ABSTRACT: Aditoprim (ADP), a new developed dihydrofolate reductase (DHFR) inhibitor, has great potential in clinical veter-inary medicine because of its greater pharmacokinetic properties than structural analogs. Preclinical toxicology studies wereperformed to assess the safety of ADP including an acute oral toxicity test, a subchronic toxicity test and five mutagenicitytests. In the acute oral toxicity test, ADP was administered singly by oral gavage to Wistar rats and Kunming mice. TheLD50 calculated was 1400 mg kg–1 body weight (BW) day–1 in rats and 1130 mg kg–1 BW day–1 in mice. In a subchronic study,Wistar rats were administered ADP at dose levels of 0, 20, 100 and 1000 mg kg–1 diet for 90 days. Significant decreases wereobserved on body weight and food efficiency in the high-dose group. Treatment-related changes in clinical serum biochem-istry were found in the medium- and high-dose groups. Significant increases in the relative weights of livers and kidneys infemales and testis in males in the 1000 mg kg–1 diet, and significant decrease in relative weights of livers in males in the100 mg kg–1 diet were noted. Histopathological observations revealed that the 1000 mg kg–1 ADP diet could induce lym-phocytic infiltration and hepatocytic necrosis near the hepatic portal area. The genotoxicity of ADP was negative in tests,such as the bacterial reverse mutation assay, mice bone marrow erythrocyte micronucleus assay, in vitro chromosomalaberration test, in vitro cho/hgprt mammalian cell mutagenesis assay and mice testicle cells chromosome aberration. Based onthe subchronic study, the no-observed-adverse-effect level for ADP was a 20 mg kg–1 diet, which is about 1.44-1.53 mg kg–1
*Correspondence to: Zong-hui Yuan, Hubei Collaborative Innovation Center forAnimal Nutrition and Feed Safety, Wuhan, Hubei, China.E-mail: [email protected]
aNational Reference Laboratory of Veterinary Drug Residues (HZAU) and MAOKey Laboratory for Detection of Veterinary Drug Residues, Wuhan, Hubei430070, China
bMOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poul-try Products, Huazhong Agricultural University, Wuhan, Hubei, 430070, China
cHubei Collaborative Innovation Center for Animal Nutrition and Feed Safety,Wuhan, Hubei, China
dDepartment of Biosciences, COMSATS Institute of Information Technology,Sahiwal, Pakistan
IntroductionAditoprim, (5-[(4-dimethylamino-3,5-dimethoxy-phenyl)methyl]pyrimidine-2,4-diamine, ADP) (Fig. 1) is a broad-spectrum bacte-riostatic agent which belongs to diaminopyrimidines. Its struc-tural analogs, such as trimethoprim (TMP), diaveridine (DVD),baquiloprim (BQP) and ormetoprim (OMP), have been used inhuman and veterinarymedicines as dihydrofolate reductase (DHFR)inhibitors in bacterial cells. Diaminopyrimidines are often in combi-nation with sulfonamides for synergistic effects against both gram-negative and gram-positive bacteria (Capasso & Supuran, 2014).The gram-negative bacteria, such as Enterobacteriuceae, Hae-mophilus and Vibrio, are highly susceptible to ADP, whereasAcinetobacter and Alcaligenes and the gram-positive bacteria suchas Staphylococci and Streptococci species are moderately suscepti-ble to ADP (Then & Keller, 1988).
It was reported that ADP exhibited a better penetration into thecytoplasm than that of TMP (Then & Keller, 1988). ADP has a broadantimicrobial spectrum, good antibacterial activity and excellentpharmacokinetics characteristics including rapid absorption, highbioavailability, strong penetration into tissue in vivo, high peakconcentrations and a long elimination half-life (Haenni et al.,1987; Iqbal et al., 1990; Lohuis et al., 1992; Riond et al., 1992; Iqbalet al., 1995). It was determined that the drug would have a broaddevelopment prospect in veterinary clinical practice.
The toxicological studies of ADP were limited. The acute oralLD50 of ADP for rats and mice was reported to be 1402 and
1130mg kg–1 BW, respectively (Stephan-Guldner, 1993). Moreover,it was reported that the subchronic toxicity of ADP was investi-gated in Beagle dogs (Wolfe et al., 1993). In this study, ADP was ad-ministered orally via gelatin capsules at 0, 10, 20, and 100 mg kg–1
BW day–1 and 0, 1, 5 and 10 mg kg–1 BW day–1 for 13 weeks. Theresults of this study indicated that ADP had an adverse effect onthe hematopoietic system in Beagle dogs at a dose as low as 10mg kg–1 and the no toxic effect level was determined to be 1 mgkg–1 BWday–1 (Wolfe et al., 1993). The limited profile could not pro-vide a safety assessment for ADP. It is necessary to perform a series
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Figure 1. Structure of aditoprim (ADP).
X. Wang et al.
of basic toxicological studies which are in accordance with preclin-ical toxicology guidelines.
In the present study, a series of studies designed according tostandard protocols were carried out, including the acute toxicitytest, the repeat dose subchronic toxicity test and a battery of fivemutagenicity tests. It could provide the general toxicity proper-ties including the no-observed-adverse-effect level and targetorgans and the determination of mutagenicity. The achieve-ments would provide scientific information for further researchto avoid the adverse reactions and toxic effects in clinical use.
