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Toxicological evaluation of ferrous N-carbamylglycinate chelate: Acute, Sub-acute toxicity and mutagenicity
Regulatory Toxicology and Pharmacology 73 (2015) 644e651
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Toxicological evaluation of ferrous N-carbamylglycinate chelate: Acute, Sub-acute toxicity and mutagenicity Dan Wan a, Xihong Zhou a, Chunyan Xie a, Xugang Shu b, Xin Wu a, *, Yulong Yin a, ** a
Hunan Provincial Engineering Research Center for Healthy Livestock and Poultry Production, Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, 410125, China b College of Chemistry and Chemical Engineering, Zhongkai University of Agriculture and Engineering, Guangzhou, 510225, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 August 2015 Received in revised form 7 September 2015 Accepted 8 September 2015 Available online 11 September 2015
Iron is an essential trace element that is vital important in various biological process. A deficiency in iron could induce public health problem e.g. anaemia, while an overload could induce ROS production, lipid peroxidation and DNA bases modifications. In the present study, a new iron fortifier was synthesized, and its acute/sub-acute toxicity was investigated. According to the improved Karber's method, the median lethal dose (LD50) of the ferrous N-carbamylglycinate in SD rat was 3.02 g/kg and the 95% confidence intervals were between 2.78 and 3.31 g/kg. No biologically significant or test substance-related differences were observed in body weights, feed consumption, clinical signs, organ weights, histopathology, ophthalmology, hematology, and clinical chemistry parameters in any of the treatment groups of ferrous N-carbamylglycinate at target concentrations corresponding to 150, 300, and 600 mg/kg/day for 28 days. The no observed adverse effect level (NOAEL) for ferrous N-carbamylglycinate was at least 600 mg/kg b.w. day in rats. In addition, no evidence of mutagenicity was found, either in vitro in bacterial reverse mutation assay or in vivo in mice bone marrow micronucleus assay and sperm shape abnormality assay. On the basis of our findings, we conclude that ferrous N-carbamylglycinate is a low-toxic substance with no genotoxicity. © 2015 Elsevier Inc. All rights reserved.
Keywords: Iron amino acid chelates Ferrous N-carbamylglycinate Acute toxicity Sub-acute toxicity Mutagenicity
1. Introduction Iron is an essential trace element in both animal and human nutrition. It plays vital roles in electron transfer reactions, gene regulation, oxygen transport and storage, proteins and enzymes function, energy metabolism as well as in redox reactions. A deficiency in iron in the diet could induce anaemia, which concerns
Abbrevations: MMS, Methyl methyl sulfonate; CP, cyclophosphamide; SD, SpragueeDawley; BMM, bone marrow micronucleus; LD50, median lethal dose; ANOVA, one-way analysis of variance; NOAEL, no observed adverse effect level; PCE, polychromatic erythrocytes; NCE, normochromatic erythrocytes; OECD, Organization for Economic Co-operation and Development; WBC, blood cell count; RBC, red blood cell count; HGB, hemoglobin concentration; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PLT, platelet counts; TP, total protein; ALB, total albumin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TCHO, total cholesterol; TG, triglyceride; Cr, creatinine; BUN, blood urea nitrogen; GLU, glucose; A/G, total albumin/globulin ratios; HE, hematoxylin and eosin. * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (X. Wu), [email protected] (Y. Yin). http://dx.doi.org/10.1016/j.yrtph.2015.09.013 0273-2300/© 2015 Elsevier Inc. All rights reserved.
about 30% of the world population (especially female and young) and decrease in immunity (Recalcati et al., 2012; Nairz et al., 2013; Wu et al., 2013; Ganz and Nemeth, 2015). Among the different strategies available today to fight against this public health problem, the development of iron fortifiers appears as the most costeffective and the most appropriate long-term approach (Fox et al., € f et al., 2002; Berhanu et al., 2014). 1998; Domello Up to date, increasing results revealed that ferrous sulfate are usually not optimum as iron complementary product. The solubility of ferrous sulfate in the presence of water has been shown to decrease linearly with time (Lee and Clydesdale, 1980), and various processing methods are known to change the chemical nature of added iron compounds (Lee et al., 1979). It easily changed by oxidation or chelated with macromolecular, e.g. proteins, phytic acid, etc, and therefore its bioavailability was reduced (Aluru et al., 2011; Torres-Fuentes et al., 2012; Petry et al., 2014). Thus, less reactive compounds such as reduced iron and ferric orthophosphate or chelates are used to fortify food with low moisture content. However, some dietary compounds, such as reducing components, stearic acid, certain amino acids (His, Glu, Asp, and Cys) and peptides enhance iron absorption and improving iron
