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Safety and clinical efficacy of tenvermectin, a novel antiparasitic 16-membered macrocyclic lactone antibiotics
Accepted Manuscript Safety and clinical efficacy of tenvermectin, a novel antiparasitic 16-membered macrocyclic lactone antibiotics
Chenzhong Fei, Rufeng She, Guiyu Li, Lifang Zhang, Wushun Fan, suhan Xia, Feiqun Xue PII: DOI: Reference:
S0928-0987(18)30071-X https://doi.org/10.1016/j.ejps.2018.02.010 PHASCI 4407
To appear in:
European Journal of Pharmaceutical Sciences
Received date: Revised date: Accepted date:
4 September 2017 17 January 2018 6 February 2018
Please cite this article as: Chenzhong Fei, Rufeng She, Guiyu Li, Lifang Zhang, Wushun Fan, suhan Xia, Feiqun Xue , Safety and clinical efficacy of tenvermectin, a novel antiparasitic 16-membered macrocyclic lactone antibiotics. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Phasci(2017), https://doi.org/10.1016/j.ejps.2018.02.010
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ACCEPTED MANUSCRIPT Safety and clinical efficacy of tenvermectin, a novel antiparasitic 16-membered macrocyclic lactone antibiotics Chenzhong Fei a, Rufeng She a, Guiyu Li b, Lifang Zhang a, Wushun Fan a, suhan Xia a, Feiqun Xue
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a. Key Laboratory for Veterinary Chemical Drugs and Pharmaceutics, Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 200241 Shanghai, P.R. China .
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b. Zhejiang Hisun Pharmaceutical Co. Ltd., 318000, Taizhou, Zhejiang Province, P.R. China
Abstract: Tenvermectin (TVM) is a novel 16-membered macrocyclic lactone antibiotics, which contains component TVM A and TVM B. However there is not any report on safety and clinical efficacy of TVM for developing as a potential drug. In order to understand the part of safety and clinical efficacy of TVM, we conducted
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the acute toxicity test, the standard bacterial reverse mutation (Ames) test and the clinical deworming test. In the acute toxicity studies, TVM, TVM A and ivermectin (IVM) were administrated once by oral gavage to mice and
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rats. Results showed that the oral LD50 values of TVM, TVM A and IVM in mice were 74.41, 106.95 and 53.06 mg/kg respectively. The oral LD50 values of TVM and TVM A in rats were determined to be 164.22 and 749.34 mg/kg respectively. TVM and IVM are moderately toxic substances, meanwhile the TVM A belongs to low toxic
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compounds, implying that the acute toxicity is highly related to the length of side chain of TVM at position C25. In the Ames test, results showed that TVM did not induce mutagenicity in Salmonella typhimurium TA97a, TA98,
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TA100, TA102 and TA1535 with and without metabolic activation system, speculating that the mutagenicity is
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probably not related to the side chain at position C25 of 16-membered macrocyclic lactone antibiotics. In the efficacy trail of TVM against swine nematodes, growing pigs natural infection of Ascaris suum and Trichuris suis
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were treated with a single subcutaneous injection 0.3 mg/kg b.w.. Results showed that TVM and IVM had excellent effect in expelling Ascaris suum, and TVM had potential efficacy against Trichuris suis, however IVM had no effect on Trichuris suis. This study suggests that the side chain of TVM at position C25 may have important biological functions, which is one of the key sites of the studies on structure-activity relationship of 16-membered macrocyclic lactone compounds. TVM is a new compound exhibited some advantages worthy of developing.
Corresponding author. Tel.: +86 02134293396. E-mail addresses: [email protected] Corresponding author. Tel.: +86 02134293460. E-mail addresses: [email protected], [email protected]
ACCEPTED MANUSCRIPT Keywords: Tenvermectin; Ivermectin; Oral LD50 values; Bacterial reverse mutation (Ames); Efficacy against nematodes; Ascaris suum; Trichuris suis
1. Introduction 16-membered macrocyclic lactone antibiotics used as potential pesticides and antiparasitic drugs have attracted great interest due to their latent high activity and a high margin of safety. Avermectin, ivermectin (IVM) and
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milbemycin are well known as representatives of 16-membered macrolide compounds. (Laing et al., 2017; Shoop et al., 1995; Wang et al., 2016). IVM was first marketed in 1981 and remains the leading worldwide antiparasitic agent for livestock today. However, as the drug resistance is growing, alternatives or new products with potential activity are urgently needed (Geurden et al., 2015; McKellar and Benchaoui, 1996; Rodriguez-Hidalgo et al., 2017;
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Rodriguez-Vivas et al., 2014).
Tenvermectin (TVM) is a kind of new 16-membered macrolide compounds isolated and purified from the
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fermentation broth of genetically engineered strain Streptomyces avermitilis MHJ1011, which consists of component TVM A (25-methyl-22,23-dihydro avermectin) and TVM B (25-methyl-22,23-dihydro avermectin). The structure of TVM is shown in Fig.1, when R=CH3 named TVM A, and when R=C2H5 named TVM B. Huang
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et al. (2015a) reported that TVM showed better pharmacological properties against Tetranychus cinnabarinus and Bursaphelenchus xylophilus compared with IVM and milbemycin. The composition of TVM A and B in
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fermentation products is close to 3: 1 (Pan et al., 2016; Wan et al., 2016). The toxicity of IVM and milbemycins is
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related to their bonds with GABA receptor in the CNS of the mammalian host (McKellar and Benchaoui, 1996; Merola and Eubig, 2012; Rodriguez-Hidalgo et al., 2017). The oral LD50 values of IVM were reported in the range
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of 10–50 mg/kg in rats and 28-30 mg/kg in mice (Campbell and Benz, 1984; Dadarkar et al., 2007). However, the information of safety and clinical efficacy against nematodes of TVM has not been reported and remained unclear to date. It is important to understand the potential risks and efficacy of TVM against parasites used as a novel compound. Accordingly, this study conducted the oral acute toxicity test in mice and rats and the standard bacterial reverse mutation test as well as a clinical deworming test with growing pigs natural infection of ascaris suum and trichuris suis to determine the efficacy of TVM against swine nematodes. These tests can preliminarily reveal the druggability of TVM and the structure-activity relationship of 16-membered macrocyclic lactone antibiotics. 2. Materials and methods 2.1. Test materials
ACCEPTED MANUSCRIPT TVM A (purity: 90.9％, lot: S140314), TVM B (purity: 93.8％, lot: S140314) and TVM injection (10mL:0.1g, lot: 141101 ) were obtained from Zhejiang Hisun Pharmaceutical Co. Ltd.; IVM injection (10mL:0.1g, lot: A130501Y) and IVM (lot: AD311/11) was purchased from Merial Co. Ltd; TVM is a mixture of TVM A and TVM B in a ratio of 3: 1 ( prepared by dissolving TVM A and B in ethanol, mixed and dried by nitrogen). Sodium carboxymethyl cellulose (CMC, Aladdin Industrial Co. Shanghai, China); Nicotinamide-adenine dinucleotide
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phosphate (NADP, Merck Co. Rahway, NJ, USA). Glucose-6-phosphate (G6P, Sigma–Aldrich, St. Louis, MO, USA). D-biotin, L-histidine, agar, potassium chloride, sodium chloride and other chemicals were purchased from Sino-pharm Chemical reagent Co. (Shanghai, China).
2.2. Acute oral toxicity study
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2.2.1. Animals
Male and female specific pathogen-free Kunming (KM) mice weighing 15 g to 17 g were purchased from the
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Shanghai Slac Laboratory Animal Centre of Chinese Academy of Sciences (SLAC CAS). Male and female specific pathogen-free Sprague–Dawley (SD) rats weighing 160 g to180 g were obtained from the SLAC CAS. The license number of mice and rats were SCXK (Hu) 2012-0002 and SCXK (Hu) 2007-0005 respectively. The mice and rats
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were quarantined in a pathogen-free, well-ventilated room in our institute to enable them to acclimatize their environment for 5 days prior to the initiation of study. The animals were housed in wire-topped polypropylene
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cages with rice husk bedding and maintained under standard laboratory conditions at a temperature of 23 ± 2 °C, a
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relative humidity of 50% ± 10% and a photoperiod of 12 h. The feed in pellet form matched the Chinese standard “Laboratory animal rats and mice feed” (GB14924.3-2010) was purchased from the SLAC CAS. The animals were
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given food and water ad libitum. All the animals used in our studies were in accordance with “Guide for the Care and Use of Laboratory Animals” NIH Publication and approved by the Shanghai Veterinary Research Institutional Animal Care Committee.
2.2.2. Experimental design To obtain the LD50 of TVM, the experiments were designed in accordance with the method provided by the Chinese Toxicology Assessment Procedures and Methods for Food Safety (Chinese Standard, GB 15193.3, 2003). Animals were fasting for 12h before administration. The dosage levels of test compounds in mice and rats were determined through a pre-testing. In this experiments, after 5 d periods of quarantine and acclimatization, healthy
ACCEPTED MANUSCRIPT mice weighing 18 g to 22 g and rats weighing 180 g to 220 g of either sex were marked and randomly divided into groups. The test compounds of different concentrations were prepared by serial dilutions using 0.5% sodium carboxymethyl cellulose solution. The volumes of suspension by oral gavages were approximately 0.2 mL/10 g weight for mice and 1 mL/100 g weight for rats. KM mice (5 males and 5 females each group) treatments with different levels of TVM, TVM A and IVM were shown in Table 1 to 3. SD rats (6 males and 6 females each group) treatment with TVM were shown in Table 4. SD rats (5 males and 5 females each group) treatment with TVM A
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were shown in Table 5. Changes in behavior and mortality of animals were observed and recorded for 14 days. Evaluations included observations of appearance, respiratory, salivation, tremors and convulsions, motor activity, gait and posture, reactivity to handling and bizarre behavior (e.g. walking backwards). These animals died during the study were necropsied, and the time of death was recorded as precisely as possible. At the end of the test,
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surviving animals were sacrificed and necropsied. The LD50 values and 95% confidence intervals were calculated
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using the Bliss method by the software of Drug and Statistics 3.0 (Bliss, 1934, 1935).