Materials and Methods
Materials
Aditoprim (molecular weight 303.36 gmol, purity 98%, lot num-ber 20121228), was obtained from the Institute of VeterinaryPharmaceuticals, Huazhong Agricultural University (Wuhan,China). Hypoxanthine aminopterin thymidine (HAT), Trypsin,Ham’s F12 and RPMI 1640 medium were provided by Hyclone(Shanghai, China). Ethylmethanesulfonate (EMS), Benzo-.alpha;-pyrene (BaP) and 6-TG were purchased from Aladdin (Shanghai,China). Fetal bovine serum (FBS) and new-born calf serum wereproduced from Hangzhou Sijiqing Biological Materials LimitedCompany (Hangzhou, China). Phenobarbital/benzoflavone (10%)-induced rat liver S9 was purchased from Platt Bio-PharmaceuticalCo., Ltd. (Wuhan, China). The rat and mice diet was procured fromthe Center of Laboratory Animals of Hubei Province (Wuhan, PRChina). All the other chemicals were of analytical grade or com-plied with the standards needed for cell culture experiments.
Animals and Treatment
Specific pathogen-free Wistar rats, Kunming mice and their feedwere purchased from the Center of Laboratory Animals of HubeiProvince. The study was approved by the Ethical Committee ofthe Faculty of Veterinary Medicine at Huazhong Agriculture Uni-versity. Use of animals in this study was in accordance with NIHPublication 85-23 ’Guide for the Care and Use of Laboratory An-imals‘ (NRC, 1996).
A barrier-maintained animal room conditioned at a tempera-ture of 22 ± 3 °C, a relative humidity of 50 ± 20% and a 12-hlight/dark cycle. The basic feed (lot number: 20130102) withoutany drugs was produced according to the Chinese standard"Laboratory animal rats and mice feed" (GB14924.3, 2010). Ani-mals received basic feed and fresh water during the 1-week ac-climatization period, housed 4 or 5 per cage with hardwoodshavings as bedding.
Acute Oral Toxicity Study
Wistar rats (8 weeks old) and Kunming mice (6 weeks old) wereused in this study. An UDP (up and down procedure) study wasperformed to evaluate the acute toxicity of ADP, in accordance
with the method provided by the Organization for Economic Co-operation and Development (OECD) Guideline No.425 (OECD,2008). Food was withdrawn overnight before administration ofstarting doses at 10.00 to 11.00 hours. With a slope factor sigmaof 0.5 and the starting doses were 440 and 360 mg kg–1 BW,respectively, the animals were administered with 0.5%carboxymethyl-cellulose (CMC) solution of ADP by oral gavage.Dose progression was stopped when one of three stopping rulesof the AOT425 StatPgm program (OECD, 2008) was satisfied.Then rats and mice that survived were observed for 14 days. Ob-servations included evaluation of skin and fur, eyes and mucousmembranes, respiratory, autonomic effects (e.g. salivation), cen-tral nervous system effects (tremors and convulsions), changesin the level of motor activity, gait and posture, reactivity to han-dling and stereotypes or bizarre behavior (e.g. self-mutilation,walking backwards). Body weights were recorded before, duringand at the end of the test. The time of death was recorded asprecisely as possible. At the end of the test, surviving animalswere weighed and sacrificed.
For acute oral toxicity, LD50 values and 95% confidence inter-vals (CI) were calculated by the software AOT 425 Stat Pgm(software source: http://www.oecd.org/document/62/0,3343,en_2649_34377_44706494_1_1_1_1,00.html).
Subchronic Toxicity Study
Experimental design. Male and female rats (5 to 6 weeks old) wererandomly assigned to four groups. The control group and thehigh-dose group consisted of 20 female rats and 20 male rats,and the low- and medium-dose group consisted of 15 female ratsand 15male rats. ADPwas administered to rats by feeding concen-trations of 20, 100 and 1000 mg kg–1 diet for 13 weeks. Five or tenanimals/group/sex were anesthetized with pentobarbital sodiumand killed at the 45th or 91th day after an overnight fast, respec-tively. The rats left in the control and high-dose groups after 90days were then administered with control feed for another 2weeks and killed as described previously to perform an examina-tion. This study followed the OECD Guideline No.408 (OECD,1998) and was conducted in compliance with FDA Good Labora-tory Practice Regulations (FDA, 2010).
Clinical observations. Before treatment, rats were individually han-dled and carefully examined for abnormal behavior and appearance.All rats were observed at least once a day for mortality or morbidity,changes in posture, changes in skin, fur, eyes, mucous membranesand behaviors. Changes in gait were assessed weekly by allowingthe animal towalk freely. Individual bodyweight datawere obtainedbefore experimental diets administration, weekly during the treat-ment periods and at necropsy (fasted). Food consumption, food ef-ficiency (g weight gain per 100 g of food consumed) andmean dailyintake of ADP (mg kg–1 day–1) were also calculated.
