and 1000 bone marrow polychromatic erythro-

197cytes (PCE) were examined from each mouse. The number of

198micronucleated PCE, PCE and normochromatic erythrocytes (NCE)

199was recorded (Kasamoto et al., 2013).

2 C. Fei et al. / Regulatory Toxicology and Pharmacology xxx (2015) xxx–xxx

YRTPH 3223 No. of Pages 6, Model 5G

29 January 2015

Please cite this article in press as: Fei, C., et al. Safety evaluation of a triazine compound nitromezuril by assessing bacterial reverse mutation, sperm abnor-

malities, micronucleus and chromosomal aberrationQ1 . Regul. Toxicol. Pharmacol. (2015), http://dx.doi.org/10.1016/j.yrtph.2015.01.011

200 2.6. Bone marrow CA test

201 The CA test was conducted according to the veterinary guideli-

202 nes on mice bone marrow chromosome aberrations test and the

203 state standard for the national standard of mammalian bone mar-

204 row cell chromosome aberration (GB15193.6-2003, PR China). Fifty

205 mice, half male and half female, were randomly divided into five

206 groups. Mice in groups 1 to 3 were treated thrice with 0.5�,

207 0.25� and 0.125� the LD50 of NZL by oral gavages at 24 h inter-

208 vals. Mice in group 4 (positive control) were administered with

209 40 mg/kg b.w. CPA, and mice in group 5 (negative control) received

210 0.5 mL of normal 0.5% CMC.

211 Four hours before sacrificing the mice, 4 mg/kg b.w. colchicine

212 was injected intraperitoneally. Mice were sacrificed by cervical dis-

213 location at 24 h after the final dose of NZL, CPA or CMC was admin-

214 istered. The femur bones of each mouse were separated and

215 cleaned of surrounding muscle tissue. Two ends of the femur were

216 cut until a small opening was visible. Approximately 5 mL of 2.2%

217 sodium citrate solution was injected into one opening of the bone

218 with a syringe. The bone marrow was flushed into a clean, dry cen-

219 trifuge tube and then centrifuged at 1000 rpm for 10 min. After dis-

220 carding the supernatant, the collected cells were disturbed by

221 gently tapping the tube that was resuspended in approximately

222 6 mL of pre-warmed hypotonic solution (0.075 mol/L KCl) for

223 20 min at 37 �C. Then, the cells were fixed with 2 mL of metha-

224 nol/acetic acid (3:1, v/v, extemporaneous) and then immediately

225 centrifuged at 1000 rpm for 10 min. The supernatant was discard-

226 ed. The cells were resuspended in another 4 mL of fixative, placed

227 at room temperature for 15 min and then centrifuged at 1000 rpm

228 for 10 min. The supernatant was discarded. The collected cells

229 were fixed again using the same method. Chromosomes were pre-

230 pared using the air-drying method and then stained with 2% Giem-

231 sa (0.01 M phosphate buffer, pH 6.8). A total of 200 well-spread

232 metaphases from each mouse were scored blindly. Additional scor-

233 ing and counting criteria were set according to Carrano and

234 Natarajan (1988) and Pacchierotti and Stocchi (2013).

235 2.7. Statistical analysis

236 Data obtained in this study are expressed as mean ± SD. The fre-

237 quencies of SA, MN and CA in the treated groups were compared

238 with those of controls in Chi-square and Fisher’s exact tests. The

239dose–response relationship was evaluated using the Cochran–Ar-

240mitage trend test. All analyses were performed using SPSS 15.0

241for Windows, and differences were considered statistically sig-

242nificant at the probability level of less than 0.05 or 0.01 for all tests.

243The assay result was considered positive when MI was above 2 for

244at least one of the test doses or the frequencies of SA, MN and CA

245showed statistically significant increases through at least two con-

246secutive dose levels, and when abnormalities increased in a dose-

247dependent manner.