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bioavailability (Storcksdieck et al., 2007). In recent years, new iron complementary products, such as ferrous EDTANa, iron chelated amino acids (Gly, Met, Lys, etc.) were commercially available in the market. A lot of studies have shown peptides chelate irons that with low molecular weights (below 500 Da) had a better bioavailability than ferrous sulfate. For instance, iron chelating peptides generated from chickpea protein (Torres-Fuentes et al., 2012) and iron chelating peptide Ser-Cys-His identified by Guo et al. (2013) exhibited high chelating activities. It was found that the total iron solubility was improved by 72% in the presence of the 1 kDa-Protein fraction following simulated gastrointestinal digestion compared to control FeSO4$7H2O solutions (O'Loughlin et al., 2015). In addition, the improved bioactivity of iron amino acid complex supplementation was also found in animals, e.g. piglets, etc. It was revealed that increases of iron amino acid complex supplementation was significantly better in the total iron content, heme-iron concentration in the muscle and skin color than ferrous sulfate supplementation in weaning piglets (Yu et al., 2000). Hence, it would be considerable to design a structural determined amino acid/amino acid derivative chelated iron to be used in the prevention of iron deficiency in both human and animals in medicinal industry and farming. Overall results of various studies indicated ferrous glycinate to be well suited for use as an iron fortificant and beneficial in the treatment of iron deficient anaemia at lower doses than those associated with ferrous sulphate preparations. When comparing the bioavailability of ferrous sulfate to ferrous glycinate in different meals, the absorption of ferrous glycinate, which partially prevented the inhibitory effect of phytates, was about twice the absorption from ferrous sulfate in human (Layrisse et al., 2000). While in broilers, it was found that the administration of Fe-Gly significantly reduced the fecal Fe concentrations and improved growth performance, iron tissue storage and antioxidant status compared to the ferrous sulfate (Ma et al., 2012). Moreover, it was reported 6 months after supplementation of bis-glycinate chelate iron and ferrous sulfate, the ferritin concentration was significantly higher in the group that received bisglycinate chelate iron in Mexican Children (Duque et al., 2014). Based on the structures of ferrous glycinate and N-carbamylglutamate (Wu et al., 2015), here we designed a new iron chelate ferrous N-carbamylglycinate as an iron fortifier, which aimed to improve the iron bioavailability (compared to inorganic irons) and reduce extra digestion (compared to macromolecule, e.g. proteins chelated irons) (Fig. 1). The safety of this compound, including acute, sub-acute and mutagenicity toxicities were evaluated in the present study. In detail, the present study was conducted to assess the potential genotoxicity and acute and sub-acute (28-day) oral toxicity of ferrous N-carbamylglycinate. All studies here were conducted in accordance with Good Laboratory Practice guidelines
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(OECD, 1995, 1997a; 1997b, 2001; FDA, 2010). 2. Materials and methods 2.1. Chemicals 2-aminofluorene (C13H11N, CAS 153-78-6), 4-Nitroquinoline Noxide (C9H6N2O3, CAS 56-57-5), cyclophosphamide (CP, C7H17Cl2N2O3P, CAS 50-18-0) were purchased from Aladdin Reagent Co., LTD (Shanghai, China). Fenaminosulf (C8H10N3NaO3S, CAS 140-56-7), Methyl methyl sulfonate (MMS) (C2H6O3S, CAS 6627-3) were purchased from J&K Scientific LTD (Shanghai, China). Phenobarbital/benzoflavone (10%)-induced rat liver S9 was purchased from Platt Bio-Pharmaceutical Co., Ltd. (Beijing, China). New-born calf serum was produced by Hangzhou Sijiqing Biological Materials Limited Company (Hangzhou, China). The rat and mice diet were procured by the Comparative Medicine Centre of Yangzhou University (Yangzhou, China). All other reagents used were of analytical grade or better, as indicated in the certificate of analysis supplied by the manufacturer. 2.2. Ferrous N-carbamylglycinate Ferrous N-carbamylglycinate (Fe, 13%) was produced by a modified method according to Li et al. (2013), as illustrated in Fig. 2. Briefly, the mixture of glycine (C2H5NO2, CAS 56-40-6) (1) and potassium cyanate (KCNO, CAS 590-28-3) in KOH/water (1/100, w/ w) was reacted for 20 h, then crystallized (0 C) and dried to give the product of N-carbamylglycinate (C3H6N2O3, CAS 462-60-2) (2). After then, the N-carbamylglycinate (2) was mixed with FeSO4 and reacted at water in 60 C for 6 h. The crude product was filtrated twice, and the filtrate was washed by ethyl alcohol/water (v/v, 95/ 100) and then dried, finally given a light green crystallized Ferrous N-carbamylglycinate (3). 2.3. Animals Specific pathogen-free (SPF) SpragueeDawley (SD) rats (8 weeks, 180e220 g) and Kunming mice (6 weeks, 18e22 g) were purchased from the Comparative Medicine Centre of Yangzhou University (Yangzhou, China) (license No.: SCXK (Su) 2007-0001). The basic feed without any drugs was produced according to the Chinese standard “Laboratory animal rats and mice feed” (GB14924.3, 2010). Rats and mice were kept in a barrier-maintained animal room conditioned at a temperature of 25 ± 3 C, a relative humidity of 50 ± 10% and a 12-h light/dark cycle. Before treatment, animals were individually handled and carefully examined for abnormal
Fig. 1. Chemical structures of ferrous glycinate (A) and ferrous N-carbamlglycinate (B).