Clinical pathology.After an overnight fast, animals were anesthetizedwith pentobarbital sodium and euthanized by exsanguinations.Blood samples were collected from the femoral vein for hematologyand serum biochemistry. The anticoagulants were potassium EDTAfor the hematology tests. For clinical chemistry, blood samples wereplaced in serum tubes at room temperature for approximately 30min to obtain the serum aliquots. After clotting, the blood tubeswere centrifuged at 940 g for 10 min with a Himac CR 21 G centri-fuge (Hitachi, Tokyo, Japan). Supernatants were decanted andstored at -20 °C for further analysis.
Acute, subchronic toxicity and mutagenicity studies of ADP
Hematology. Hematological measurements were performed with aCoulter HmX Hematology Analyzer (Beckman Coulter Inc., Fullerton,CA, USA). Hematological evaluations included red blood cell count(RBC), hemoglobin concentration (HGB), hematocrit (HCT), meancorpuscular volume (MCV), mean corpuscular hemoglobin (MCH),mean corpuscular hemoglobin concentration (MCHC), red cell vol-ume distribution (RDW), blood platelet hematocrit (PCT), meanplatelet volume (MPV), platelet distribution width (PDW) and whiteblood cell count (WBC).
Serum biochemistry. Serum chemistry was assessed using aSynchron Clinical System CX4 (Beckman Coulter, Brea, CA, USA) ac-cording to the manufacturer’s instructions (Beijing Leadman Bio-chemistry Technology Co. Ltd, Beijing, China). Parameters includedalanine aminotransferase (ALT), aspartate aminotransferase (AST), al-kaline phosphatase (ALP), total protein (TP), albumin (ALB), glucose(GLU), blood urea nitrogen (BUN), total cholesterol (TCHO), creati-nine (Cr), triglyceride (TG), total bilirubin (TBL), sodium (Na), potas-sium (K), chloride (Cl) and calcium (Ca).
Histopathological examinations. All the organs/tissues were carefullyexamined macroscopically and gross lesions were recorded. Duringnecropsies, the organs of each animal including the brain, heart,lungs, kidneys, liver, spleen, prostate, adrenal glands, testicles (epi-didymis), ovary and uterus were weighed separately. Organ/bodyweight ratios were calculated based on the fasted animal’s bodyweight. The organs from each animal, with the exception of testes,were preserved in 10% neutral-buffered formalin and slides wereprepared for histopathological examination. Histopathological ex-amination was conducted using the routine paraffin embeddingtechnique. Sections of 5 μm thickness stained with hematoxylinand eosin (H&E) were examined under lightmicroscopy formorpho-logical alterations (Hutchings et al., 2004). Tissues from other groupswere examined as necessary to determine NOAEL in target organs.
Calculations of drug intakes. The dose levels (mg kg–1 BW day–1)were calculated using the nominal concentration of drugs in thediet, the mean daily food consumption and the body weight forthe week. The equation was (mean weekly food consumption ofeach rat/7)/(the body weight for the week) × nominal concentrationof drugs in the diet.
Statistical analysis. For the 90-day study, statistical analyzes wereconducted by comparing the treatment groups with the controlgroup using the SPSS 17.0 program (SPSS Inc., Chicago, IL, USA).Levene’s test was used to examine the homogeneity of variances.If the variance was homogeneous, the data was analyzed withone-way analysis of variance (ANOVA); otherwise they were ana-lyzed by the Kruskal–Wallis non-parametric ANOVA. If the variancewas significant (P ≤ 0.05), Dunnett’s test was used to identify thestatistical significance of the individual groups. Histopathologicalfindings were subjected to Fisher’s exact probability test. Thepre-set two-sided P-value of<0.05 or 0.01 was taken as statisticallysignificant. Values are expressed as means with their standard er-rors (Lindamood et al., 1992; Lafranconi et al., 1994; Takeda &Flood, 2002).
Mutagenicity Studies
Bacterial reverse mutation assay. A bacterial reverse mutation as-say [Bacterial Reverse Mutation Test (FDA, 2000a)] was per-formed to evaluate the mutagenicity of ADP, with and without
S9, using the following five Salmonella strains as prescribed inthe OECD guideline No.471 (OECD, 1997a) and Redbook 2000:IV.C.1.a. TA97a, TA98, TA100, TA102 and TA1535. All strains ex-cept TA100 (Platt Bio-Pharmaceutical Co., Ltd., Wuhan, PR China)were provided by the China Center for type culture collectionand checked for maintenance of genetic markers prior to thestudy. Owing to the to antimicrobial effect of ADP on Salmonellastrains, the highest concentration of the 1 μg per plate, with andwithout S9, was selected. Concentrations tested were 0, 0.0625,0.125, 0.25, 0.5 and 1 μg per plate. Strain-specific positive con-trols tested without S9 were 9-aminoacridine (TA97a), 2-nitrofluorene (TA98), mitomycin C (TA102) and sodium azide(TA100 and TA1535). 2-aminofluorene was used as a positivecontrol for all strains tested with S9. Test solutions were preparedin dimethylsulfoxide (DMSO) as serial dilutions to deliver the re-quired concentration in a constant volume. The assay tubeswere pre-incubated at 37 °C for 20 min before plating onto min-imal agar. Three test plates per concentration were incubated at37 °C for 48 h and then counted. The criteria for a positive re-sponse were a ≥ two-fold increase in the average plate countcompared with the solvent control for at least one concentrationlevel and a dose response over the range of tested concentra-tions in at least one strain with or without S9.