2483. Results

249The mutagenic activities of NZL are summarized in Table 1. Sig-

250nificant bacteriotoxicity was observed on strain TA97 at concentra-

251tions of 200 and 1000 lg/plate, both in the presence and absence of

252the S9 activation system. Bacteriotoxicity was also found on strain

253TA100 at 1000 lg/plate in the absence of the S9 activation system

254(P < 0.01). Regardless of whether the S9 mix was present, the num-

255ber of revertants induced in strains TA97 and TA1535 were not sig-

256nificantly different than those of the negative (solvent) control at

257any NZL treatment concentration (P > 0.05). The number of rever-

258tants induced at NZL concentrations of 40–1000 lg/plate increased

259significantly in strains TA98 and TA100, both in the presence and

260absence of the S9 activation system. Most MIs were above 2 and

261the response was dose-dependent. According to the Ames test cri-

262teria, NZL was positive both in the presence and absence of the S9

263system for S. typhimurium TA98 and TA100.

264The SA test revealed that the abnormal sperms were primarily

265amorphous and lacked hooks. Table 2 shows the effects of different

266NZL concentrations on sperm morphology of the mice at 5 weeks

267after the first administration. The numbers of abnormal sperms

268in the mice treated with NZL at 96, 192 and 385 mg/kg b.w. were

26920.6 ± 2.0, 21.2 ± 2.6 and 24.8 ± 8.2, respectively. The numbers of

270abnormal sperms in the negative and positive control groups were

27121.0 ± 2.5 and 63.8 ± 19.8, respectively. The positive control group

272showed a significant increase in the number of abnormal sperm

273heads (P < 0.01). No significant difference in the number of abnor-

274mal sperm heads was observed between the NZL groups and the

275negative control group (P > 0.05). Since NZL is widely distributed

276in liver, kidney, testis, brain and other tissues. This result indicates

277that NZL has no significant effect on sperm morphology.

Table 1

Mutagenic activity and mutagenic index (MI) of NZL in TA97, TA98, TA100 and TA1535 Salmonella typhimurium strains in the absence (�S9) and in the presence (+S9) of

metabolization.

Group S9 TA97 TA98 TA100 TA1535

Rev/plate MI Rev/plate MI Rev/plate MI Rev/plate MI

NC-S9mix � 98 ± 7 22 ± 4 153 ± 6 13 ± 4

PC-S9mix � 1230 ± 173 12.55 1099 ± 132 49.95 1399 ± 186 9.14 765 ± 93 58.85

1000 lg/plate � 3 ± 4 0.03 196 ± 25 8.91 11 ± 6 0.07 14 ± 5 1.08

200 lg/plate � 34 ± 5 0.35 98 ± 2 4.45 695 ± 15 4.54 13 ± 3 1.00

40 lg/plate � 99 ± 7 1.01 40 ± 1 1.82 292 ± 10 1.91 17 ± 5 1.31

8 lg/plate � 97 ± 7 0.99 18 ± 2 0.82 183 ± 5 1.20 12 ± 4 0.92

NC + S9mix + 99 ± 15 19 ± 3 132 ± 12 13 ± 2

PC + S9mix + 1342 ± 125 13.56 1128 ± 113 59.37 1445 ± 157 10.95 345 ± 46 56.53

1000 lg/plate + 4 ± 1 0.40 201 ± 30 10.58 1342 ± 46 10.17 12 ± 2 0.92

200 lg/plate + 33 ± 8 0.33 99 ± 24 5.21 768 ± 33 5.82 14 ± 5 1.08

40 lg/plate + 94 ± 10 0.95 56 ± 13 2.95 340 ± 22 2.58 14 ± 3 1.08

8 lg/plate + 98 ± 8 0.99 23 ± 4 1.21 177 ± 21 1.34 15 ± 3 1.15

�S9, without metabolic activation; +S9, with metabolic activation (500 lL of 10% S9 mix/plate); S9, an Aroclor-1254 induced rat liver; NC, negative (solvent) controls; PC,

positive controls; Rev, number of revertants.