2.3. Bacterial reverse mutation assay
TVM was assayed via the Ames test by standard plate-incorporation assay according to OECD Guideline (471,
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1997) and China standard for Ames (GB 15193.4-2003). Histidine requiring Salmonella typhimurium (TA97a, lot: 4366D; TA98, lot: 4367D; TA100, lot: 4370D; TA102, lot: 4372D and TA1535, lot: 4369D) used in this study
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were obtained from Molecular Toxicology Inc. (Boone, NC, USA). The preliminary Ames test was performed both with and without the S9 activation system. The metabolic activation system (S9 mix) was freshly prepared before
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each test by use of an Aroclor-1254 induced rat liver fraction (S9, lot: 3369, MolTox™, Boone, NC, USA). TVM
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were dissolved in Dimethylsulfoxide (DMSO, 99.99% of purity, Sigma–Aldrich Chemical Co.) to be a final concentration of 50.0 mg/mL that was assigned for the highest test concentration (50000 ug/mL) and serially diluted with DMSO for lower test concentrations (16667, 5556, 1852, 617, 206, 69 ug/mL). The mutagenicity assay was carried out in both the presence and absence of metabolic activation system along with the negative and positive controls as described in OECD Guideline No. 471. The positive control materials were purchased from Sigma–Aldrich Chemicals Co. as follows: TA97a (1.0 ug/plate, ICR-191, cas:17070-45-0, lot: SLBD9266V), TA98 (1.0 ug/plate, 2-nitrofluorene, cas:607-57-8, lot: S43858V), TA100 and TA1535 (2.0 ug/plate, Sodium azide, cas:26628-22-8, lot: 063M7386), TA102 (1.0 ug/plate, methyl methanesulfonate, cas:66-27-3, lot:87596LJ), all stains plus S9 (30.0 ug/plate for TA102 and 3.0 ug/plate for others, 2-Aminoanthracene, cas:613-13-8,
ACCEPTED MANUSCRIPT lot:STBD3302V). Each treatment was made in triplicate. Test plates per concentration were incubated at 37℃ for 48 h and then counted. The mutagenic index (MI) was calculated for each dose as the average number of revertants per plate divided by the average number of revertants per plate of the negative (solvent) control. For test strains TA97a, TA98, TA100 and TA102, a sample was considered positive when MI was above 2 for at least one of the test doses, and when the response was dose dependent. The MI for a positive response of strain TA1535 was more than 3 for at least one concentration level and showed dose-response relationship with or without S9 (Biso et al.,
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2010).
2.4. Efficacy of TVM against swine nematodes 2.4.1 Test animals
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16 growing pigs parasite-infected naturally, weighing 30–35 kg (females or castrated males), were purchased from individual farmers and housed in the swine barn. Each pig was selected by two consecutive days fecal
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samples examined positive significantly for nematode worm eggs, and one of them selected randomly was necropsied for confirming Ascaris suum and Trichuris suis infection. Pigs were individually ear-tagged and fed a corn, soybean-based complete ground diet formulated for the growth stage. Pens were separated from each other by
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solid concrete and provided with nipple waterer and self-feeder. Pigs were observed daily during the trial. 2.4.2. Experimental design
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15 pigs were randomly blocked into two groups, 10 pigs for TVM treatment group and 5 pigs for IVM treatment
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control. On Day 2 and 1 before adminstration and day 7 and 8, day 14 and 15, day 21 and 22, day 28 and 29 after dose, fecal samples were taken from each pig and the faecal egg counts (FEC) were determined using an modified
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McMaster technique with a sensitivity of 30 eggs per gram of faeces (EPG) (Rinaldi et al., 2011). The eggs of Ascaris suum and Trichuris suis were identified by Morphological characteristic structures (Hennessy et al., 2006; Peña-Espinoza et al., 2016). Pigs were administrated TVM and IVM with a single subcutaneous injection 0.3 mg/kg body weight (recommended dose of IVM), and sacrificed on day 30 after treatment. The intestinal contents and lungs were examined for nematodes according to standard parasitological procedures (Hennessy et al., 2006). Adult and larval worms were collected, identified and counted by standard parasitological techniques for confirming the effectiveness. 2.4.3 Drug efficacy criteria
ACCEPTED MANUSCRIPT The efficacy of treatment was evaluated by FEC of each pig before and after treatments, or between different treatments. The faecal egg counts reduction rate (FECR%) and the fecal eggs positive rate were calculated and compared. The data of FEC were transformed to log (EPG+1) and subjected to a two-way analysis of variance using the software of Drug and Statistics 3.0 and treatment effect was tested for significance at the 0.05 level (Andre et al., 2016).
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3. Results 3.1. Acute toxicity study 3.1.1 Acute toxicity of IVM
Mice in control groups dosed with IVM, some adverse effects such as hypoactivity, debility, curl up and
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trembling were obviously observed 4 h after the oral administration. These effects became more serious until to death in higher dose groups. Most of the death occurred between 12 and 24 h after administration. The results of
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oral acute toxicity of IVM in mice summarized in Table 1. The oral LD50 values of IVM were determined to be 53.06 mg/kg b.w. with 95% confidence intervals ranging from approximately 48.72 to 58.34 mg/kg. 3.1.2 Acute toxicity of TVM
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The poisoning symptoms of animals mainly occurred at 2-3 hours after administration. The mice and rats exhibited restless, debility, curl up, depression, ataxia, shortness of breath, trembling and death. In lower dose
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groups, mildly poisoned animals gradually increased the activities and recovered mostly 2-4 h after administration.
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In higher dose groups, severe symptoms of poisoning and death were clearly observed and the degree of poisoning symptoms was directly proportional to the dosage. The peak of death time of mice and rats was during 8-12 h after
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administration and all deaths occurred within 1 day after administration. No obvious organ abnormalities were visible in the animal necropsy. The results of the oral acute toxicity of TVM were summarized in Table 2 and 3. The oral LD50 values of TVM in mice and rats were determined to be 74.41 mg/kg b.w. and 164.22 mg/kg b.w. ,with 95% confidence intervals ranging from approximately 65.99 to 83.42 mg/kg and 135.50 to 201.36 mg/kg, respectively. 3.1.3 Acute toxicity of TVM A The results of the oral acute toxicity of TVM A in mice and rats were summarized in Table 4 and 5. Adverse effects such as hypoactivity, debility, curl up, shortness of breath, trembling and death were obviously observed at 3 h after administration and these effects were more serious and persistent at higher doses. The animals with mild
ACCEPTED MANUSCRIPT poisoning symptoms generally recovered after 12 h. Times of the death of mice and rats induced by severe side effects were mainly at 8 to 36 h after administration. Most surviving animals gradually recovered the feeding and activities at 3th day after administration. However one moribund mouse died at 4th day after administration and one rat appeared delayed death at 10th day after administration. No distinctly organ abnormalities were seen in the autopsy of mice and rats. According to dose levels and the numbers of dead animals, the oral LD50 values of TVM A in mice and rats were determined to be 106.95 mg/kg and 749.34 mg/kg, with 95% confidence intervals ranging
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from approximately 103.78 to 110.22 mg/kg and 653.86 to 858.87 mg/kg, respectively.
3.2. Bacterial reverse mutation assay
The test material TVM was suspended in DMSO and the concentrations of drug ranged from 6.8 to 5000 ug/plate
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in the presence and absence of the S9 mix. The results of the S. typhimurium reverse mutation assay in the presence and absence of metabolic activation are shown in Table 6. The numbers of revertants for the vehicle controls in the
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presence and absence of the S9 mix for all test strains were in the scope of the negative controls and the numbers of revertants increased remarkably in all positive control compared to negative control. The mutagenicity index (MI) for all positive control materials were above 3. The range of MIs for the test material in the presence of the S9 mix
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for strains TA97a, TA98, TA100, TA102 and TA1535 were 0.8-1.1, 0.8-0.9, 1.0-1.1, 1.1 and 0.9-1.1, respectively. The range of MIs in absence of the S9 mix for strains TA97a, TA98, TA100, TA102 and TA1535 were 1.0-1.2,
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0.9-1.2, 0.9-1.1, 0.9-1.0 and 1.0-1.4, respectively. Amounts of the particle tended to precipitate in the plates but did
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not to obscure the background at the concentration of 1667 ug/plate and 5000ug/plate. The number of revertants in the level of treatment were not significantly higher than that of revertants in negative control. MIs were not
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observed to increase more than two fold with the S. typhimurium TA97a, TA98, TA100, TA102 and TA1535 strains after treatment with or without S9 mix. There was no evidence of cytotoxicity in all bacterial systems used in mutation assay.
3.3 Efficacy of TVM against swine nematodes The clinical adverse reactions were not observed after therapy. Results of scolecology examinations for each pig before and after administration are shown in table 7 to 8. One week after TVM treatment, the FEC of Ascaris suum and Trichuris suis decreased significantly (p<0.05), but egg positive rate did not decrease obviously. Two weeks after administration, the FECR% and positive rate of Ascaris suum all decreased obviously (p<0.05). Four weeks
ACCEPTED MANUSCRIPT after administration, the negative rate of Ascaris eggs and Trichuris eggs were 100% and 99.43%, suggesting that TVM had potential effect against Ascaris suum and Trichuris suis. Meanwhile, pigs after IVM treatment, the FECR% and positive rate of Ascaris suum all decreased obviously (p<0.05), but did not decrease significantly for Trichuris suis (p>0.05), indicating that IVM had good efficacy against Ascaris suum but limited effect on Trichuris suis. Necropsy found roundworms in one pig for each group and whipworms in two pigs in TVM groups and five pigs in IVM groups respectively. This study revealed that TVM and IVM had similar good efficacy against Ascaris and
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only TVM had potential efficacy against Trichuris.