Mice bone marrow erythrocyte micronucleus assay. This study wasperformed in accordance with the OECD Guideline No. 475(OECD, 1997b) for principles of Good Laboratory Practices(GLP) and Redbook 2000: IV.C.1.d. mammalian erythrocyte mi-cronucleus test (FDA, 2000b). Fifty 9-week-old SPF Kunmingmice were randomly divided into five groups each consistingof 10 mice (five males and five females). Cyclophosphamide(i.p. 40 mg kg–1 BW) was administered 6 h before samplingas a positive control and CMC solution was used as a negativecontrol. In this dose-response study, ADP was administeredtwice in 30 h with a 24-h interval at a dose level of 141.5,282.5, and 565 mg kg–1 BW through oral gavage. Six hoursafter the last treatment, all the animals were euthanized toobtain cell suspensions from the femur bone marrow. Bonemarrows were flushed with 1 ml of newborn calf serum toobtain cell suspensions. One drop of the mixture was smearedon the clean slide, air dried, fixed with 95% methanol for10 min and stained with Giemsa stain. All slides were coded toensure that the evaluation was blinded. Micronucleus frequencieswere determined for each animal by counting 1000 of polychro-matic erythrocytes (PCE) and the micronucleus occurrence rateper one thousand PCE was recorded. The ratio of polychromaticerythrocytes (PCE) to normochromatic erythrocytes (NCE) wasdetermined for each animal by counting a total of 1000 erythro-cytes. The micronucleus occurrence rate and PCE/NCE ratio ofeach group were compared using SPSS 17.0 software.
In vitro chromosomal aberration test. The potential of ADP to in-duce structural and numerical chromosome aberrations was eval-uated in Chinese hamster lung fibroblast cells (V79). The study wasperformed in accordance with the OECD Guideline No. 476 (OECD,1997c) and Redbook 2000: IV.C.1.b in vitro mammalian chromo-somal aberration test (FDA, 2003). V79 cells were procured fromATCC and cultured in RAPI 1640 supplemented with 10% fetal bo-vine serum (FBS), 1.0 mmol L-glutamine, 100 IU ml–1 penicillin and100 μg ml–1 streptomycin. The cultures were grown in a 5% CO2
atmosphere at 37 °C. The maximum concentration tested in thepresent study was based on the solubility of ADP in DMSO and
the preliminary cytotoxicity test. Eventually, the maximum con-centration selected was 300 μg ml–1 based on the solubility ofADP in the test system, and other concentrations (150, 75 and37.5 μg ml–1) selected were separated by a factor of 2. A vehiclecontrol was set with 0.5% DMSO. Mitomycin C and cyclophospha-mide were used as the positive controls for cultures incubated inthe absence and presence of S9, respectively. In the absence ofS9, cultures were incubated with the test compound for 6 and 18h, respectively, washed, placed in fresh culturemedium containing0.3 μg ml–1 colchicine and incubated for an additional 4 h. In thepresence of S9, cultures were incubated with the test compoundfor 6 h. After incubation, the cells were washed and placed infreshly prepared culture medium containing colchicine and ex-posed for 4 h before harvesting. The cells were trypsinized, incu-bated in 0.075 M KCl for 20–25 min at 37 °C and fixed twice withice-cold 1:3 glacial acetic acid: methanol. Then suspensions werecentrifuged at 230 g for 5 min. Supernatant was discarded andthe cells were resuspended in 0.1–0.2 ml of cold fixative. Cell sus-pensions in fixative (20 μl) were dropped on chilled slides and slideswere dried in the air. The slides were stained with 10% Geimsa for20 min and washed with fresh water. Two hundred well-spreadmetaphases were scored per concentration and control.
In vitro CHO/hgprt mammalian cell mutagenesis assay. To evaluatethe potential of ADP to induce mutations at the hgprt locus in cul-tured mammalian cells, CHO-K1 cells were used for this assay. Re-moval of pre-existing hgprt- mutants in cultures were done bygrowth for 2 days in Ham’s F12 medium supplemented with 10%FBS and HAT. The test was performed according to the procedureof O’Neill and Hsie (1979) and O’Neill et al. (1977, 1982). In this test,cells were treated for 6 h in different concentrations of ADP and thecontrol materials with and without S9, and then allowed an expres-sion time of 7–8 days; the cells were then replated. After the periodof expression of the mutant phenotype, mutant colonies were se-lected in the following manner. Each culture flask was trypsinizedand the cells were seeded in new flasks as follows: (i) 5 plates eachwith 200 cells and without the purine analogue 6-thioguanine; and(ii) 5 plates each with 2 × 105 cells and with 6-thioguanine (10 μgml–1). After 8 days, selection was determined by the formation ofcolonies resistant to the purine analog. Next, the colonies werefixed with methanol for 15 min and stained with Giemsa for 20min. Giemsa was poured off, and visible colonies were counted.Methylcholanthrene (3-MCA) was used as a positive control withS9 and EMS was used as positive control without S9. The negativecontrol was 0.5% DMSO. The concentrations of ADP for the assayranged from 37.5 to 300 μg ml–1 with an interval of 2. The colonycounts obtained were used to calculate: (i) cloning efficiency (CE)in absolute values (number of colonies formed divided by the num-ber of cells seeded); (ii) the number of mutant colonies observed ineach treatment; (iii) the absolute CE observed after selection; and(iv) the mutation frequency (MF) (number of mutant colonies di-vided by the total number of cells seeded × the absolute CE afterselection).