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Fig. 2. Synthesis of ferrous N-carbamlglycinate: (i) 0.5 equiv KCNO, KOH/water (1/100, w/w), room temperature, stirred, 20 h (ii) 0.5 equiv FeSO4, water (pH 6.0), 60 C, stirred, 6 h.
behavior and appearance (Ruan et al., 2014; Tang et al., 2014). All animals had a one-week acclimatization period before experiment started and received basic feed and fresh water freely during the experiment period (Ren et al., 2014). Use of animals was in accordance with “Guide for the Care and Use of Laboratory Animals” NIH Publication (NRC, 1996). All the studies were approved by the Ethical Committee of the Faculty of Veterinary Medicine at Yangzhou University. 2.4. Experimental design 2.4.1. Acute oral toxicity study Feed of SpragueeDawley (SD) rats (10 males and 10 females) was withdrawn overnight before administration of starting doses at 12 h. An acute, one-day oral toxicity study was conducted in accordance with the Organization for Economic Co-operation and Development (OECD) Guideline 423 (OECD, 2001). Oral acute study for calculating median lethal dose (LD50) was performed according to modified Karber's method. In this method, the Ferrous N-carbamylglycinate was suspended in 0.5% carboxymethyl-cellulose solution and exposed to a single dose of the Ferrous N-carbamylglycinate 2020, 2420, 2900, 3480, 4170 and 5000 mg/kg b.w. by oral gavage, respectively, which were determined through respective pretesting. Control groups were administered 0.5% carboxymethylcellulose solution equally. Then rats that survived were observed for another 14 days. Observations included evaluation of skin and fur, eyes and mucous membranes, respiratory, autonomic effects (e.g. salivation), central nervous system effects (tremors and convulsions), changes in the level of motor activity, gait and posture, reactivity to handling and stereotypes or bizarre behavior (e.g. selfmutilation, walking backwards). The time of death was recorded as precisely as possible. At the end of the test, surviving animals were sacrificed (Ruan et al., 2013). All studies were conducted in accordance with FDA Good Laboratory Practice guidelines (FDA, 2010). 2.4.2. Sub-acute toxicity study SD rats (8 weeks old, 110e140 g, 40 male and 40 female) were randomly assigned to four groups (0, 150, 300, and 600 mg/kg b.w./ day, 28 days) by a computer-generated (weight-ordered) randomization procedure (Ren et al., 2013a). Each group consisted of 10 female rats and 10 male rats. Dietary Ferrous N-carbamylglycinate concentrations were adjusted weekly according to the average male body weights for each dosage group from the previous week. All rats were observed at least once a day for mortality or morbidity, changes in posture, changes in skin, fur, eyes, mucous membranes and behaviors (Ren et al., 2013b). Changes in gait were assessed weekly by allowing the animal to walk freely (Chen et al., 2014). All rats were anesthetized with pentobarbital sodium and killed at the 28th day after an overnight fast. The endpoints included clinical
and ophthalmological observations, body weight and feed consumption values, feed efficiency (g weight gain per 100 g of food consumed), a functional observational battery, motor activity, hematology and coagulation assessment, serum chemistry levels, and clinical pathology (Zeng et al., 2008). During necropsies, all the organs/tissues were carefully examined, macroscopically and gross lesions were recorded. The organs of each animal including the heart, lung, kidneys, liver, spleen, testicles (epididymis), ovary and adrenal glands were weighed separately (Kong et al., 2009). Organ/body weight ratios were calculated based on the fasted animal's body weight. The organs from each animal, with the exception of testes, were preserved in 10% neutral-buffered formalin and slides were prepared for histopathological examination using the routine paraffin embedding technique. Sections of 5 mm thickness stained with hematoxylin and eosin (HE) were examined under light microscopy for morphological alterations by a board-certified veterinary pathologist. Tissues from other groups were examined as necessary to determine no observed adverse effect level (NOAEL) in target organs. 2.4.3. Mutagenicity studies Bacterial reverse mutation assay was performed to evaluate the mutagenicity of Ferrous N-carbamylglycinate, with and without S9, using the following four Salmonella strains as prescribed in the OECD guideline No.471 (OECD, 1997a): TA97a, TA98, TA100, and TA102. All strains were provided by Chinese (Jiangsu) Center for Disease Control and Prevention for type culture collection and checked for maintenance of genetic markers prior to the study. Test solutions were prepared in water as serial dilutions to deliver the required concentration in a constant volume. 2-aminofluorene (10 mg per plate) was used as a positive control for all strains tested with S9. Fenaminosulf (50 mg per plate) was used as a positive control for TA97 and TA98 strains tested without S9. 4Nitroquinoline N-oxide (0.5 mg per plate) was used as a positive control for TA100 strain tested without S9. Methyl methyl sulfonate (MMS) (1 mL per plate) was used as a positive control for TA102 strain tested without S9. The assay tubes were pre-incubated at 37 C for 20 min before plating onto minimal agar. Three test plates per concentration were incubated at 37 C for 48 h and then counted. The criteria for a positive response were a more than two-fold increase in the average plate count compared with the solvent control for at least one concentration level and a dose response over the range of tested concentrations in at least one strain with or without S9. Mice bone marrow erythrocyte micronucleus assay was performed in accordance with the OECD Guideline No. 474 (OECD, 1997b) for principles of Good Laboratory Practices (GLP). Fifty 810 week-old SPF Kunming mice (18e22 g) were randomly divided
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2.5. Statistical analyses
Table 1 The results acute test of ferrous N-carbamylglycinate in rats. Groups
Number of rats
Dosage (mg/kg b.w.)