4. Discussion
The molecule of TVM，IVM and milbemycin differs from each other only at one position. The TVM differs
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from IVM at position C25 substituent group and differs from milbemycin at position C13 substituent group. IVM contains at least 80% of 22, 23-dihydroavermectin B1a and less than 20% 22, 23-dihydroavermectin B1b. The
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main active ingredient is the 22, 23-dihydroavermectin B1a (Huang et al., 2015b; McKellar and Benchaoui, 1996; Wan et al., 2016). TVM is a mixture of TVM A and TVM B in a ratio of 3:1, which is expected to reduce the production cost substantially. However, it is necessary to understand the pharmacological properties of different
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components during research process, which can provide the references for developing other similar new drugs showing practical significance (Arsic et al., 2017; Wan et al., 2017a; Wan et al., 2017b). Therefore, this
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preliminary study evaluated the acute toxicity of TVM and components TVM A respectively.
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Acute toxicity tests were used to compare relatively acute hazards of chemicals, especially for new compounds that no other toxicology data are available (Wu et al., 2014). The LD50 values of drugs are generally the beginning
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of safety evaluation. It is also an important reference criteria on the classification of relative toxicity of drug compounds (Copplestone, 1988; OECD, 2001). As the result of acute toxicity test is influenced by many factors, such as the strains and ages of experimental animals, the environment and the time of fasting, the results of different laboratories are quite different (Dadarkar et al., 2007; Wu et al., 2015). Therefore, this experiments set up the similar drug IVM as a control, and the LD50 value of TVM acquired under the same conditions for IVM can objectively compared the difference of acute toxicity of both. The relative toxicity classification of different structures of new compounds is helpful for understanding the relationship between structure and toxicity, which is conducive to the development of new drugs and further selection of target compounds.
ACCEPTED MANUSCRIPT In this acute toxicity test, there were no obvious organ abnormalities on necropsy. The poisoning symptoms pointed to neurotoxicity and the influence of central nervous may be the reason of death of animals, consistent with reports of the acute toxicity symptoms of similar drug (Campbell and Benz, 1984; McKellar and Benchaoui, 1996). The calculated LD50 value of TVM, TVM A and IVM for KM mice were 74.41, 106.95 and 53.06 mg/kg respectively, indicating that the relative toxicity of TVM was lower than that of IVM, and the new compound with directional modification had some advantages (Huang et al., 2015a). The results of acute toxicity for SD rats
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showed that the acute toxicity of TVM A was significantly lower than that of TVM, and the oral LD50 values of TVM and TVM A were 164.22 and 749.39 mg/kg respectively, According to the World Health Organization’s recommended classification of the relative toxicity of chemicals, TVM is a moderately toxic substance while TVM A belongs to a low toxic compound (Copplestone, 1988; Dadarkar et al., 2007). Since TVM is a mixture of TVM A
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and B in a ratio of 3:1, it is speculated that the relative toxicity of TVM B is higher, and TVM B is the main reason that the acute toxicity of TVM was significantly higher than that of TVM A.
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The molecule of TVM A and TVM B differs at position C25 substituent with methyl or ethyl respectively, and the difference of acute toxicity obviously suggests that the site may have important biological functions, which is one of the key sites studied on structure-activity relationship. The side chain of C25 in TVM is consistent with the
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similar drug milbemycin and shorter than IVM. It is reported that milbemycins is similar to TVM with lower toxicity (McKellar and Benchaoui, 1996; Shoop et al., 1995). Therefore, it is implied that the length of side chain at
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needed to reveal the reason.
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position C25 is highly related to the toxicity of 16-membered macrocyclic lactone compounds. Further studies are
The Ames test has been used worldwide as an initial screening to determine the mutagenic potential of new
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chemicals and drugs. The mutagenicity for chemicals is a crucial factor for druggability and an important basis for drug screening (Fei et al., 2015; OECD, 1997; Shim et al., 2008). TVM had no mutagenicity to Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 with and without metabolic activation system, consistent with the reports of the same kind of drugs (Campbell and Benz, 1984; Campbell et al., 1983; Sutherland and Campbell, 1990). However, there are many factors affecting the inherent toxicity of the compounds. Therefore, a series of mutagenicity and genotoxicity tests are needed to further evaluate the safety of TVM on the basis of determination of the more valuable components (Jena et al., 2002). TVM is a novel compound and its structure differs from IVM only at position C25 substituent group, speculating that the mutagenicity is probably not related to the side chain at position C25 of 16-membered macrocyclic lactone antibiotics.
ACCEPTED MANUSCRIPT IVM is a first-line deworming drug, and ascariasis and trichuriasis are the most serious parasitic diseases in swine (Laing et al., 2017). The activity of TVM against T. cinnabarinus in vitro is higher than that of IVM, and the activity of TVM against ascariasis and trichuriasis in vivo has not been reported (Huang et al., 2015a). For evaluating the anthelmintic activity of TVM preliminarily, growing pigs that naturally infected with Ascaris suum and Trichuris suis were screened and confirmed for this experiment. The dosage of TVM and IVM were all set to 0.3 mg/kg b.w. with a single subcutaneous injection, which is the recommended dose of IVM (Campbell and Benz,
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1984). It is more convenient to compare the activity of these two similar compounds. The results showed that TVM and IVM with a single subcutaneous injection 0.3 mg/kg b.w. had good effect in expelling Ascaris suum，and TVM also had potential effect against Trichuris suis. The result of TVM against trichuriasis is not completely consistent with the reports on the role of IVM (Campbell and Benz, 1984; Campbell et al., 1983; Laing et al., 2017).
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Because of the restrictions of clinical animal quantities, further studies are needed to verify the effectiveness of TVM against Trichuris suis infection.
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TVM had similar safety and anthelmintic activity to IVM. However, the activity of TVM against trichuriasis occurs prior to that of IVM. Huang et al. (2015a) also reported the enhance activities of TVM against T. cinnabarinus and B. xylophilus. Because of the difference only at position C25 substituent groups, we speculated
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that the action mechanisms of both drugs are roughly the same. Recent studies confirmed that glutamate gated chloride channel is a more important target for these kinds of drug, and the GABA receptor is a secondary target.
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The biological activity of these compounds is different in higher animals, showing that there may exist other targets
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of action (Feng et al., 2002; Wang et al., 2016). It can be postulated that the targets of action of TVM is different from that of IVM (Prichard et al., 2012).The focus of further studies should be verify the safety and effectiveness of
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TVM and evaluated the cross-resistance between TVM and IVM (George, 2017; Prichard et al., 2012). TVM may have difference target and medicinal basis worthy of developing as a new drug.
5. Conclusion The acute toxicity of TVM is lower than that of IVM. TVM and IVM were all moderately toxic substance according to the classification of the relative toxicity. Meanwhile, the acute toxicity of TVM A was significantly lower than that of TVM, which belongs to a low toxic compound, indicating that the length of side chain of TVM at position C25 is highly related to the toxicity of these kinds of compounds. The Ames test results were all negative for Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 with and without metabolic
ACCEPTED MANUSCRIPT activation system, illustrating that TVM may have no genotoxicity. TVM and IVM with a single subcutaneous injection 0.3 mg/kg b.w. had excellent efficacy against Ascaris suum, and only TVM had potential effect on Trichuris suis. This study suggests that TVM had some advantages and the structure of TVM at position C25 may have important biological functions. TVM has a medicinal basis worthy of developping, especially component
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TVM A has higher security and deserves further study.
Acknowledgements
This study was supported by The National Key Research and Development Program of China (2016YFD0501303) and The Special Fund for Agro-Scientific Research in the Public Interest (201303038–9).
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Campbell, W.C., Fisher, M.H., Stapley, E.O., Albers-Schonberg, G., Jacob, T.A., 1983. Ivermectin: a potent new antiparasitic agent. Science (New York, N.Y.) 221, 823-828. Copplestone, J.F., 1988. The development of the WHO Recommended Classification of Pesticides by Hazard. Bulletin of The World Health Organization 66, 545-551. Dadarkar, S.S., Deore, M.D., Gatne, M.M., 2007. Comparative evaluation of acute toxicity of ivermectin by two methods after single subcutaneous administration in rats. Regul Toxicol Pharmacol 47, 257-260.
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Huang, J., Chen, A.-L., Zhang, H., Yu, Z., Li, M.-H., Li, N., Lin, J.-T., Bai, H., Wang, J.-D., Zheng, Y.-G., 2015a. Gene replacement for the generation of designed novel avermectin derivatives with enhanced acaricidal and nematicidal activities. Applied and environmental microbiology 81, 5326-5334.
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Merola, V.M., Eubig, P.A., 2012. Toxicology of avermectins and milbemycins (macrocylic lactones) and the role of P-glycoprotein in dogs and cats. Vet Clin North Am Small Anim Pract 42, 313-333, vii. OECD Guideline 471, 1997. OECD Guideline for the testing of chemicals. Bacterial Reverse Mutation Test. OECD Guideline 420, 2001. OECD Guideline for the testing of chemicals. Acute Oral Toxicity. Pan, J.J., Wan, X., Zhang, S.Y., Huang, J., Zhang, H., Chen, A.L., Wang, J.D., 2016. Three new 16-membered macrolide compounds from a genetically engineered strain S. avermitilis MHJ1011. Bioorg Med Chem Lett 26, 3376-3379. Peña-Espinoza, M., Thamsborg, S.M., Denwood, M.J., Drag, M., Hansen, T.V., Jensen, V.F., Enemark, H.L., 2016. Efficacy of ivermectin against gastrointestinal nematodes of cattle in Denmark evaluated by different methods for analysis of faecal egg count reduction. International Journal for Parasitology: Drugs and Drug Resistance 6, 241-250.