Positive controls without S9 activation were fenaminosulf (50 lg/plate) for strains TA97 and TA98, sodium azide (1.5 lg/plate) for strain TA100 and sodium azide

(50 lg/plate) for strain TA1535.

Positive controls with S9 activation were 2-amino-fluorene (10 lg/plate) for strains TA97, TA98 and TA100, 2-amino-anthracene (10 lg/plate) for strain TA1535.

MI, mutagenicity index: number of revertants induced in the sample/number of spontaneous revertants in the negative (solvent) controls.

C. Fei et al. / Regulatory Toxicology and Pharmacology xxx (2015) xxx–xxx 3

YRTPH 3223 No. of Pages 6, Model 5G

29 January 2015

Please cite this article in press as: Fei, C., et al. Safety evaluation of a triazine compound nitromezuril by assessing bacterial reverse mutation, sperm abnor-

malities, micronucleus and chromosomal aberrationQ1 . Regul. Toxicol. Pharmacol. (2015), http://dx.doi.org/10.1016/j.yrtph.2015.01.011

278 Table 3 shows the effects of NZL on bone marrow micronucleus

279 after 30 h of treatment. No differences in sensitivity to micronucle-

280 us inducers were observed between the male and female mice. No

281 significant differences in the frequencies of micronucleated ery-

282 throcyte cell counts were observed between the NZL treatment

283 groups and the negative control group (P > 0.05). Additionally, no

284 significant differences were found among the three dose levels

285 (P > 0.05). The micronucleated erythrocyte cell counts were higher

286 in the positive control group than in the NZL groups and the nega-

287 tive control group (P < 0.01). The PCE and NCE ratios of all groups

288 were within the normal range (0.81–1.05), with no statistically sig-

289 nificant difference compared to the control groups. This indicates

290 that NZL at the three doses exhibit no cytotoxicity or obvious inhi-

291 bition of PCE formation.

292 Table 4 shows the effects of chromosomal aberrations on Kun-

293 ming mice at 72 h after NZL treatment. The average percentages

294 of the total number of chromosomal aberrations in the negative

295 and positive control groups were 0.89‰ and 5.67‰, respectively.

296 In the NZL groups, the average percentage of the total number of

297 chromosomal aberrations ranged from 0.88‰ to 0.93‰. The num-

298 ber of chromosomal aberrations, including chromatid and chromo-

299 some breaks, acentric fragments, dicentric chromosomes and

300 chromatid exchanges, was significantly higher in the positive con-

301 trol group than in the negative control group and the NZL groups

302 (P < 0.01). No significant difference in the percentage of chromoso-

303 mal aberrations was observed between the NZL groups and the

304 negative control group, and no significant differences were

305 observed among the three dose levels (P > 0.05).

3064. Discussion

307Rigorous safety evaluation is necessary to determine whether

308the triazine compound NZL is safe to use as a novel anticoccidial

309drug. Information on genotoxicity is a key component of risk

310assessment of chemicals in general. In this study, potential geno-

311toxicity of the novel triazine drug NZL was examined in bone mar-

312row samples by conducting the Ames test, and the SA, MN and CA

313tests, as recommended by various regulatory agencies (Chinese

314FDA, U.S. FDA, among others).

315The Ames test has been used worldwide as an initial screen to

316determine the mutagenic potential of new chemicals and drugs.