Deaths
Death rate (%)
1 2 3 4 5 6
10 10 10 10 10 10
5000 4170 3480 2900 2420 2020
10 9 7 3 1 0
100 90 70 30 10 0
into five groups each consisting of 10 mice (five males and five females). Cyclophosphamide (CP, i.p. 40 mg/kg BW) was administered 6 h before sampling as a positive control and water was used as a negative control. In this doseeresponse study, Ferrous N-carbamylglycinate was administered twice in 30 h with a 24-h interval at a dose level of 375, 750, and 1500 mg/kg BW through oral gavage. Six hours after the last treatment, all the animals were euthanized to obtain cell suspensions from the femur bone marrow. Bone marrows were flushed with 1 mL of newborn calf serum to obtain cell suspensions. One drop of the mixture was smeared on a clean slide, air dried, fixed with 95% methanol for 10 min and stained with Giemsa stain. All slides were coded to ensure that the evaluation was blinded. Micronucleus frequencies were determined for each animal by counting 1000 of polychromatic erythrocytes (PCE) and the micronucleus occurrence rate per one thousand PCE was recorded. The ratio of PCE to normochromatic erythrocytes (NCE) was determined for each animal by counting a total of 1000 erythrocytes. The micronucleus occurrence rate and PCE/NCE ratio of each group were compared using SPSS 17.0 software. Sperm shape abnormality assay was performed in accordance with the principles of Good Laboratory Practices (FDA, 2010) and Chinese standard (GB15193.7-2003). Fifty mice were randomly divided into five groups of 10 each. Ferrous N-carbamylglycinate (oral. 375, 750, and 1500 mg/kg BW) and cyclophosphamide (CP, oral. 40 mg/kg BW), as a positive control were administered for five days with a 24-h interval at a dose level of through oral gavage. Water was used as a negative control. Mice were sacrificed after the last treatment by cervical dislocation. Both of the cauda epididymises were dissected out in a plate with 1.5 mL normal saline then cut twice into pieces. After stirred for 3 min, it was filtered, and smears were made according to the standard protocol for sperm morphology assay. A total of 1000 sperms per animal were scored under a microscope with 40 10 magnification. Sperm head abnormalities were determined as having either normal or abnormal morphology. According to the Chinese standard (GB15193.7-2003), a “hookless head” does not have a spherical spot at the tip of the sperm head; a “banana” has a banana-like form; an “amorphous head” lacks the usual hook and is deformed; and a “folded sperm” is folded on itself.
Statistical analyzes were conducted by comparing the treatment groups with the control group using the SPSS 17.0 program (SPSS Inc., Chicago, USA). Levene's test was used to examine the homogeneity of variances. If the variance was homogeneous, the data was analyzed with one-way analysis of variance (ANOVA); otherwise they were analyzed by the KruskaleWallis non-parametric ANOVA. If the variance was significant (<0.05), Dunnett's test was used to identify the statistical significance of the individual groups. For histopathological findings and abnormal sperm morphology, they were subjected to Fisher's exact probability test. The pre-set two-sided P-value of <0.05 or 0.01 was taken as statistically significant. Values are expressed as mean ± SD. 3. Results 3.1. Acute toxicity During the acute experiment period, the rats were found motionless and breathless, suddenly jumped and squealed. Their furs were messy and gray loose stool was around the anus. Except for the 2020 mg/kg b.w. group, some rats were found dead in other groups (Table 1). Particularly, the rats in the highest dosed group (5000 mg/kg b.w.) were found dead within 4e16 h after administration. All rats died were dissected and submitted to the pathoanatomical analysis. At post-mortem, no obvious changes were noted except the gastrointestinal tracts which were distended and filled with reddish-brown liquid and gray contents. For the 14-day observation, most rats got a recovery. LD50 values and its 95% confidence intervals of the Ferrous N-carbamylglycinate in SD rats were calculated according to improved Karber's method. The results indicated that the LD50 values of the Ferrous N-carbamylglycinate in SD rats were 3.02 g/kg and the 95% confidence intervals were between 2.78 and 3.31 g/kg. 3.2. Sub-acute toxicity No deaths or obvious clinical signs were found in any groups throughout the sub-acute experimental period. The body weight changes, feed intakes and efficiency were summarized in Table 2, but no significant difference (p > 0.05) was found in each group. Except for GLU concentration in serum, no significant difference (p > 0.05) was observed in any other hematology and serum biochemical parameters (Table 3). No significant difference in organ weight of the investigated tissues, including heart, liver, lung, spleen, kidney, ovary, testis and adrenals in both male and females in the 150 mg/kg b.w., 300 mg/kg b.w. and 600 mg/kg b.w. diet groups was found (Table 4). And no treatment related
Table 2 Effect of the 28-day ferrous N-carbamylglycinate repeated dosage on body weight and feed consumption in rats. Groups (n ¼ 5) Control Initial body weight (g) Final body weight (g) Body weight gain (g) Total feed consumption (g) Feed consumption rate
Male Female Male Female Male Female Male Female Male Female
132.50 117.20 351.70 230.80 219.20 113.60 6623 4277 33.10 26.56
FeN-150 ± ± ± ± ± ±
7.09 5.14 28.94 7.42 30.52 6.88
133.00 129.80 354.10 226.30 221.10 105.70 6012 3550 36.78 29.77
± ± ± ± ± ±
FeN-300 5.85 8.47 21.49 14.68 21.06 12.50
131.90 134.40 333.80 222.00 201.90 106.00 5890 3779 30.88 28.05
FeN, ferrous N-carbamylglycinate; FeN-150, 150 mg/kg BW/day; FeN-300, 300 mg/kg BW/day; FeN-600, 600 mg/kg BW/day.