ACCEPTED MANUSCRIPT Prichard, R., Ménez, C., Lespine, A., 2012. Moxidectin and the avermectins: Consanguinity but not identity. International Journal for Parasitology: Drugs and Drug Resistance 2, 134-153. Rinaldi, L., Coles, G.C., Maurelli, M.P., Musella, V., Cringoli, G., 2011. Calibration and diagnostic accuracy of simple flotation, McMaster and FLOTAC for parasite egg counts in sheep. Veterinary Parasitology 177, 345-352. Rodriguez-Hidalgo, R., Perez-Otanez, X., Garces-Carrera, S., Vanwambeke, S.O., Madder, M., Benitez-Ortiz, W., 2017. The current status of resistance to alpha-cypermethrin, ivermectin, and amitraz of the cattle tick (Rhipicephalus microplus) in Ecuador. PloS one 12, e0174652.
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Wan, X., Zhang, S.Y., Zhang, H., Zhai, J., Huang, J., Chen, A.L., Wang, J.D., 2017b. New tenvermectin analogs obtained by microbial conversion with Saccharopolyspora erythraea. J Antibiot (Tokyo) 70, 190-192.
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Wang, C.-X., Ding, R., Jiang, S.-T., Tang, J.-S., Hu, D., Chen, G.-D., Lin, F., Hong, K., Yao, X.-S., Gao, H., 2016. Aldgamycins J–O, 16-Membered Macrolides with a Branched Octose Unit from Streptomycetes sp. and Their Antibacterial Activities. Journal of Natural
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Wu, H., Xiao, W., Zhang, K., Xue, F., Zhang, C., Yan, M., Wang, X., Jiang, S., 2014. Acute and subchronic toxicity of arprinocid in Sprague-Dawley rats. Regul Toxicol Pharmacol 69, 487-495. Wu, X., Wan, D., Xie, C., Li, T., Huang, R., Shu, X., Ruan, Z., Deng, Z., Yin, Y., 2015. Acute and sub-acute oral toxicological evaluations and mutagenicity of N-carbamylglutamate (NCG). Regul Toxicol Pharmacol 73, 296-302.
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Figure 1. The structures of tenvermectin. A: R=CH3; B: R=CH2CH3
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Figure 2. The structures of ivermectin. B1a: R= CH2CH3; B1b: R= CH3
Figure 3. The structures of milbemycin oxime. A3: R=CH3; A4: R=CH2CH3
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Table 1 Oral acute toxicity of ivermectin by gavage in mice Males Females Weight/g
Number of dead/dosed
Weight/g
Number of dead/dosed
Mortality %
34 38
19.7±0.41 19.6±0.84
0/5 1/5
19.0±0.58 19.2±1.43
0/5 2/5
0% 30%
43 49
19.7±0.79 20.2±0.72
0/5 2/5
19.1±0.15 20.4±1.76
2/5 0/5
20% 20%
55 63
20.0±1.40 19.6±0.65
3/5 3/5
19.4±0.23 19.8±0.74
3/5 4/5
60% 70%
71
20.0±0.40
5/5
20.7±0.76
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Dose mg/kg
5/5
100%
Table 2 Oral acute toxicity of tenvermectin by gavage in mice Males
Females
Weight/g
Number of dead/dosed
Weight/g
Number of dead/dosed
Mortality %
42 50
18.9±0.88 18.8±0.89
0/5 1/5
19.6±0.67 19.3±0.66
0/5 0/5
0% 10%
61 73
21.4±0.47 21.4±0.68
2/5 4/5
19.2±0.55 20.9±0.64
1/5 2/5
30% 60%
87 105
19.0±0.64 19.2±0.89
5/5 5/5
18.7±0.58 19.8±1.22
3/5 3/5
80% 80%
125
19.0±0.72
5/5
20.2±0.25
5/5
100%
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Dose mg/kg
Table 3 Oral acute toxicity of tenvermectin by gavage in rats Females
Number of dead/dosed
Weight/g
Number of dead/dosed
Mortality %
80
195±7.6
1/6
189±1.4
0/6
8%
100
199±6.6
0/6
189±4.7
2/6
17%
125
196±7.1
3/6
189±4.6
3/6
50%
156
198±9.0
3/6
190±3.7
3/6
50%
195
197±5.2
3/6
191±4.6
5/6
67%
244
204±3.5
3/6
193±5.4
6/6
75%
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Weight/g
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Males
Dose mg/kg
Table 4 Oral acute toxicity of tenvermectin A by gavage in mice Males
Females
Weight/g
Number of dead/dosed
Weight/g
Number of dead/dosed
Mortality %
90
19.3±0.71
0/5
18.9±0.22
0/5
0%
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Dose mg/kg 96
20.5±0.16
0/5
19.3±0.41
1/5
10%
103
19.9±0.53
2/5
18.5±0.26
0/5
20%
110
19.8±0.36
2/5
19.0±0.33
4/5
60%
118
20.6±0.45
5/5
19.1±0.47
5/5
100%
Table 5 Oral acute toxicity of tenvermectin A by gavage in rats Males
Females
Dose mg/kg
Weight/g
Number of dead/dosed
Weight/g
Number of dead/dosed
Mortality %
580
198±7.8
2/5
189±2.4
0/5
20%
680 798
203±7.6 206±7.5
2/5 2/5
191±4.7 189±6.2
2/5 4/5
40% 60%
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198±9.3
3/5
193±3.8
5/5
80%
1100
207±5.2
3/5
192±3.6
5/5
80%
Table 6 Mutagenic activity and mutagenic index (MI) of tenvermectin in TA97a, TA98, TA100, TA102 and TA1535 Salmonella typhimurium strains in the absence (− S9) and in the presence (+ S9) of metabolization. TA97a
TA98
TA100
TA102
TA1535
S 9
Rev/plate
NC -S9mix
-
82.7±2.31
PC -S9mix
-
499±60.92
6. 0
238±4.36
10. 2
694±38.43
6.0
1594.7±65.4 9
5. 4
557.7±37.0 7
49. 4
6.86 ug/plate
-
85.7±6.03
1. 2
29±3.46
0.9
115.3±6.81
0.9
303±6
0. 9
12.3±0.58
1.1
20.58 ug/plate
-
91.3±7.23
1. 0
271.73
1.1
121.7±5.13
0.9
301±5
0. 9
16.3±2.31
1.1
61.73 ug/plate
-
92.3±5.77
1. 0
25.33.79
1.2
121.3±4.51
1.0
295.3±9.5
1. 0
14.7±3.21
1.2
185.19 ug/plate
-
94±3
1. 1
25.3±6.43
1.1
115.3±3.06
1.0
281±11
1. 0
11±1.73
1.0
555.56 ug/plate
-
84±6.08
1. 1
27.3±6.81
1.1
119±3.46
1.1
299±6.24
1. 0
13.3±3.79
1.3
1666.67 ug/plate
-
80.7±8.14
1. 1
25±6.08
1.2
106.3±1.15
1.1
268.7±4.16
1. 0
12±4
1.4
5000 ug/plate
-
101±6.93
1. 0
20.7±2.52
1.2
109.3±1.53
1.0
272.7±3.21
1. 0
12.3±1.53
1.1
NC -S9mix
+
112±7.94
PC -S9mix
+
675.7±33.5 0
6. 0
6.86 ug/plate
+
118.3±6.66
0. 8
20.58 ug/plate
+
110.3±2.52
0. 9
61.73 ug/plate
+
120.3±11.9 3
0. 9
185.19 ug/plate
+
99.7±3.06
555.56 ug/plate
+
97.3±7.57
1666.67 ug/plate
+
100.7±6.43
5000 ug/plate
+
92.3±11.06
Rev/plate
MI
42±3
MI
MI
294±10.39
112±3
Rev/plate
MI
11.3±1.53
262±7.81
15.3±3.06
39. 2
1840±55.83
16. 4
1864±164.84
7. 1
605.7±36.5 6
39. 6
31.3±0.58
0.9
124±3.46
1.1
278.7±6.43
1. 1
16.3±2.08
1.1
34.3±4.16
0.9
128±1
1.0
284.7±15.04
1. 1
14.3±3.06
1.1
32±5
0.9
120.31.15
1.1
290.7±6.66
1. 1
14.7±1.53
0.9
32.3±1.15
0.8
116.39.29
1.0
275.7±5.86
1. 1
16±2.65
1.1
1. 1
37.3±2.31
0.8
123±6.24
1.1
284.7±15.18
1. 1
14.3±3.21
1.0
1. 0
36.3±3.06
0.8
115.3±11.5 9
1.1
285.7±13.58
1. 1
16.3±2.52
0.9
1. 1
38±6.08
0.8
124.7±1.15
1.1
298.7±7.77
1. 1
16.3±3.06
1.1
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1645.3±139.5 8
Rev/plate
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116±7.55
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23.3±6.11
Rev/plate
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MI
0. 9
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-S9, without metabolic activation; +S9, with metabolic activation (500 uL 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 ICR-191 (1.0 μg/plate) for strains TA97a, 2-nitrofluorene (1.0 μg /plate) for strain TA98, methyl methanesulfonate (1.0 ug/plate) for strain TA102, sodium azide (2.0 μg /plate) for strain TA100 and TA1535. Positive controls with S9 activation were 2-aminoanthracene (3.0μg / plate) for strains TA97, TA98, TA100 and TA1535, 2aminoanthracene (30.0μg / plate) for strain TA102 MI, mutagenicity index: number of revertants induced in the sample/number of spontaneous revertants in the negative (solvent) controls.