317Our results showed that NZL did not induce mutagenesis at any

318concentration on strains TA97 and TA1535, regardless of whether

319the S9 mix was present. NZL induced mutagenesis in the frameshift

320mutation-detecting strain TA98 (DNA target –C–G–C–G–C–G–C–

321G), both with and without the S9 mix. NZL also induced mutage-

322nesis in the TA100 strain, which detects base pair substitutions

323of a leucine [GAG] by proline [GGG], but did not induce mutage-

324nesis in the corresponding isogenic strain TA1535 Q4(McCarroll

325et al., 2010; Mortelmans and Zeiger, 2000). These results suggest

326that NZL may induce frameshift mutations and base pair substitu-

327tions. However, the structurally related drugs diclazuril and toltra-

328zuril were tested negative. The genotoxic mechanism by which

329NZL induces these mutations is unclear. For following up on posi-

330tive in vitro gene mutation results, both the transgenic rodent gene

331mutation assay and the Comet assay are suitable (Kirkland and

332Speit, 2008). These studies can help understand the mechanism

333of action of NZL and the impact of DNA repair mechanisms, which

334may also have tumorigenic potential. (Lambert et al., 2005; McCar-

335roll et al., 2010; Sasaki et al., 2000).

Table 2

Effects of NZL on sperm morphology 35 days after exposure in Kunming mice.

Groups No. of mice Types and number of abnormal sperms Means SA SA (%) P values

Lack hook Banana like Amorphous Folded Two tails

CPA 50 mg/kg b.w (positive control) 10 156 83 332 51 16 63.8 ± 19.8 6.38 <0.01

0.5% CMC (negative control) 10 64 24 100 20 2 21.0 ± 2.5 2.10

NZL, 96 mg/kg b.w 10 64 20 104 16 2 20.6 ± 2.0 2.06 >0.05

NZL, 192 mg/kg b.w 10 66 22 102 20 2 21.2 ± 2.6 2.12 >0.05

NZL, 385 mg/kg b.w 10 70 26 126 28 4 24.8 ± 8.2 2.54 >0.05

NZL, nitromezuril; SA, sperm abnormality; and CPA, cyclophosphamide; CMC, sodium carboxy-methyl cellulose.

Table 3

Effects of bone marrow micronucleus in Kunming mice after administrated with NZL 30 hours.

Chemical Dose (mg/kg b.w) No. of mice No. of MNPCEs/1000PCEs P values PCEs/NCEs P values

CPA (positive control) 50 10 11.62 ± 3.85 <0.01 0.81 ± 0.18 >0.05

0.5% CMC (negative control) 0 10 1.87 ± 1.45 1.05 ± 0.23

NZL 96 10 1.56 ± 1.13 >0.05 0.84 ± 0.25 >0.05

192 10 1.56 ± 1.01 >0.05 0.97 ± 0.25 >0.05

385 10 2.00 ± 1.00 >0.05 0.83 ± 0.24 >0.05

NZL, Nitromezuril; CPA, cyclophosphamide; CMC, sodium carboxy-methyl cellulose; MNPCE, PCE with one or more micronucleus; PCE, polychromatic erythrocyte; and NCE,

normochromatic erythrocyte.

Table 4

Effects of chromosomal aberrations in Kunming mice after dosing NZL 72 h.

Chemical Dose (mg/kg) No. of mice Metaphases cells observed Total No. of aberrant cells CA (‰) P values

CPA 40 10 2000 35 5.67 ± 1.49 <0.01

CMC 0 10 2000 5 0.89 ± 0.31

NZL 96 10 2000 7 0.93 ± 0.58 >0.05

192 10 2000 6 0.88 ± 0.88 >0.05

385 10 2000 9 0.93 ± 1.05 >0.05

NZL, nitromezuril; CPA, cyclophosphamide (positive control); CMC, sodium carboxy-methyl cellulose (Negative control); and CA, chromosomal aberrations.

4 C. Fei et al. / Regulatory Toxicology and Pharmacology xxx (2015) xxx–xxx

YRTPH 3223 No. of Pages 6, Model 5G

29 January 2015

Please cite this article in press as: Fei, C., et al. Safety evaluation of a triazine compound nitromezuril by assessing bacterial reverse mutation, sperm abnor-

malities, micronucleus and chromosomal aberrationQ1 . Regul. Toxicol. Pharmacol. (2015), http://dx.doi.org/10.1016/j.yrtph.2015.01.011

336 The mouse spermmorphology assay developed byWyrobek has

337 been widely adopted for evaluating mammalian germ cell muta-

338 gens. In the present study, we analyzed sperm head abnormalities

339 at 5 weeks after NZL treatment. Sperm cells, particularly early pri-

340 mary spermatocytes and spermatogonia, were exposed to the drug.