Mice testicle cells chromosome aberration test. To evaluate the poten-tial of ADP to induce chromosome aberration on germ cells in vivo,a mice testicle cells chromosome aberration test was performed ac-cording to the GB 15193.1-2003 (MOH, 2003) and OECD GuidelineNo.483 (OECD, 1997d). Nine-week-old SPF Kunmingmice were ran-domly divided into five groups. The negative control, positive con-trol and ADP treatment group (141.5, 282.5 and 565 mg kg–1 BW)were set. The test substance was administered daily by gavage for
5 consecutive days, whereas cyclophosphamide was administeredby intraperitoneal injection. Mice were sacrificed and smear slideswere made with testicle cells 12 day after the first treatment. Thenthe air-dried slides were stained with Giemsa for 10–15 min. Allslides were coded to ensure that evaluation was blinded. The ratiosof chromosome aberrations in testicle cells were determined foreach animal by counting 500 cells. The mean±SD of ratios of chro-mosome aberrations in each group were compared using SPSS17.0 software.
Results
Acute Toxicity
The toxic signs and symptoms were observed in male andfemale rats at 1400 and 2000 mg kg–1 BW It included rough hairsand hair coat, depression, eyes or nose bleeding and hind limbparalysis. At post-mortem, no obvious changes were notedexcept for some minor pin-point hemorrages on the liver. Thestomachs were distended and filled with yellow and whitegranular material. For the 14-day observation, most rats got arecovery in the high-dose level. Experimental sequence and out-come of the acute test in rats by the up and down procedure areshown in in Supplementary Table S1 and S2. Similar symptomswere observed in male and female mice at the high-dose levelafter administration, but there were no mortalities during thesubsequent 14-day observation period. Experimental sequenceand outcome of the acute test in mice by the up and downprocedure are shown in Supplmentary Table S3 and S4.
Eventually, the LD50 values of ADP were calculated by softwareAOT 425 statpgm. It was determined to be 1400 and 1130mg/kg b.w./day with a 95% confidence interval ranging from954.9-1750 and 404.3-1560 for both female and male rats, respec-tively. The LD50 values for female and male mice were 1130 and1373 mg/kg b.w./day with 95% confidence interval ranging from859.2-1900 and 779.2-2880 mg/kg b.w./day, respectively.
90-Day Feeding Study
Clinical signs and mortality. No deaths or obvious clinical signswere found in any groups throughout the experimental period,except one male rat in the high-dose group was found dead atthe beginning of this study.
Body weight changes, food and ADP intakes and food efficiency.Compared with the control group, the body weight in high-dosegroup significantly decreased during the treatment period fromthe 5th week (Fig. 2A), and the food efficiency depressed espe-cially in the first 4 weeks (Fig. 2B). Compared with the controlgroup, no significant difference was noted in the ADP groups onfood intakes throughout the experimental time (Fig. 2C). Therewere no significant differences in the body weight, food intakesand food efficiency in the 1000 mg kg–1 diet group when com-pared with the control group during the recovery period. Inmalerats, theee mean daily ADP intakes at 20, 100 and 1000 mg kg–1 dietcalculated after estimation of mean values of food consumption forthe entire experimental period were 1.44, 7.26 and 78.87 mg kg–1
BW day–1, respectively (Table 1). In female rats, the intakes of ADPwere 1.53, 7.42 and 78.21 mg kg–1 BW day–1, respectively.
Hematology and serum biochemical changes. Hematology data arepresented in Table 2. No significant changes were observed for each
Acute, subchronic toxicity and mutagenicity studies of ADP
group in hematology onmale and female rats killed on the 45th day.A significant decrease in red blood cell counts was found in 100 mgkg–1 diet in female rats, which was not dose-related toxic effect. Dur-ing the recovery period, there were no significant differences in he-matology at the high-dose group when compared with the controlgroup. In male rats, a decrease in TG was found in the 100 mgkg–1 dose group on the 45th day, and increases of TP, ALB, ALT andAST were observed in 1000 mg kg–1 diet on the 90th day of experi-ment. In female rats, increases in TP and Cr in 100mg kg–1 diet groupand ALT, AST and Cr in the 1000 mg kg–1 diet group were observedon the 90th day of the experiment (Table 3). A significant increase
in ALP in female rats and significant increases in TCHOand TG inmalerats were noted in the 1000 mg kg–1 diet during the recovery period(data not shown).
Organ weight, organ/body weight ratios and macroscopic observa-tion. On the 90th day, there were significant decreases in organweight of the liver in males at the 100 and 1000 mg kg–1 diet. Sig-nificant increases in relative weights of the liver in females in the100 and 1000 mg kg–1 diet, and a significant decrease in relativeweights of liver in males at the 100 mg kg–1 diet were noted whencompared with the negative control group (Table 4). There weresignificant decreases in organ weight of heart in males at the 100mg kg–1 diet. Significant increases in relative weights of heart, kid-neys and brain in females and testis in males at the 1000 mg kg–1
diet were noted when compared with the negative control group.Because no histopathological changes were observed in these or-gans, it seemed that the changes of absolute or relative weightsof these organs had nothing to do with the test substance. Therewere no significant changes in organ weight and the organ weightratio in the 1000 mg kg–1 diet when compared with the controlgroup after the recovery period.