ACCEPTED MANUSCRIPT Table 7 Ascaris suum examinations for each pig before and after administration Tenvermectin，n=10 Ivermectin，n=5 Time of collection positive rate of eggs Mean FEC(epg) positive rate of eggs Mean FEC(epg) Before administration
100%
1 week after administration
80%
173
2 weeks after administration
30%
3 weeks after administration 4 weeks after administration
100%
1842
20%
15*
5*
0%
0*
0%
0*
0%
0*
0%
0*
0%
0*
FECR%
3114 *a
100.0%
100.0%
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The asterisk （*） indicates the significantly differences of FEC between administration before and after in the columes (P < 0.05); The lowercase letter（a） indicates the significantly different of FEC between groups treatment with TVM and IVM in the rows (P < 0.05).
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FEC (epg), feacal egg counts (eggs per gram of faeces); FECR%, faecal egg counts reduction rate.
Before administration
70%
1 week after administration
10%
2 weeks after administration
20%
4 weeks after administration
5
*a
80%
1422
60%
348
23*a
80%
270
20%
30*a
80%
705
10%
6*a
80%
840
99.43%
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FECR%
1056
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3 weeks after administration
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Table 8 Trichuris suis examinations for each pig before and after administration Tenvermectin，n=10 Ivermectin，n=5 Time of collection positive rate of eggs Mean FEC(epg) positive rate of eggs Mean FEC(epg)
40.93%
The asterisk （*） indicates the significantly differences of FEC between administration before and after in the columes (P < 0.05);
< 0.05).
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The lowercase letter（a） indicates the significantly different of FEC between groups treatment with TVM and IVM in the rows (P
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FEC (epg), feacal egg counts (eggs per gram of faeces); FECR%, faecal egg counts reduction rate.
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Graphical abstract
Chenzhong Fei, Rufeng She, Guiyu Li, Lifang Zhang, Wushun Fan, suhan Xia, Feiqun Xue PII: DOI: Reference:
S0928-0987(18)30071-X https://doi.org/10.1016/j.ejps.2018.02.010 PHASCI 4407
To appear in:
European Journal of Pharmaceutical Sciences
Received date: Revised date: Accepted date:
4 September 2017 17 January 2018 6 February 2018
Please cite this article as: Chenzhong Fei, Rufeng She, Guiyu Li, Lifang Zhang, Wushun Fan, suhan Xia, Feiqun Xue , Safety and clinical efficacy of tenvermectin, a novel antiparasitic 16-membered macrocyclic lactone antibiotics. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Phasci(2017), https://doi.org/10.1016/j.ejps.2018.02.010
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Safety and clinical efficacy of tenvermectin, a novel antiparasitic 16-membered macrocyclic lactone antibiotics Chenzhong Fei a, Rufeng She a, Guiyu Li b, Lifang Zhang a, Wushun Fan a, suhan Xia a, Feiqun Xue
a
a. Key Laboratory for Veterinary Chemical Drugs and Pharmaceutics, Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 200241 Shanghai, P.R. China .
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b. Zhejiang Hisun Pharmaceutical Co. Ltd., 318000, Taizhou, Zhejiang Province, P.R. China
Abstract: Tenvermectin (TVM) is a novel 16-membered macrocyclic lactone antibiotics, which contains component TVM A and TVM B. However there is not any report on safety and clinical efficacy of TVM for developing as a potential drug. In order to understand the part of safety and clinical efficacy of TVM, we conducted
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the acute toxicity test, the standard bacterial reverse mutation (Ames) test and the clinical deworming test. In the acute toxicity studies, TVM, TVM A and ivermectin (IVM) were administrated once by oral gavage to mice and
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rats. Results showed that the oral LD50 values of TVM, TVM A and IVM in mice were 74.41, 106.95 and 53.06 mg/kg respectively. The oral LD50 values of TVM and TVM A in rats were determined to be 164.22 and 749.34 mg/kg respectively. TVM and IVM are moderately toxic substances, meanwhile the TVM A belongs to low toxic
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compounds, implying that the acute toxicity is highly related to the length of side chain of TVM at position C25. In the Ames test, results showed that TVM did not induce mutagenicity in Salmonella typhimurium TA97a, TA98,
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TA100, TA102 and TA1535 with and without metabolic activation system, speculating that the mutagenicity is
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probably not related to the side chain at position C25 of 16-membered macrocyclic lactone antibiotics. In the efficacy trail of TVM against swine nematodes, growing pigs natural infection of Ascaris suum and Trichuris suis
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were treated with a single subcutaneous injection 0.3 mg/kg b.w.. Results showed that TVM and IVM had excellent effect in expelling Ascaris suum, and TVM had potential efficacy against Trichuris suis, however IVM had no effect on Trichuris suis. This study suggests that the side chain of TVM at position C25 may have important biological functions, which is one of the key sites of the studies on structure-activity relationship of 16-membered macrocyclic lactone compounds. TVM is a new compound exhibited some advantages worthy of developing.
Corresponding author. Tel.: +86 02134293396. E-mail addresses: [email protected] Corresponding author. Tel.: +86 02134293460. E-mail addresses: [email protected], [email protected]
ACCEPTED MANUSCRIPT Keywords: Tenvermectin; Ivermectin; Oral LD50 values; Bacterial reverse mutation (Ames); Efficacy against nematodes; Ascaris suum; Trichuris suis
1. Introduction 16-membered macrocyclic lactone antibiotics used as potential pesticides and antiparasitic drugs have attracted great interest due to their latent high activity and a high margin of safety. Avermectin, ivermectin (IVM) and
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milbemycin are well known as representatives of 16-membered macrolide compounds. (Laing et al., 2017; Shoop et al., 1995; Wang et al., 2016). IVM was first marketed in 1981 and remains the leading worldwide antiparasitic agent for livestock today. However, as the drug resistance is growing, alternatives or new products with potential activity are urgently needed (Geurden et al., 2015; McKellar and Benchaoui, 1996; Rodriguez-Hidalgo et al., 2017;
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Rodriguez-Vivas et al., 2014).
Tenvermectin (TVM) is a kind of new 16-membered macrolide compounds isolated and purified from the
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fermentation broth of genetically engineered strain Streptomyces avermitilis MHJ1011, which consists of component TVM A (25-methyl-22,23-dihydro avermectin) and TVM B (25-methyl-22,23-dihydro avermectin). The structure of TVM is shown in Fig.1, when R=CH3 named TVM A, and when R=C2H5 named TVM B. Huang
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et al. (2015a) reported that TVM showed better pharmacological properties against Tetranychus cinnabarinus and Bursaphelenchus xylophilus compared with IVM and milbemycin. The composition of TVM A and B in
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fermentation products is close to 3: 1 (Pan et al., 2016; Wan et al., 2016). The toxicity of IVM and milbemycins is
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related to their bonds with GABA receptor in the CNS of the mammalian host (McKellar and Benchaoui, 1996; Merola and Eubig, 2012; Rodriguez-Hidalgo et al., 2017). The oral LD50 values of IVM were reported in the range
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of 10–50 mg/kg in rats and 28-30 mg/kg in mice (Campbell and Benz, 1984; Dadarkar et al., 2007). However, the information of safety and clinical efficacy against nematodes of TVM has not been reported and remained unclear to date. It is important to understand the potential risks and efficacy of TVM against parasites used as a novel compound. Accordingly, this study conducted the oral acute toxicity test in mice and rats and the standard bacterial reverse mutation test as well as a clinical deworming test with growing pigs natural infection of ascaris suum and trichuris suis to determine the efficacy of TVM against swine nematodes. These tests can preliminarily reveal the druggability of TVM and the structure-activity relationship of 16-membered macrocyclic lactone antibiotics. 2. Materials and methods 2.1. Test materials
ACCEPTED MANUSCRIPT TVM A (purity: 90.9％, lot: S140314), TVM B (purity: 93.8％, lot: S140314) and TVM injection (10mL:0.1g, lot: 141101 ) were obtained from Zhejiang Hisun Pharmaceutical Co. Ltd.; IVM injection (10mL:0.1g, lot: A130501Y) and IVM (lot: AD311/11) was purchased from Merial Co. Ltd; TVM is a mixture of TVM A and TVM B in a ratio of 3: 1 ( prepared by dissolving TVM A and B in ethanol, mixed and dried by nitrogen). Sodium carboxymethyl cellulose (CMC, Aladdin Industrial Co. Shanghai, China); Nicotinamide-adenine dinucleotide
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phosphate (NADP, Merck Co. Rahway, NJ, USA). Glucose-6-phosphate (G6P, Sigma–Aldrich, St. Louis, MO, USA). D-biotin, L-histidine, agar, potassium chloride, sodium chloride and other chemicals were purchased from Sino-pharm Chemical reagent Co. (Shanghai, China).
2.2. Acute oral toxicity study
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2.2.1. Animals
Male and female specific pathogen-free Kunming (KM) mice weighing 15 g to 17 g were purchased from the
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Shanghai Slac Laboratory Animal Centre of Chinese Academy of Sciences (SLAC CAS). Male and female specific pathogen-free Sprague–Dawley (SD) rats weighing 160 g to180 g were obtained from the SLAC CAS. The license number of mice and rats were SCXK (Hu) 2012-0002 and SCXK (Hu) 2007-0005 respectively. The mice and rats
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were quarantined in a pathogen-free, well-ventilated room in our institute to enable them to acclimatize their environment for 5 days prior to the initiation of study. The animals were housed in wire-topped polypropylene
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cages with rice husk bedding and maintained under standard laboratory conditions at a temperature of 23 ± 2 °C, a
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relative humidity of 50% ± 10% and a photoperiod of 12 h. The feed in pellet form matched the Chinese standard “Laboratory animal rats and mice feed” (GB14924.3-2010) was purchased from the SLAC CAS. The animals were
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given food and water ad libitum. All the animals used in our studies were in accordance with “Guide for the Care and Use of Laboratory Animals” NIH Publication and approved by the Shanghai Veterinary Research Institutional Animal Care Committee.