341 Different types of abnormal sperm heads were observed at differ-

342 ent test concentrations. However, the number of abnormal sperms

343 observed after NZL treatment was not significantly different com-

344 pared to the control groups. At 96, 192 and 385 mg/kg b.w., NZL

345 had no significant effect on sperm morphology. Based on these

346 results, NZL was deemed non-genotoxic.

347 The mouse bone marrowMN assay developed by Schmid (1975)

348 is frequently used as a reliable in vivo assay system to assess muta-

349 genicity of chemicals, regardless of the endpoint of mutagenicity

350 (Kasamoto et al., 2013). Micronucleus frequency is often used to

351 assess the capacity of target chemicals to induce structural and

352 numerical chromosomal aberrations. Chromosomal aberration is

353 one of the main endpoints indicating genotoxicity and muta-

354 genicity of chemicals. Good correlation has been found between

355 chromosomal aberration induction and micronucleus induction.

356 Micronucleus frequencies and chromosomal alterations in bone

357 marrow cells are also used in cancer screens in high risk human

358 populations (Han et al., 2007). In the present study, mouse femoral

359 marrow cells tested negative for MN and CA after NZL treatment at

360 96, 192 and 385 mg/kg b.w. These clearly negative results indicate

361 that NZL does not exhibit genotoxicity. However, the positive

362 result for S. typhimurium TA98 and TA100 in the Ames test indicate

363 that NZL is a potential mammalian carcinogen.

364 Themutagenic effects of NZL were completely different in in vit-

365 ro and in vivo assays. In vitro cell assays that evaluate drug geno-

366 toxicity do not completely replicate the in vivo ADME (absorption,

367 distribution, metabolism and excretion) properties of the drug, and

368 false positive rates are usually high in these assays (Guguen-

369 Guillouzo and Guillouzo, 2010). In vivo assessment of genotoxicity

370 is important for understanding and interpreting a positive result

371 obtained in an vitro test. Such data can also provide a better under-

372 standing of the ADME of a test compound. NZL has reported three

373 metabolites in in vitro metabolism study (Zhang et al., 2014). The

374 proposed metabolic pathways could be expected to play key roles

375 in toxicology. Determining whether these metabolites have any

376 pharmacologic and toxicologic activities requires further investiga-

377 tion. These data and the results will help to improve and optimize

378 the design of compounds.

379 In summary, the doses used in this study were 385, 192 and

380 96 mg/kg, which were greater than 100 times the recommended

381 clinical dose, and the results were negative at all concentrations.

382 However, the Ames test results were positive for S. typhimurium

383 TA98 and TA100, regardless of whether the S9 mix was present.

384 The mutagenic effects of NZL were completely different in in vitro

385 and in vivo assays. The metabolic pathways of NZL could be

386 expected to play key roles in toxicology. Overall, our results indi-

387 cate that NZL is relative safe but the genotoxicity is equivocal

388 and inconclusive. Additional studies should be performed to fur-

389 ther validate these results.

390 5. Conclusions

391 In this study, mutagenicity and genotoxicity of the new triazine

392 compound NZL were evaluated. Mice treated with NZL at 96, 192

393 and 385 mg/kg b.w. tested negative for SA, MN and CA. However,

394 the Ames test results were positive for S. typhimurium TA98 and

395 TA100, and negative for TA97 and TA1535. The mutagenic effects

396 of NZL were completely different in in vitro and in vivo assays.

397 Overall, our results indicate that the genotoxicity of NZL is equivo-

398 cal and inconclusive. Additional studies should be performed to

399further validate these results. NZL is a potential novel anticoccidial

400agent suitable for further development.