± ± ± ± ± ±
FeN-600 5.74 8.26 39.41 12.09 37.87 9.96
132.70 116.30 344.50 222.30 211.80 106.00 6290 4280 33.67 23.36
± ± ± ± ± ±
7.20 3.74 15.72 10.76 18.81 12.17
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Table 3 Effects of ferrous N-carbamylglycinate on hematology and serum clinical chemistry parameters of rats in the 28-day sub-acute test. Groups (n ¼ 5) Control 9
WBC (10 /L)
Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female
12
RBC(10 /L) HGB (g/L) HCT (%) MCV (fL) MCH (pg) MCHC (g/L) PLT (109/L) ALT (U/L) AST (U/L) CHO (mmol/L) TG (mmol/L) CRE (mmol/L) GLu (mmol/L) TP (g/L) ALB (g/L)
7.53 4.69 7.50 7.11 145.90 135.00 42.69 40.55 55.20 55.01 18.22 18.17 340.00 332.90 579.40 515.70 58.80 55.20 144.80 117.10 1.23 1.13 1.84 0.83 33.81 38.50 8.15 8.15 60.75 56.61 17.31 18.07
FeN-150 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.75 1.09 0.23 0.28 4.63 7.13 1.72 1.74 2.32 4.03 0.97 0.68 12.27 7.45 134.95 74.75 8.07 11.61 26.97 16.82 0.28 0.37 0.83 0.42 11.39 7.81 0.51 0.78 6.39 1.60 1.02 0.94
8.26 5.73 7.41 7.27 135.30 136.70 40.11 40.10 54.21 55.39 18.28 18.86 337.30 340.80 614.60 545.10 62.50 56.00 151.20 138.50 1.29 1.17 1.63 1.10 35.59 37.91 9.62 9.73 63.43 59.49 19.35 18.41
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
FeN-300 3.84 1.82 0.38 0.57 8.03 5.10 1.92 1.00 1.76 3.32 0.77 1.06 5.46 6.96 64.98 54.34 8.14 7.26 26.25 18.76 0.23 0.16 0.85 0.29 10.98 6.35 1.50* 1.12* 10.62 7.29 3.78 3.74
8.18 4.99 7.78 7.11 150.80 136.00 43.95 40.38 56.56 56.75 18.62 18.73 343.10 337.30 652.10 481.10 60.50 61.70 140.50 133.60 1.31 1.24 1.68 1.11 34.18 41.63 8.73 9.75 63.65 60.57 17.31 18.84
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
FeN-600 1.10 1.71 0.43 0.31 8.01 7.63 2.20 2.75 1.56 2.25 0.84 0.49 9.42 10.73 191.99 149.44 9.07 7.97 14.69 20.91 0.18 0.16 0.71 0.43 8.24 7.70 0.81 2.57* 4.54 6.20 1.37 1.68
8.11 4.66 7.63 7.02 146.40 135.00 42.66 41.20 55.95 57.81 18.70 18.75 343.30 328.10 546.80 515.70 53.10 48.70 140.70 110.80 1.13 1.17 1.93 0.96 27.17 43.50 8.60 9.22 60.28 56.29 18.07 18.89
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.17 0.99 0.22 0.25 5.08 3.43 1.35 1.65 2.09 1.73 0.75 0.64 10.01 11.73 102.66 86.79 9.04 13.80 17.19 33.30 0.09 0.37 0.45 0.25 3.51 7.39 1.11 1.12* 2.15 2.48 0.59 1.15
* Significantly different from the control group at p < 0.05. FeN, Ferrous N-carbamylglycinate; FeN-150, 150 mg/kg BW/day; FeN-300, 300 mg/kg BW/day; FeN-600, 600 mg/kg BW/day; WBC, white blood cell count; RBC, red blood cell count; HGB, hemoglobin concentration; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PLT, platelet counts; TP, total protein; ALB, total albumin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TCHO, total cholesterol; TG, triglyceride; Cr, creatinine; BUN, blood urea nitrogen; GLU, glucose; A/G, total albumin/globulin ratio.
macroscopical changes in rats or histopathological changes in liver, spleen, heart, lung, ovary and testis were found in each group.
3.3. Mutagenicity No increase > two-fold in the number of revertants was observed with the S. typhimurium TA97a, TA98, TA100, TA102 and
Table 4 Effects of the 28-day ferrous N-carbamylglycinate treatment on weight of rat organ. Groups (n ¼ 5) Control Heart Liver Spleen Lung Kidney Adrenals Testis Ovary
Male Female Male Female Male Female Male Female Male Female Male Female
0.34 0.36 4.40 3.53 0.22 0.26 0.40 0.47 0.78 0.75 0.16 0.17 0.98 0.06
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.02 0.02 0.29 0.27 0.03 0.04 0.03 0.06 0.05 0.03 0.02 0.04 0.10 0.01
4. Disscussion
FeN-150 0.35 0.37 4.16 3.58 0.23 0.25 0.38 0.52 0.79 0.72 0.15 0.18 1.01 0.06
TA1535 strains after treatment with Ferrous N-carbamylglycinate at 0.005, 0.05, 0.5 and 5 mg/plate compared with the negative control (Table 5). No statistically significant changes (p > 0.05) in the ratios of MN-PCE to PCE was found in the Ferrous N-carbamylglycinate (370, 750, or 1500 mg/kg b.w./day) treatment groups (Table 6). No significant difference (p > 0.05) in the percentage of abnormal sperm was found in Ferrous N-carbamylglycinate treated groups (Table 7). These results above indicated that the Ferrous N-carbamylglycinate can be classified as a low-toxic substance without genotoxicity.