2.2.2. Experimental design To obtain the LD50 of TVM, the experiments were designed in accordance with the method provided by the Chinese Toxicology Assessment Procedures and Methods for Food Safety (Chinese Standard, GB 15193.3, 2003). Animals were fasting for 12h before administration. The dosage levels of test compounds in mice and rats were determined through a pre-testing. In this experiments, after 5 d periods of quarantine and acclimatization, healthy
ACCEPTED MANUSCRIPT mice weighing 18 g to 22 g and rats weighing 180 g to 220 g of either sex were marked and randomly divided into groups. The test compounds of different concentrations were prepared by serial dilutions using 0.5% sodium carboxymethyl cellulose solution. The volumes of suspension by oral gavages were approximately 0.2 mL/10 g weight for mice and 1 mL/100 g weight for rats. KM mice (5 males and 5 females each group) treatments with different levels of TVM, TVM A and IVM were shown in Table 1 to 3. SD rats (6 males and 6 females each group) treatment with TVM were shown in Table 4. SD rats (5 males and 5 females each group) treatment with TVM A
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were shown in Table 5. Changes in behavior and mortality of animals were observed and recorded for 14 days. Evaluations included observations of appearance, respiratory, salivation, tremors and convulsions, motor activity, gait and posture, reactivity to handling and bizarre behavior (e.g. walking backwards). These animals died during the study were necropsied, and the time of death was recorded as precisely as possible. At the end of the test,
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surviving animals were sacrificed and necropsied. The LD50 values and 95% confidence intervals were calculated
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using the Bliss method by the software of Drug and Statistics 3.0 (Bliss, 1934, 1935).
2.3. Bacterial reverse mutation assay
TVM was assayed via the Ames test by standard plate-incorporation assay according to OECD Guideline (471,
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1997) and China standard for Ames (GB 15193.4-2003). Histidine requiring Salmonella typhimurium (TA97a, lot: 4366D; TA98, lot: 4367D; TA100, lot: 4370D; TA102, lot: 4372D and TA1535, lot: 4369D) used in this study
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were obtained from Molecular Toxicology Inc. (Boone, NC, USA). The preliminary Ames test was performed both with and without the S9 activation system. The metabolic activation system (S9 mix) was freshly prepared before
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each test by use of an Aroclor-1254 induced rat liver fraction (S9, lot: 3369, MolTox™, Boone, NC, USA). TVM
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were dissolved in Dimethylsulfoxide (DMSO, 99.99% of purity, Sigma–Aldrich Chemical Co.) to be a final concentration of 50.0 mg/mL that was assigned for the highest test concentration (50000 ug/mL) and serially diluted with DMSO for lower test concentrations (16667, 5556, 1852, 617, 206, 69 ug/mL). The mutagenicity assay was carried out in both the presence and absence of metabolic activation system along with the negative and positive controls as described in OECD Guideline No. 471. The positive control materials were purchased from Sigma–Aldrich Chemicals Co. as follows: TA97a (1.0 ug/plate, ICR-191, cas:17070-45-0, lot: SLBD9266V), TA98 (1.0 ug/plate, 2-nitrofluorene, cas:607-57-8, lot: S43858V), TA100 and TA1535 (2.0 ug/plate, Sodium azide, cas:26628-22-8, lot: 063M7386), TA102 (1.0 ug/plate, methyl methanesulfonate, cas:66-27-3, lot:87596LJ), all stains plus S9 (30.0 ug/plate for TA102 and 3.0 ug/plate for others, 2-Aminoanthracene, cas:613-13-8,
ACCEPTED MANUSCRIPT lot:STBD3302V). Each treatment was made in triplicate. Test plates per concentration were incubated at 37℃ for 48 h and then counted. The mutagenic index (MI) was calculated for each dose as the average number of revertants per plate divided by the average number of revertants per plate of the negative (solvent) control. For test strains TA97a, TA98, TA100 and TA102, a sample was considered positive when MI was above 2 for at least one of the test doses, and when the response was dose dependent. The MI for a positive response of strain TA1535 was more than 3 for at least one concentration level and showed dose-response relationship with or without S9 (Biso et al.,
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2010).
2.4. Efficacy of TVM against swine nematodes 2.4.1 Test animals
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16 growing pigs parasite-infected naturally, weighing 30–35 kg (females or castrated males), were purchased from individual farmers and housed in the swine barn. Each pig was selected by two consecutive days fecal
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samples examined positive significantly for nematode worm eggs, and one of them selected randomly was necropsied for confirming Ascaris suum and Trichuris suis infection. Pigs were individually ear-tagged and fed a corn, soybean-based complete ground diet formulated for the growth stage. Pens were separated from each other by
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solid concrete and provided with nipple waterer and self-feeder. Pigs were observed daily during the trial. 2.4.2. Experimental design
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15 pigs were randomly blocked into two groups, 10 pigs for TVM treatment group and 5 pigs for IVM treatment
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control. On Day 2 and 1 before adminstration and day 7 and 8, day 14 and 15, day 21 and 22, day 28 and 29 after dose, fecal samples were taken from each pig and the faecal egg counts (FEC) were determined using an modified
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McMaster technique with a sensitivity of 30 eggs per gram of faeces (EPG) (Rinaldi et al., 2011). The eggs of Ascaris suum and Trichuris suis were identified by Morphological characteristic structures (Hennessy et al., 2006; Peña-Espinoza et al., 2016). Pigs were administrated TVM and IVM with a single subcutaneous injection 0.3 mg/kg body weight (recommended dose of IVM), and sacrificed on day 30 after treatment. The intestinal contents and lungs were examined for nematodes according to standard parasitological procedures (Hennessy et al., 2006). Adult and larval worms were collected, identified and counted by standard parasitological techniques for confirming the effectiveness. 2.4.3 Drug efficacy criteria
ACCEPTED MANUSCRIPT The efficacy of treatment was evaluated by FEC of each pig before and after treatments, or between different treatments. The faecal egg counts reduction rate (FECR%) and the fecal eggs positive rate were calculated and compared. The data of FEC were transformed to log (EPG+1) and subjected to a two-way analysis of variance using the software of Drug and Statistics 3.0 and treatment effect was tested for significance at the 0.05 level (Andre et al., 2016).
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3. Results 3.1. Acute toxicity study 3.1.1 Acute toxicity of IVM
Mice in control groups dosed with IVM, some adverse effects such as hypoactivity, debility, curl up and
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trembling were obviously observed 4 h after the oral administration. These effects became more serious until to death in higher dose groups. Most of the death occurred between 12 and 24 h after administration. The results of
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oral acute toxicity of IVM in mice summarized in Table 1. The oral LD50 values of IVM were determined to be 53.06 mg/kg b.w. with 95% confidence intervals ranging from approximately 48.72 to 58.34 mg/kg. 3.1.2 Acute toxicity of TVM
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The poisoning symptoms of animals mainly occurred at 2-3 hours after administration. The mice and rats exhibited restless, debility, curl up, depression, ataxia, shortness of breath, trembling and death. In lower dose
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groups, mildly poisoned animals gradually increased the activities and recovered mostly 2-4 h after administration.
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In higher dose groups, severe symptoms of poisoning and death were clearly observed and the degree of poisoning symptoms was directly proportional to the dosage. The peak of death time of mice and rats was during 8-12 h after
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administration and all deaths occurred within 1 day after administration. No obvious organ abnormalities were visible in the animal necropsy. The results of the oral acute toxicity of TVM were summarized in Table 2 and 3. The oral LD50 values of TVM in mice and rats were determined to be 74.41 mg/kg b.w. and 164.22 mg/kg b.w. ,with 95% confidence intervals ranging from approximately 65.99 to 83.42 mg/kg and 135.50 to 201.36 mg/kg, respectively. 3.1.3 Acute toxicity of TVM A The results of the oral acute toxicity of TVM A in mice and rats were summarized in Table 4 and 5. Adverse effects such as hypoactivity, debility, curl up, shortness of breath, trembling and death were obviously observed at 3 h after administration and these effects were more serious and persistent at higher doses. The animals with mild
ACCEPTED MANUSCRIPT poisoning symptoms generally recovered after 12 h. Times of the death of mice and rats induced by severe side effects were mainly at 8 to 36 h after administration. Most surviving animals gradually recovered the feeding and activities at 3th day after administration. However one moribund mouse died at 4th day after administration and one rat appeared delayed death at 10th day after administration. No distinctly organ abnormalities were seen in the autopsy of mice and rats. According to dose levels and the numbers of dead animals, the oral LD50 values of TVM A in mice and rats were determined to be 106.95 mg/kg and 749.34 mg/kg, with 95% confidence intervals ranging
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from approximately 103.78 to 110.22 mg/kg and 653.86 to 858.87 mg/kg, respectively.