4016. Uncited references

402Degrandi et al. (2010), Föllmann et al. (2013), Hayashi et al.

403(1984) and O’Brien et al. (2014). Q5

404Acknowledgments

405This work was financially supported by the Q6National Natural

406Science Foundation of China Q7(31272607), the Special Fund for

407Agro-Scientific Research in the Public Interest (201303038-1) and

408the National High Technology Research and Development Program

409of China (863 program) (2011AA10A214). The authors express

410their gratitude to Dr. Shijin Pu of Yangzhou University for support

411in conducting the chromosomal aberration tests.

412References

413Biso, F.I. et al., 2010. Assessment of DNA damage induced by extracts, fractions and414isolated compounds of Davilla nitida and Davilla elliptica (Dilleniaceae). Mutat.415Res. 702, 92–99.416Carrano, A.V., Natarajan, A.T., 1988. Consideration for population monitoring using417cytogenetic techniques. Mutat. Res. 204, 379–406.418Conway, D. et al., 2001. Efficacy of diclazuril in comparison with chemical and419ionophorous anticoccidials against Eimeria spp. in broiler chickens in floor pens.420Poult. Sci. 80, 426–430.421Degrandi, T.H. et al., 2010. Evaluation of the cytotoxicity, genotoxicity and422mutagenicity of diphenyl ditelluride in several biological models.423Mutagenesis 25, 257–269.424Fan, C.Z. et al., 2014. Subchronic toxicity experiment of nitromezuril to chickens.425Chin. Vet. Sci. 44, 326–330 (in Chinese).426Fei, C.Z. et al., 2012. General toxicology studies of a new anticoccidial compound427nitromezuril in mice. Chin. J. Vet. Drug. 46, 13–15 (in Chinese).428Fei, C.Z. et al., 2013a. Anticoccidial effects of a novel triazine nitromezuril in broiler429chickens. Vet. Parasitol. 198, 39–44.430Fei, C.Z. et al., 2013b. Subacute toxicity testing of a new triazines nitromezuril in431mice. Chin. Vet. Sci. 43, 84–90 (in Chinese).432Fernandez, M.L. et al., 2012. High-throughput screening with the Eimeria tenella433CDC2-related kinase2/cyclin complex EtCRK2/EtCYC3a. Microbiology 158,4342262–2271.435Föllmann, W. et al., 2013. Ames test. In: Hughes, S.M. (Ed.), Brenner’s Encyclopedia436of Genetics, second ed. Academic Press, San Diego, pp. 104–107.437Guguen-Guillouzo, C., Guillouzo, A., 2010. General review on in vitro hepatocyte438models and their applications. Methods Mol. Biol. 640, 1–40.439Han, E.H. et al., 2007. Eugenol inhibit 7,12-dimethylbenz[a]anthracene-induced440genotoxicity in MCF-7 cells: bifunctional effects on CYP1 and NAD(P)H: quinine441oxidoreductase. FEBS Lett. 581, 749–756.442Hayashi, M. et al., 1984. Kinetics of micronucleus formation in relation to443chromosomal aberrations in mouse bone marrow. Mutat. Res. 127, 129–137.444Hwang, E., Kim, G., 2012. Safety evaluation of Zanthoxylum piperitum-derived445essential oil by assessing micronucleus abnormalities, mutagenicity, and446chromosomal aberration. Food Res. Int. 47, 267–271.447Jena, G.B. et al., 2002. Genotoxicity testing, a regulatory requirement for drug448discovery and development: impact of ICH guidelines. Indian J. Pharm. 34, 86–44999.450Kamath, G.H., Rao, K.S., 2013. Genotoxicity guidelines recommended by451International Conference of Harmonization (ICH). Methods Mol. Biol. 1044,452431–458.453Kasamoto, S. et al., 2013. In vivo micronucleus assay in mouse bone marrow and454peripheral blood. Methods Mol. Biol. 1044, 179–189.455Kreiner, T. et al., 2011. Influence of toltrazuril treatment on parasitological456parameters and health performance of piglets in the field – an Austrian457experience. Vet. Parasitol. 183, 14–20.458Lambert, I.B. et al., 2005. Detailed review of transgenic rodent mutation assays.459Mutat. Res. 590, 1–280.460Li, A.P., 2004. A comprehensive approach for drug safety assessment. Chem. Biol.461Interact. 150, 27–33.462Ma, D. et al., 2014. A bioisosteres’ application as internal standard for simultaneous463determination of three triazine anticoccidial chemicals by UPLC. J. Liq.464Chromatogr. Related Technol. 37, 786–802.465Merino, A. et al., 2010. Drug profiling: knowing where it hits. Drug Discovery Today46615, 749–756.467Mitchell, I.d.G., Skibinski, D.O.F., 2012. Analysis of genotoxicity data in a regulatory468context. Methods Mol. Biol. 817, 399–417.469O’Brien, J.M. et al., 2014. Transgenic rodent assay for quantifying male germ cell470mutant frequency. J. Vis. Exp. (e51576)471OECD Guideline 471, 1997. Bacterial reverse mutation test. OECD guideline for472testing of chemicals.