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.03 0.03 0.36 0.35 0.03 0.04 0.10 0.06 0.05 0.05 0.03 0.04 0.07 0.02
FeN-300 0.37 0.37 4.09 3.67 0.20 0.25 0.50 0.44 0.78 0.72 0.15 0.15 1.05 0.06
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.03 0.02 0.48 0.23 0.02 0.03 0.22 0.15 0.05 0.11 0.03 0.03 0.23 0.01
FeN-600 0.30 0.36 4.26 3.71 0.21 0.26 0.39 0.51 0.76 0.79 0.16 0.22 0.94 0.06
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.08 0.04 0.28 0.27 0.02 0.03 0.04 0.04 0.04 0.04 0.04 0.16 0.20 0.01
No statistically significant differences were found. FeN, ferrous N-carbamylglycinate; FeN-150, 150 mg/kg BW/day; FeN-300, 300 mg/kg BW/day; FeN-600, 600 mg/ kg BW/day.
In the present study, a series of preclinical trials were performed to evaluate the safety of ferrous N-carbamylglycinate, including acute toxicity, sub-acute (28-day oral) toxicity and mutagenicity tests. All the tests provided a comprehensive safety assessment for Ferrous N-carbamylglycinate and could contribute to the development of Ferrous N-carbamylglycinate to be used in future studies. In the earlier studies, the acute oral LD50 of ferrous glycinate were 560 mg iron/kg body weight of rat (Jeppsen and Borzelleca, 1999; Jeppsen, 2001). However, the present study showed that the LD50 of the ferrous N-carbamlglycinate was 3.02 g/kg, equaled to 392 mg iron/kg body weight of rat. The results of the present study on acute oral toxicity were similar but slightly lower than ferrous glycinate in the former research. It indicated the N-carbamlglycinate chelates may have a better bioavailability than glycinate chelate in rats. The results of acute oral toxicity in this study
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Table 5 Mean number of revertants ±SD in the presence or absence of metabolic activation (n ¼ 3, mean ± SD). Treatment
TA97
TA98
(S9) FeN 0.03 FeN 0.3 FeN 3 FeN 30 Water DMSO Fenaminosulf (500 mg/mL) 2-AF (1 mg/mL) 4-nitroquinoline N-oxide (50 mg/mL) MMS (10 mL/mL)
þ (S9)
130.00 135.20 121.68 131.00 132.00 128.10 708.00
± ± ± ± ± ± ±
5.18 3.96 2.36 5.55 4.90 5.42 90.51a
114.55 125.66 119.00 125.33 120.33 130.32 e
± ± ± ± ± ±
5.73 6.21 4.96 5.25 6.23 5.12
TA100
(S9)
þ (S9)
35.00 ± 2.16 33.96 ± 3.08 38.68 ± 3.25 34.33 ± 2.88 39.55 ± 1.96 32.88 ± 0.92 1145.33 ± 48.45a
32.33 34.00 34.67 40.00 35.67 35.67 e
TA102
(S9)
± ± ± ± ± ±
0.94 2.16 2.05 4.97 1.25 2.46
151.67 155.00 150.66 145.25 162.32 151.34 e
þ (S9) ± ± ± ± ± ±
9.69 8.49 8.89 7.38 7.24 4.27
178.67 166.67 171.00 169.67 173.67 177.00 e
(S9) ± ± ± ± ± ±
11.08 11.61 9.04 17.23 6.31 9.42
275.33 281.33 279.67 274.00 277.38 278.67 e
e e
1348.33 ± 88.24a e e e
1245.67 ± 68.37a e 1698.33 ± 162.45a e e 688.67 ± 98.87a e e
e
e
e
e
e
e
þ (S9) ± ± ± ± ± ±
11.27 6.80 6.94 11.66 18.00 8.99
289.26 296.67 284.00 280.00 263.33 272.67 e
± ± ± ± ± ±
8.90 12.04 16.00 13.07 14.08 8.38
1520.00 ± 108.24a e
1428.00 ± 98.43a e
a Number of revertant colonies induced was double or more in the sample/number of spontaneous in the negative control. 2-AF, 2-aminofluorene; MMS, methyl methyl sulfonate; FeN, ferrous N-carbamylglycinate; FeN 0.03, 100 mL of FeN at the concentration of 0.03 mg/mL was added to the plate; FeN 0.3, 100 mL of FeN at the concentration of 0.3 mg/mL was added to the plate; FeN 3, 100 mL of FeN at the concentration of 3 mg/mL was added to the plate; FeN 30, 100 mL of FeN at the concentration of 30 mg/mL was added to the plate.