3.2. Bacterial reverse mutation assay
The test material TVM was suspended in DMSO and the concentrations of drug ranged from 6.8 to 5000 ug/plate
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in the presence and absence of the S9 mix. The results of the S. typhimurium reverse mutation assay in the presence and absence of metabolic activation are shown in Table 6. The numbers of revertants for the vehicle controls in the
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presence and absence of the S9 mix for all test strains were in the scope of the negative controls and the numbers of revertants increased remarkably in all positive control compared to negative control. The mutagenicity index (MI) for all positive control materials were above 3. The range of MIs for the test material in the presence of the S9 mix
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for strains TA97a, TA98, TA100, TA102 and TA1535 were 0.8-1.1, 0.8-0.9, 1.0-1.1, 1.1 and 0.9-1.1, respectively. The range of MIs in absence of the S9 mix for strains TA97a, TA98, TA100, TA102 and TA1535 were 1.0-1.2,
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0.9-1.2, 0.9-1.1, 0.9-1.0 and 1.0-1.4, respectively. Amounts of the particle tended to precipitate in the plates but did
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not to obscure the background at the concentration of 1667 ug/plate and 5000ug/plate. The number of revertants in the level of treatment were not significantly higher than that of revertants in negative control. MIs were not
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observed to increase more than two fold with the S. typhimurium TA97a, TA98, TA100, TA102 and TA1535 strains after treatment with or without S9 mix. There was no evidence of cytotoxicity in all bacterial systems used in mutation assay.
3.3 Efficacy of TVM against swine nematodes The clinical adverse reactions were not observed after therapy. Results of scolecology examinations for each pig before and after administration are shown in table 7 to 8. One week after TVM treatment, the FEC of Ascaris suum and Trichuris suis decreased significantly (p<0.05), but egg positive rate did not decrease obviously. Two weeks after administration, the FECR% and positive rate of Ascaris suum all decreased obviously (p<0.05). Four weeks
ACCEPTED MANUSCRIPT after administration, the negative rate of Ascaris eggs and Trichuris eggs were 100% and 99.43%, suggesting that TVM had potential effect against Ascaris suum and Trichuris suis. Meanwhile, pigs after IVM treatment, the FECR% and positive rate of Ascaris suum all decreased obviously (p<0.05), but did not decrease significantly for Trichuris suis (p>0.05), indicating that IVM had good efficacy against Ascaris suum but limited effect on Trichuris suis. Necropsy found roundworms in one pig for each group and whipworms in two pigs in TVM groups and five pigs in IVM groups respectively. This study revealed that TVM and IVM had similar good efficacy against Ascaris and
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only TVM had potential efficacy against Trichuris.
4. Discussion
The molecule of TVM，IVM and milbemycin differs from each other only at one position. The TVM differs
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from IVM at position C25 substituent group and differs from milbemycin at position C13 substituent group. IVM contains at least 80% of 22, 23-dihydroavermectin B1a and less than 20% 22, 23-dihydroavermectin B1b. The
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main active ingredient is the 22, 23-dihydroavermectin B1a (Huang et al., 2015b; McKellar and Benchaoui, 1996; Wan et al., 2016). TVM is a mixture of TVM A and TVM B in a ratio of 3:1, which is expected to reduce the production cost substantially. However, it is necessary to understand the pharmacological properties of different
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components during research process, which can provide the references for developing other similar new drugs showing practical significance (Arsic et al., 2017; Wan et al., 2017a; Wan et al., 2017b). Therefore, this
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preliminary study evaluated the acute toxicity of TVM and components TVM A respectively.
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Acute toxicity tests were used to compare relatively acute hazards of chemicals, especially for new compounds that no other toxicology data are available (Wu et al., 2014). The LD50 values of drugs are generally the beginning
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of safety evaluation. It is also an important reference criteria on the classification of relative toxicity of drug compounds (Copplestone, 1988; OECD, 2001). As the result of acute toxicity test is influenced by many factors, such as the strains and ages of experimental animals, the environment and the time of fasting, the results of different laboratories are quite different (Dadarkar et al., 2007; Wu et al., 2015). Therefore, this experiments set up the similar drug IVM as a control, and the LD50 value of TVM acquired under the same conditions for IVM can objectively compared the difference of acute toxicity of both. The relative toxicity classification of different structures of new compounds is helpful for understanding the relationship between structure and toxicity, which is conducive to the development of new drugs and further selection of target compounds.
ACCEPTED MANUSCRIPT In this acute toxicity test, there were no obvious organ abnormalities on necropsy. The poisoning symptoms pointed to neurotoxicity and the influence of central nervous may be the reason of death of animals, consistent with reports of the acute toxicity symptoms of similar drug (Campbell and Benz, 1984; McKellar and Benchaoui, 1996). The calculated LD50 value of TVM, TVM A and IVM for KM mice were 74.41, 106.95 and 53.06 mg/kg respectively, indicating that the relative toxicity of TVM was lower than that of IVM, and the new compound with directional modification had some advantages (Huang et al., 2015a). The results of acute toxicity for SD rats
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showed that the acute toxicity of TVM A was significantly lower than that of TVM, and the oral LD50 values of TVM and TVM A were 164.22 and 749.39 mg/kg respectively, According to the World Health Organization’s recommended classification of the relative toxicity of chemicals, TVM is a moderately toxic substance while TVM A belongs to a low toxic compound (Copplestone, 1988; Dadarkar et al., 2007). Since TVM is a mixture of TVM A
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and B in a ratio of 3:1, it is speculated that the relative toxicity of TVM B is higher, and TVM B is the main reason that the acute toxicity of TVM was significantly higher than that of TVM A.
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The molecule of TVM A and TVM B differs at position C25 substituent with methyl or ethyl respectively, and the difference of acute toxicity obviously suggests that the site may have important biological functions, which is one of the key sites studied on structure-activity relationship. The side chain of C25 in TVM is consistent with the
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similar drug milbemycin and shorter than IVM. It is reported that milbemycins is similar to TVM with lower toxicity (McKellar and Benchaoui, 1996; Shoop et al., 1995). Therefore, it is implied that the length of side chain at
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needed to reveal the reason.
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position C25 is highly related to the toxicity of 16-membered macrocyclic lactone compounds. Further studies are
The Ames test has been used worldwide as an initial screening to determine the mutagenic potential of new
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chemicals and drugs. The mutagenicity for chemicals is a crucial factor for druggability and an important basis for drug screening (Fei et al., 2015; OECD, 1997; Shim et al., 2008). TVM had no mutagenicity to Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 with and without metabolic activation system, consistent with the reports of the same kind of drugs (Campbell and Benz, 1984; Campbell et al., 1983; Sutherland and Campbell, 1990). However, there are many factors affecting the inherent toxicity of the compounds. Therefore, a series of mutagenicity and genotoxicity tests are needed to further evaluate the safety of TVM on the basis of determination of the more valuable components (Jena et al., 2002). TVM is a novel compound and its structure differs from IVM only at position C25 substituent group, speculating that the mutagenicity is probably not related to the side chain at position C25 of 16-membered macrocyclic lactone antibiotics.
ACCEPTED MANUSCRIPT IVM is a first-line deworming drug, and ascariasis and trichuriasis are the most serious parasitic diseases in swine (Laing et al., 2017). The activity of TVM against T. cinnabarinus in vitro is higher than that of IVM, and the activity of TVM against ascariasis and trichuriasis in vivo has not been reported (Huang et al., 2015a). For evaluating the anthelmintic activity of TVM preliminarily, growing pigs that naturally infected with Ascaris suum and Trichuris suis were screened and confirmed for this experiment. The dosage of TVM and IVM were all set to 0.3 mg/kg b.w. with a single subcutaneous injection, which is the recommended dose of IVM (Campbell and Benz,
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1984). It is more convenient to compare the activity of these two similar compounds. The results showed that TVM and IVM with a single subcutaneous injection 0.3 mg/kg b.w. had good effect in expelling Ascaris suum，and TVM also had potential effect against Trichuris suis. The result of TVM against trichuriasis is not completely consistent with the reports on the role of IVM (Campbell and Benz, 1984; Campbell et al., 1983; Laing et al., 2017).
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Because of the restrictions of clinical animal quantities, further studies are needed to verify the effectiveness of TVM against Trichuris suis infection.
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TVM had similar safety and anthelmintic activity to IVM. However, the activity of TVM against trichuriasis occurs prior to that of IVM. Huang et al. (2015a) also reported the enhance activities of TVM against T. cinnabarinus and B. xylophilus. Because of the difference only at position C25 substituent groups, we speculated
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that the action mechanisms of both drugs are roughly the same. Recent studies confirmed that glutamate gated chloride channel is a more important target for these kinds of drug, and the GABA receptor is a secondary target.
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The biological activity of these compounds is different in higher animals, showing that there may exist other targets
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of action (Feng et al., 2002; Wang et al., 2016). It can be postulated that the targets of action of TVM is different from that of IVM (Prichard et al., 2012).The focus of further studies should be verify the safety and effectiveness of
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TVM and evaluated the cross-resistance between TVM and IVM (George, 2017; Prichard et al., 2012). TVM may have difference target and medicinal basis worthy of developing as a new drug.
5. Conclusion The acute toxicity of TVM is lower than that of IVM. TVM and IVM were all moderately toxic substance according to the classification of the relative toxicity. Meanwhile, the acute toxicity of TVM A was significantly lower than that of TVM, which belongs to a low toxic compound, indicating that the length of side chain of TVM at position C25 is highly related to the toxicity of these kinds of compounds. The Ames test results were all negative for Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 with and without metabolic
ACCEPTED MANUSCRIPT activation system, illustrating that TVM may have no genotoxicity. TVM and IVM with a single subcutaneous injection 0.3 mg/kg b.w. had excellent efficacy against Ascaris suum, and only TVM had potential effect on Trichuris suis. This study suggests that TVM had some advantages and the structure of TVM at position C25 may have important biological functions. TVM has a medicinal basis worthy of developping, especially component
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TVM A has higher security and deserves further study.
Acknowledgements
This study was supported by The National Key Research and Development Program of China (2016YFD0501303) and The Special Fund for Agro-Scientific Research in the Public Interest (201303038–9).
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Products 79, 2446-2454.