C. Fei et al. / Regulatory Toxicology and Pharmacology xxx (2015) xxx–xxx 5

YRTPH 3223 No. of Pages 6, Model 5G

29 January 2015

Please cite this article in press as: Fei, C., et al. Safety evaluation of a triazine compound nitromezuril by assessing bacterial reverse mutation, sperm abnor-

malities, micronucleus and chromosomal aberrationQ1 . Regul. Toxicol. Pharmacol. (2015), http://dx.doi.org/10.1016/j.yrtph.2015.01.011

473 Otubanjo, O.A., Mosuro, A.A., 2001. An in vivo evaluation of induction of abnormal474 sperm morphology by some anthelmintic drugs in mice. Mutat. Res. 497, 131–475 138.476 Pacchierotti, F., Stocchi, V., 2013. Analysis of chromosome aberrations in somatic477 and germ cells of the mouse. Methods Mol. Biol. 1044, 147–163.478 Peek, H.W., Landman, W.J., 2011. Coccidiosis in poultry: anticoccidial products,479 vaccines and other prevention strategies. Vet. Q. 31, 143–161.480 Sasaki, Y.F. et al., 2000. The comet assay with multiple mouse organs: comparison of481 comet assay results and carcinogenicity with 208 chemicals selected from the482 IARC monographs and U.S. NTP carcinogenicity database. Crit. Rev. Toxicol. 30,483 629–799.

484Schmid, W., 1975. The micronucleus test. Mutat. Res. 31, 9–15.485Wang, C. et al., 2013. Nuclear translocation and accumulation of glyceraldehyde-3-486phosphate dehydrogenase involved in diclazuril-induced apoptosis in Eimeria487tenella. Vet. Res. 44, 29–38.488Wyrobek, A.J., Bruce, W.R., 1975. Chemical induction of sperm abnormalities in489mice. Cell. Biol. 72, 4425–4429.490Zhang, K. et al., 2014. Identification of in vitro metabolites of a new anticoccidial491drug nitromezuril using HepG2 cells, rat S9 and primary hepatocytes by liquid492chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom.49328, 1723–1734.

494

6 C. Fei et al. / Regulatory Toxicology and Pharmacology xxx (2015) xxx–xxx

YRTPH 3223 No. of Pages 6, Model 5G

29 January 2015

Please cite this article in press as: Fei, C., et al. Safety evaluation of a triazine compound nitromezuril by assessing bacterial reverse mutation, sperm abnor-

malities, micronucleus and chromosomal aberrationQ1 . Regul. Toxicol. Pharmacol. (2015), http://dx.doi.org/10.1016/j.yrtph.2015.01.011