Histopathological findings. Inflammatory infiltration of many lym-phocytes near the hepatic portal area, hepatocytic necrosis andkaryopyknosis of liver cells were observed for male and female ratsfed the 100 and 1000 mg ADP kg–1 diet (Fig. 3). In the 100 mg kg–1
ADP, aditoprim; ADP20, 20 mg kg–1 diet; ADP100, 100 mg kg–1 diet; ADP1000, 1000 mg kg–1 diet.* Significantly different from the control group at P < 0.05.
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diet group of ADP, the severity and incidence of the pathologicalphenomena were significantly lower than that of 1000mg kg–1 dietgroup (Table 5). During the recovery period, the severity of inflam-matory infiltration in the high-dose group was less than that onthe 90th day. Evaluation of other organs /tissues did not revealany other histopathological changes.
Mutagenicity Studies
Bacterial reverse mutation assay. No increase > two-fold in thenumber of revertants was observed with the S. typhimuriumTA97a, TA98, TA100, TA102 and TA1535 strains after treatmentwith ADP at levels of 0.0625, 0.125, 0.25, 0.5 and 1 μg plate–1
compared with the negative control. However, the number of re-vertants in the positive controls was more than two-fold com-pared with the negative control with and without S9. Theseresults suggested that ADP was not mutagenic in this test atthe dose level of 0.0625 to 1 μg plate–1 (Table 6).
Mice bone marrow erythrocyte micronucleus assay. Comparedwith the control group, there were no statistically signficantchanges found in the ADP treatment groups in the ratios ofMN-PCE to PCE (Table 7). However, the positive control groupshowed a statistical difference in the ratios of MN-PCE to PCE.Additionally, the ratios of PCE/NCE in each group ranged from0.6 to 1.2. The results indicated that ADP was not mutagenicfor mice in vivo.
In vitro chromosomal aberration test. There was no significantincrease in the ratio of chromosome aberrations for each ofthe ADP-administered group after 6-h treatment with andwithout S9 compared with the negative control. Moreover,no significant increase in the ratio of chromosome aberrationswas observed for each of ADP-administered group after aprolonged treatment time of 18 h. However, the positive con-trols, mitomycin C (-S9) and cyclophosphamide (+S9), inducedsignificant increases in the ratio of chromosome aberrationscompared with the negative control (Table 8). These results in-dicated that, ADP was not mutagenic for V79 cells in the testconditions of this study.
In vitro CHO/hgprt mammalian cell mutagenesis assay. The mu-tation frequency of the negative control with and without S9was 5.04 and 4.52 per 1 × 106 cells, respectively. No significantincreases were observed in the mutation frequency of the ADPtreatment groups when compared with the negative controlwith and without S9. These results indicated that, ADP couldnot cause hgprt gene mutation on CHO-K1 cells in the testconditions of this study (Table 9).
Mice testicle cells chromosome aberration test. The results ofthis test for the treatment and control groups are summarizedin Table 8. Compared with the negative control group, therewere no statistically significant changes found in the ADPtreatment groups in the ratios of chromosome aberration
J. Appl. Toxicol. 2015Wiley & Sons, Ltd.
Table 3. Serum clinical chemistry of rats sacrificed on the 90th day in the subchronic test of ADP(mean ± SD)
ADP, aditoprim; ADP20, 20 mg kg–1 diet; ADP100, 100 mg kg–1 diet; ADP1000, 1000 mg kg–1 diet.* Significantly different from the control group at P < 0.05.
Acute, subchronic toxicity and mutagenicity studies of ADP
(Table 10). However, the positive control group showed a statisticaldifference in the ratios of chromosome aberration. The results indi-cated that ADP was not mutagenic to germ cells for mice in vivo.
DiscussionIn the present study, a series of preclinical trials were performedto evaluate the safety of ADP, including acute toxicity, sub-chronic (90-day oral) toxicity and mutagenicity tests. All the testsprovided a comprehensive safety assessment for ADP and couldcontribute to the development of ADP to be used in futurestudies.
In an earlier study, an acute oral LD50 of ADP for rats and micewas reported to be 1402 and 1130 mg kg–1 BW, respectively(Stephan-Guldner, 1993). In the present study, the LD50 of ADP
for rats and mice was 1400 and 1130 mg kg–1 BW day–1 with a95% CI ranging from 954.9 to 1750 and 859.2 to 1900 mg kg–1
BW, respectively. The results of the present study on acute oral tox-icity were in accordance with the former research. An acute oralLD50 in the range of 1500 to 1850 mg kg–1 BW for rats and 1910to 3960 mg kg–1 BW for mice was reported for TMP (EMEA,1997a). The acute oral LD50 of BQP was approximately 500–1000mg kg–1 BW (EMEA, 1997b). The results of acute oral toxicity in thisstudy indicated that ADP could be classified as category 4 (300 ~2000 mg kg–1), which was similar to TMP and BQP, based on theGHS (Globally Harmonized System of Classification and Labellingof Chemicals) system (GHS, 2005). According to the provision ofa low-toxic substance (501~5000 mg kg–1) of an acute toxicity testin Chinese Procedures for Toxicological Assessment of Food (MOH,2003), ADPwas considered to be a low-toxic substance. During the
ADP, aditoprim; ADP20, 20 mg kg–1 diet; ADP100, 100 mg kg–1 diet; ADP1000, 1000 mg kg–1 diet.* Significantly different from the control group at P < 0.05.