Wu, H., Xiao, W., Zhang, K., Xue, F., Zhang, C., Yan, M., Wang, X., Jiang, S., 2014. Acute and subchronic toxicity of arprinocid in Sprague-Dawley rats. Regul Toxicol Pharmacol 69, 487-495. Wu, X., Wan, D., Xie, C., Li, T., Huang, R., Shu, X., Ruan, Z., Deng, Z., Yin, Y., 2015. Acute and sub-acute oral toxicological evaluations and mutagenicity of N-carbamylglutamate (NCG). Regul Toxicol Pharmacol 73, 296-302.
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Figure 1. The structures of tenvermectin. A: R=CH3; B: R=CH2CH3
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Figure 2. The structures of ivermectin. B1a: R= CH2CH3; B1b: R= CH3
Figure 3. The structures of milbemycin oxime. A3: R=CH3; A4: R=CH2CH3
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Table 1 Oral acute toxicity of ivermectin by gavage in mice Males Females Weight/g
Number of dead/dosed
Weight/g
Number of dead/dosed
Mortality %
34 38
19.7±0.41 19.6±0.84
0/5 1/5
19.0±0.58 19.2±1.43
0/5 2/5
0% 30%
43 49
19.7±0.79 20.2±0.72
0/5 2/5
19.1±0.15 20.4±1.76
2/5 0/5
20% 20%
55 63
20.0±1.40 19.6±0.65
3/5 3/5
19.4±0.23 19.8±0.74
3/5 4/5
60% 70%
71
20.0±0.40
5/5
20.7±0.76
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Dose mg/kg
5/5
100%
Table 2 Oral acute toxicity of tenvermectin by gavage in mice Males
Females
Weight/g
Number of dead/dosed
Weight/g
Number of dead/dosed
Mortality %
42 50
18.9±0.88 18.8±0.89
0/5 1/5
19.6±0.67 19.3±0.66
0/5 0/5
0% 10%
61 73
21.4±0.47 21.4±0.68
2/5 4/5
19.2±0.55 20.9±0.64
1/5 2/5
30% 60%
87 105
19.0±0.64 19.2±0.89
5/5 5/5
18.7±0.58 19.8±1.22
3/5 3/5
80% 80%
125
19.0±0.72
5/5
20.2±0.25
5/5
100%
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Dose mg/kg
Table 3 Oral acute toxicity of tenvermectin by gavage in rats Females
Number of dead/dosed
Weight/g
Number of dead/dosed
Mortality %
80
195±7.6
1/6
189±1.4
0/6
8%
100
199±6.6
0/6
189±4.7
2/6
17%
125
196±7.1
3/6
189±4.6
3/6
50%
156
198±9.0
3/6
190±3.7
3/6
50%
195
197±5.2
3/6
191±4.6
5/6
67%
244
204±3.5
3/6
193±5.4
6/6
75%
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Dose mg/kg
Table 4 Oral acute toxicity of tenvermectin A by gavage in mice Males
Females
Weight/g
Number of dead/dosed
Weight/g
Number of dead/dosed
Mortality %
90
19.3±0.71
0/5
18.9±0.22
0/5
0%
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Dose mg/kg 96
20.5±0.16
0/5
19.3±0.41
1/5
10%
103
19.9±0.53
2/5
18.5±0.26
0/5
20%
110
19.8±0.36
2/5
19.0±0.33
4/5
60%
118
20.6±0.45
5/5
19.1±0.47
5/5
100%
Table 5 Oral acute toxicity of tenvermectin A by gavage in rats Males
Females
Dose mg/kg
Weight/g
Number of dead/dosed
Weight/g
Number of dead/dosed
Mortality %
580
198±7.8
2/5
189±2.4
0/5
20%
680 798
203±7.6 206±7.5
2/5 2/5
191±4.7 189±6.2
2/5 4/5
40% 60%
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198±9.3
3/5
193±3.8
5/5
80%
1100
207±5.2
3/5
192±3.6
5/5
80%
Table 6 Mutagenic activity and mutagenic index (MI) of tenvermectin in TA97a, TA98, TA100, TA102 and TA1535 Salmonella typhimurium strains in the absence (− S9) and in the presence (+ S9) of metabolization. TA97a
TA98
TA100
TA102
TA1535
S 9
Rev/plate
NC -S9mix
-
82.7±2.31
PC -S9mix
-
499±60.92
6. 0
238±4.36
10. 2
694±38.43
6.0
1594.7±65.4 9
5. 4
557.7±37.0 7
49. 4
6.86 ug/plate
-
85.7±6.03
1. 2
29±3.46
0.9
115.3±6.81
0.9
303±6
0. 9
12.3±0.58
1.1
20.58 ug/plate
-
91.3±7.23
1. 0
271.73
1.1
121.7±5.13
0.9
301±5
0. 9
16.3±2.31
1.1
61.73 ug/plate
-
92.3±5.77
1. 0
25.33.79
1.2
121.3±4.51
1.0
295.3±9.5
1. 0
14.7±3.21
1.2
185.19 ug/plate
-
94±3
1. 1
25.3±6.43
1.1
115.3±3.06
1.0
281±11
1. 0
11±1.73
1.0
555.56 ug/plate
-
84±6.08
1. 1
27.3±6.81
1.1
119±3.46
1.1
299±6.24
1. 0
13.3±3.79
1.3
1666.67 ug/plate
-
80.7±8.14
1. 1
25±6.08
1.2
106.3±1.15
1.1
268.7±4.16
1. 0
12±4
1.4
5000 ug/plate
-
101±6.93
1. 0
20.7±2.52
1.2
109.3±1.53
1.0
272.7±3.21
1. 0
12.3±1.53
1.1
NC -S9mix
+
112±7.94
PC -S9mix
+
675.7±33.5 0
6. 0
6.86 ug/plate
+
118.3±6.66
0. 8
20.58 ug/plate
+
110.3±2.52
0. 9
61.73 ug/plate
+
120.3±11.9 3
0. 9
185.19 ug/plate
+
99.7±3.06
555.56 ug/plate
+
97.3±7.57
1666.67 ug/plate
+
100.7±6.43
5000 ug/plate
+
92.3±11.06
Rev/plate
MI
42±3
MI
MI
294±10.39
112±3
Rev/plate
MI
11.3±1.53
262±7.81
15.3±3.06
39. 2
1840±55.83
16. 4
1864±164.84
7. 1
605.7±36.5 6
39. 6
31.3±0.58
0.9
124±3.46
1.1
278.7±6.43
1. 1
16.3±2.08
1.1
34.3±4.16
0.9
128±1
1.0
284.7±15.04
1. 1
14.3±3.06
1.1
32±5
0.9
120.31.15
1.1
290.7±6.66
1. 1
14.7±1.53
0.9
32.3±1.15
0.8
116.39.29
1.0
275.7±5.86
1. 1
16±2.65
1.1
1. 1
37.3±2.31
0.8
123±6.24
1.1
284.7±15.18
1. 1
14.3±3.21
1.0
1. 0
36.3±3.06
0.8
115.3±11.5 9
1.1
285.7±13.58
1. 1
16.3±2.52
0.9
1. 1
38±6.08
0.8
124.7±1.15
1.1
298.7±7.77
1. 1
16.3±3.06
1.1
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1645.3±139.5 8
Rev/plate
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116±7.55
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23.3±6.11
Rev/plate
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-S9, without metabolic activation; +S9, with metabolic activation (500 uL 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 ICR-191 (1.0 μg/plate) for strains TA97a, 2-nitrofluorene (1.0 μg /plate) for strain TA98, methyl methanesulfonate (1.0 ug/plate) for strain TA102, sodium azide (2.0 μg /plate) for strain TA100 and TA1535. Positive controls with S9 activation were 2-aminoanthracene (3.0μg / plate) for strains TA97, TA98, TA100 and TA1535, 2aminoanthracene (30.0μg / plate) for strain TA102 MI, mutagenicity index: number of revertants induced in the sample/number of spontaneous revertants in the negative (solvent) controls.
ACCEPTED MANUSCRIPT Table 7 Ascaris suum examinations for each pig before and after administration Tenvermectin，n=10 Ivermectin，n=5 Time of collection positive rate of eggs Mean FEC(epg) positive rate of eggs Mean FEC(epg) Before administration
100%
1 week after administration
80%
173
2 weeks after administration
30%
3 weeks after administration 4 weeks after administration
100%
1842
20%
15*
5*
0%
0*
0%
0*
0%
0*
0%
0*
0%
0*
FECR%
3114 *a
100.0%
100.0%
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The asterisk （*） indicates the significantly differences of FEC between administration before and after in the columes (P < 0.05); The lowercase letter（a） indicates the significantly different of FEC between groups treatment with TVM and IVM in the rows (P < 0.05).
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FEC (epg), feacal egg counts (eggs per gram of faeces); FECR%, faecal egg counts reduction rate.
Before administration
70%
1 week after administration
10%
2 weeks after administration
20%
4 weeks after administration
5
*a
80%
1422
60%
348
23*a
80%
270
20%
30*a
80%
705
10%
6*a
80%
840
99.43%
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FECR%
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3 weeks after administration
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Table 8 Trichuris suis examinations for each pig before and after administration Tenvermectin，n=10 Ivermectin，n=5 Time of collection positive rate of eggs Mean FEC(epg) positive rate of eggs Mean FEC(epg)
40.93%
The asterisk （*） indicates the significantly differences of FEC between administration before and after in the columes (P < 0.05);
< 0.05).
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The lowercase letter（a） indicates the significantly different of FEC between groups treatment with TVM and IVM in the rows (P
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FEC (epg), feacal egg counts (eggs per gram of faeces); FECR%, faecal egg counts reduction rate.
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Graphical abstract