microscopic findings were found. The NOAEL of ferrous glycinate was at least 500 mg ferrous glycinate chelate/kg body weight/day, the highest dose tested (Jeppsen and Borzelleca, 1999). Moreover, an in vivo toxicity study of Iron N-(2-hydroxy acetophenone) glycinate (FeNG) did not reveal any mortality or symptoms of toxicity, including pathological changes, body weight gain/loss pattern, organ weight, haematological or biochemical parameters in treated groups as compared to the control group of animals (Ganguly et al., 2012). Here in the present 28-day sub-acute study, no significant difference in organ weight and treatment related macroscopical or histopathological changes were found in liver or any other tissues in Ferrous N-carbamylglycinate treated rats. Moreover, no significant difference was found in hepatic function related serum AST, ALT, CHO and TG concentration. Therefore, the NOAEL was at least 600 mg for ferrous N-carbamylglycinate chelate/kg body weight/ day in rats, the highest dose tested in the present study. Iron-mediated formation of free radicals, caused various modifications to DNA bases, enhanced lipid peroxidation, and altered calcium and sulfhydryl homeostasis (Valko et al., 2005; Guo et al., 2014). Lipid peroxides, formed by the attack of radicals on polyunsaturated fatty acid residues of phospholipids, can further react with redox irons and finally produce mutagenic and carcinogenic malondialdehyde, 4-hydroxynonenal and other exocyclic DNA adducts. Therefore, the mutagenicity of the ferrous N-carbamylglycinate was investigated both in vivo and in vitro. Fortunately, the results indicated that the ferrous N-carbamylglycinate was not mutagenic in the bacterial reverse mutation assay, mice bone marrow erythrocyte micronucleus assay and sperm shape abnormality assay. Since previous studies did not investigate the potential genotoxicity of iron amino acid chelates, the results of the present study indicated the class of these compounds probably be not genotoxic. In summary, the results of the acute oral toxicity study (LD50
Table 6 Effect of ferrous N-carbamylglycinate on bone marrow polychromatic erythroblast micronuclei (MN) in mice (n ¼ 10, mean ± SD). Treatment (mg/kg BW/day, 5 days)
PCE
FeN
10 10 10 10 10
CP Water
375 750 1500 40
PCE/NCE
MN/PCE
P value
MN MN (%, mean ± SD) 000 000 000 000 000
1.18 1.21 1.09 0.88 1.20
± ± ± ± ±
0.10 0.06 0.09 0.11 0.07
11 12 14 168 10
1.02 1.19 1.20 4.79 1.00
± ± ± ± ±
0.57 1.98 2.89 4.87 0.55
<0.01
FeN, ferrous N-carbamylglycinate; CP, cyclophosphamide; NCE, normochromatic erythrocyte; PCE, polychromatic erythrocyte.
indicated that ferrous N-carbamlglycinate could be classified as category 5 (2000e5000 mg/kg), based on the Globally Harmonized System of Classification and Labelling of Chemicals system (GHS, 2005). According to the provision of a low-toxic substance (501e5000 mg/kg) of an acute toxicity test in Chinese Procedures for Toxicological Assessment of Food (MOH, 2003), ferrous N-carbamlglycinate was considered to be a low-toxic substance. Iron is an essential element required for growth and survival of almost every organism. Chronic toxicity of iron consumption can be associated with a high dietary iron intake. Cases of acute iron toxicity are relatively rare and mostly related to hepatoxicity (Tenenbein, 2001). The excessive intestinal absorption of iron resulted in lipid peroxidation and hepatocellular organelles damage, such as mitochondria and lysosomes, which was thought to contribute to hepatocyte necrosis and apoptosis, and ultimately lead to the development of hepatic fibrogenesis (Ramm and Ruddell, 2005). In an early subchronic 90-day toxicity study of ferrous glycinate, there were no biologically or statistically significant compound-related and dose-dependent macroscopic or
Table 7 Effect of ferrous N-carbamylglycinate on spermhead morphology in mice (n ¼ 10, mean ± SD). Treatment (mg/kg BW/day, 5 days)
Number of animals
Number of sperms
FeN
10 10 10 10 10
10 10 10 10 10
Water CP
375 750 1500 40
1000 1000 1000 1000 1000
Total abnormalities Hookless
Banana
Amorphous
Folded
Double-headed
Double-tailed
55 46 46 52 125
20 22 22 21 68
106 102 104 100 302
7 8 9 6 18
5 4 2 0 15
4 2 3 2 16
FeN, ferrous N-carbamylglycinate; CP, cyclophosphamide.