Figure 3. Histopathological results of the liver in the repeated dose oral toxicity study on rats. (A) control group ×400 hematoxylin and eosin(H&E); (B) 1000 mg kg–1 diet group ×400 H&E. Inflammatory infiltration and hepatocytic necrosis of liver cells were marked with arrows.
X. Wang et al.
observation, central nervous system (CNS) toxicity (depression andhind limb paralyzed) was found in the high-dose group, which wassimilar to the acute toxicity of BQP reported (EMEA, 1997b).
In the subchronic toxicity study, the terminal female and malebody weights in the high-dose group were significantly decreasedcompared with the control group. However, the decrease in thefood consumption in the high-dose group was insignificantthroughout the whole period. Therefore, it was presumed thatthe decrease of body weight in the high-dose group was as a re-sult of the toxicity effect caused directly by the intake of ADP..
In an earlier report, Wolfe et al. (1993) performed a subchronictoxicity study in Beagle dogs to assess the safety of ADP. The re-sults indicated that ADP had an adverse effect on the hematopoi-etic system (reduced erythrocyte count, hematocrit, hemoglobin
and platelets) in Beagle dogs at a dose as low as 10 mg kg–1 andthe no toxic effect level was determined to be 1 mg kg–1 BWday–1 (Wolfe et al., 1993). However, no treatment-related changeswere found in hematology throughout the subchronic study ofADP in Wistar rats even at the highest dose group, indicating thatthe different toxic effect of ADP on the hematopoietic systemmight be due to the species specificity in themetabolism betweenrats and dogs. TMP was reported to cause depression andmatura-tion defects of hemopoiesis, which consisted of falls in the concen-tration of hemoglobin and the number of red cells, neutrophils,lymphocytes and platelets. These adverse reactions were ob-served in dogs administered for 6 days with 135 mg kg–1 BWand monkeys administered singly with 300 mg kg–1 BW (Bushby& Hitchings, 1968). Because TMP and ADP had toxic effects on
J. Appl. Toxicol. 2015Wiley & Sons, Ltd.
Table 5. Histopathology of liver of Wistar rats sacrificed on day 90 of subchronic feeding of ADP
Dose
90-day feeding test (n = 10) Recovery period (n = 5)
ADP, aditoprim; ADP100, 100 mg kg–1 diet; ADP1000, 1000 mg kg–1 diet.* Significantly different from the control group at P < 0.05 by Fisher’s exact test.a A large number of inflammatory cell infiltration.b A small amount of inflammatory cell infiltration.
Table 6. Results of the bacterial reverse mutation test of ADP
DrugConcentrationμg per plate
Revertant colonies per plate (mean ± standard deviation)
ADP, aditoprim; DMSO, dimethylsulfoxide.* Number of revertant colonies induced was double or more in the sample/number of spontaneous in the negative control.
Table 7. Micronucleated polychromatic erythrocytes (MN-PCEs) in mice bone marrow after treatmentwith ADP
ADP, aditoprim; CMC, carboxymethyl-cellulose; CP, cyclophosphamide; NCE, normochromatic erythro-cytes; PCE, polychromatic erythrocytes.* Significantly different from the negative control value (P < 0.01).
Acute, subchronic toxicity and mutagenicity studies of ADP
ADP, aditoprim; CE, cloning efficiency; DMSO, dimethylsulfoxide; EMS, ethylmethanesulfonate; 3-MCA, 3-methylcholanthrene;HGPRT, hypoxanthine-Guanine-phosphoribosyl transferase; MF: mutant frequency calculated as NMC/(CE × 2 × 105); NMC, Numberof mutant clones; RCS, relative cell survival as a % of the negative control.* Mutation frequency of the HGPRT gene was three times or more than the spontaneous mutation rate.
Table 10. Results of the mice testicle chromosome aberration test of ADP
Concentration (mg kg–1 BW) Counts Sex chromosomesmonomer
Autosomalmonomer
Fracture fragment Trans-location Distortion cellrate (%)
CMC - 500 18 0 3 0 0.6ADP 141.5 500 12 2 2 1 0.6
282.5 500 19 5 2 0 0.4565 500 15 3 3 1 0.8
CP 40 500 28 9 34* 10* 8.8*
ADP, aditoprim; CMC, carboxymethyl-cellulose; CP, cyclophosphamide.* Significantly different from the negative control value (P < 0.01).
X. Wang et al.
the hematopoietic system, similar effects should be emphasized inother risk evaluations of ADP, such as a chronic toxicity study in rats.
The significant increase in serum ALT and AST in both gendersin the medium- and high-dose groups was observed when com-pared with the control group. ALT is an enzyme present in hepa-tocytes and usually helps to detect chronic liver diseases bymonitoring their concentrations. AST, which is most commonlyassociated with the liver, catalyzes the conversion of alanine topyruvate and glutamate and is released into the blood whenthe liver cell is damaged (Mehta et al., 2009). A high level of
ALT and AST in the serum indicated that the exposure of a highconcentration of ADP might be harmful to the liver. The level ofALB, which is synthesized in the liver, reflects the synthetic func-tion of the liver. An increased ALB concentration in serum-indicated disorders happened in the liver. While TP is closely as-sociated with ALB, this reflects the comprehensive function ofthe liver. In addition, a significant increase in the serum Cr wasonly observed in the females, indicating that females might bemore susceptible to ADP due to gender differences in themetabolism.
J. Appl. Toxicol. 2015Wiley & Sons, Ltd.
Acute, subchronic toxicity and mutagenicity studies of ADP
Significant changes in the organ to body weight ratios of liverwere observed in the high-dose group, when compared with thenegative controls. This indicated that ADP might have some ad-verse effects on the liver. Hepatotoxicity might be concluded asone of main side effects of diaminopyrimidines. In earlier re-ports, TMP was reported to induce fatty changes in the liver ofmonkeys in oral repeated dose toxicity studies (EMEA, 1997a).Hepatotoxicity was found to be caused by administration ofBQP, which consisted of the accumulation of hemosiderin in he-patic cells and signs of inflammation and necrosis (EMEA,1997b). In the present study, the fluctuations in serum biochem-ical parameters, such as the increases of TP, ALB, AST and ALTlevels, were regarded to be of toxicological relevance on theliver. Furthermore, pathological changes in accordance withthe former research of similar drugs were observed only in theliver in the high-dose group, indicating that the main target or-gan for toxicity of ADP was the liver in the study.
In the mutagenicity studies, a modified battery of tests wasperformed in accordance with the FDA and OECD guidelinesfor the testing of chemicals on genotoxicity. These tests werefollowed: bacterial reverse mutation assay, mice bone marrowerythrocyte micronucleus assay, in vitro chromosomal aberrationtest, in vitro CHO/hgprt mammalian cell mutagenesis assay, andmice testicle cells chromosome aberration test. The results ob-tained in these tests did not reveal any genotoxic effects of ADP.
In an earlier study, Ono et al (1997) evaluated the mutagenic-ity of DVD and TMP, which were similar to ADP in structure. DVDwas negative in the umu test and the reverse mutation testswith and without S9, whereas it induced structural chromosomeaberrations in cultured Chinese hamster CHL cells in the absenceof S9, but not in the presence of S9. The results of bone marrowmicronucleus tests in mice and rats administered with DVD werenegative. The results of the comet assay for DVD were positive inthe liver, kidney, lung and spleen cells, but not in bone marrowcells. TMP did not reveal any genetic action in all tests con-ducted (Ono et al., 1997). However, the genotoxicity of TMPwas in controversy with different mutagenicity tests. Papiset al. (2011) determined the genotoxicity of TMP in fish andmammalian cells by Comet and micronucleus assays. In contrast,it was concluded that TMP was genotoxic and fish cells in gen-eral were more sensitive to the genotoxic effects, and it was pre-sumed that inhibiting the action of dihydrofolate reductase,oxidative stress could contribute to the adverse effects (Papiset al., 2011). Binelli et al. (2009) evaluated the potentialgenotoxicity of TMP on hemocytes using the single cell gel elec-trophoresis assay. The results indicated that TMP had moderategenotoxic effects at a concentration level of 0.2–5 μM (Binelliet al., 2009). The European Union (EU) had reported that BQP,which is similar to ADP in pharmacokinetics, was not mutagenicin a series of genotoxicity tests, including Ames test, fluctuationassays, CHO-hgprt test, clastogenicity test, in vivo mouse micro-nucleus test and mouse dominant lethal test (EMEA, 1997b). Itwas therefore concluded that the mutagenic effects thresholdsand targets of diaminopyrimidines can work out by further re-search using different techniques, different biological materialsand different genetic endpoints. In the present work, the muta-genicity of ADP was assessed by prescriptive protocols and theresults were negative, indicating the relationship between thestructure and genotoxicity of diaminopyrimidines was muchmore complex.
In summary, the results of the acute oral toxicity study indi-cated that ADP can be classified as category 4 or is a low-toxic
substance according to GHS or the Chinese chemical classifica-tion system, respectively. Under the stated experimental condi-tions, any of the toxicity was not observed associated with themaximum dose or concentration, with the existing technicalmeans or detecting indexes which is the critical findings to de-termine the no-observed-adverse-effect level (NOAEL). Basedon the subchronic study, the target organ of ADP was the liverand the NOAEL of ADP was a 20 mg kg–1 diet, which was approx-imately 1.44 mg kg–1 BW day–1 in males and 2.3 mg kg–1 BWday–1 in females. The results of the mutagenicity studies suggestthat ADP is not genotoxic. The results of the present study pro-vided comprehensive safety information of ADP, which furtherhelp research in clinical use.
Acknowledgments
This work was supported by Grants from the 2014 National RiskAssessment of Quality and Safety of Livestock and Poultry Prod-ucts (GJFP2014007) and National 863 Program of China(2011AA10A214).
Conflict of InterestThe authors did not report any conflict of interest.
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