Mean abnormalities 20.08 18.20 19.00 18.00 50.80
± ± ± ± ±
5.20 5.36 6.20 2.05 16.42
Abnormalities ratio 1.97 1.84 1.86 1.81 5.44
P value
p＜0.01
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D. Wan et al. / Regulatory Toxicology and Pharmacology 73 (2015) 644e651
3.02 g/kg) indicated that ferrous N-carbamylglycinate can be classified as category 5 or is a low-toxic substance according to GHS or the Chinese chemical classification system, respectively. Based on the sub-chronic study, the NOAEL of ferrous N-carbamylglycinate was 600 mg/kg b.w. day, which was the highest dose we investigated. In addition, it is not genotoxic. The results here provided comprehensive safety information of ferrous N-carbamylglycinate, which would further help researches in clinical use. Conflicts of interest The authors declared no conflicts of interest. Acknowleagement This research was granted by National Science Foundation of China (31330075, 31110103909), National Key Technology Research and Development Program of the Ministry of Science and Technology of China (2012BAD39B00), the Chinese Academy of Sciences comprehensive strategic cooperation project of Guangdong Province (2013B091500095, 2013B090900007), Science and Technology Planning Project of Guangdong Province (2013B091500095, 2013B090900007, 2013B070704081) and Guangzhou Industry Research Collaborative innovation major special (201508020048). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.yrtph.2015.09.013. References Aluru, M.R., Rodermel, S.R., Reddy, M.B., 2011. Genetic modification of low phytic acid 1-1 maize to enhance iron content and bioavailability. J. Agric. Food Chem. 59 (24), 12954e12962. Berhanu, G., Mesfin, A., Kebebu, A., Whiting, S.J., Henry, C.J., 2014. Household food processing methods to enhance iron and zinc bioavailability in formulated haricot bean and maize complementary food. Afr. J. Food Sci. 8 (4), 190e195. Chen, S., Liu, S., Zhang, F., Ren, W.K., Li, N.Z., Yin, J., Duan, J.L., Peng, Y.Y., Liu, G., Yin, Y.L., Wu, G.Y., 2014. Effects of dietary L-glutamine supplementation on specific and general defense responses in mice immunized with inactivated Pasteurella multocida vaccine. Amino Acids 46, 2365e2375. € f, M., Lo € nnerdal, B., Abrams, S.A., Hernell, O., 2002. Iron absorption in Domello breast-fed infants: effects of age, iron status, iron supplements, and complementary foods. Am. J. Clin. Nutr. 76 (1), 198e204. Duque, X., Martinez, H., Vilchis-Gil, J., Mendoza, E., Flores-Hern andez, S., Mor an, S., Navarro, F., Roque-Evangelista, V., Serrano, A., Mera, R.M., 2014. Effect of supplementation with ferrous sulfate or iron bis-glycinate chelate on ferritin concentration in Mexican schoolchildren: a randomized controlled trial. Nutr. J. 13 (1), 71. FDA., 2010. Good Laboratory Practice Regulations 21 CFR Part 58. Docket No. FDA2010-N-0548. Fox, T.E., Eagles, J., Fairweather-Tait, S.J., 1998. Bioavailability of iron glycine as a fortificant in infant foods. Am. J. Clin. Nutr. 67 (4), 664e668. Ganguly, A., Chakraborty, P., Banerjee, K., Chatterjee, S., Basu, S., Sarkar, A., Chatterjee, M., Choudhuri, S.K., 2012. Iron N-(2-hydroxy acetophenone) glycinate (FeNG), a non-toxic glutathione depletor circumvents doxorubicin resistance in Ehrlich ascites carcinoma cells in vivo. Biometals 25 (1), 149e163. Ganz, T., Nemeth, E., 2015. Iron homeostasis in host defence and inflammation. Nat. Rev. Immunol. 15 (8), 500e510. GHS (Globally Harmonized System of Classification and Lablling of Chemicals), 2005. Chapter 3. Health Hazards. (3-2-1) Acute Toxicity. Guo, L., Harnedy, P.A., Li, B., Hou, H., Zhang, Z., Zhao, X., FitzGerald, R.J., 2014. Food protein-derived chelating peptides: biofunctional ingredients for dietary mineral bioavailability enhancement. Trends Food Sci. Technol. 37 (2), 92e105. Guo, L., Hou, H., Li, B., Zhang, Z., Wang, S., Zhao, X., 2013. Preparation, isolation and identification of iron-chelating peptides derived from Alaska pollock skin. Process Biochem. 48 (5), 988e993. Jeppsen, R.B., 2001. Toxicology and safety of Ferrochel and other iron amino acid chelates. Arch. Latinoam. Nutr. 51 (1 Suppl. 1), 26e34. Jeppsen, R.B., Borzelleca, J.F., 1999. Safety evaluation of ferrous bisglycinate chelate. Food Chem. Toxicol. 37 (7), 723e731. Kong, X.F., Zhang, Y.Z., Yin, Y.L., Wu, G.Y., Zhou, H.J., Tan, Z.L., Yang, F., Bo, M.J.,
Huang, R.L., Li, T.J., Geng, M.M., 2009. Chinese Yam polysaccharide enhances growth performance and cellular immune response in weanling rats. J Sci Food Agr. 89 (12), 2039e2044. n, M.A., Arguello, F., Llovera, D., Layrisse, M., García-Casal, M.N., Solano, L., Baro Ramírez, J., Leets, I., Tropper, E., 2000. Iron bioavailability in humans from breakfasts enriched with iron bis-glycine chelate, phytates and polyphenols. J. Nutr. 130 (9), 2195e2199. Lee, K., Clydesdale, F.M., 1980. Chemical changes of iron in food and drying processes. J. Food Sci. 45 (3), 711e715. Lee, K., Clydesdale, F.M., Tannenbaum, S.R., 1979. Iron sources used in food fortification and their changes due to food processing. Crit. Rev. Food Sci. Nutr. 11 (2), 117e153. Li, J., Shu, X., Zhang, M., Wu, X., Fan, M., Lai, R., Xie, Q., 2013. Synthesis and characterizations of Iron II complex with N-carbamyl glycinate. J. Zhongkai Univ. Agric. Eng. 26 (4), 16e19 (In Chinese). 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Molecular characterization of Whey protein hydrolysate fractions with ferrous chelating and enhanced iron solubility capabilities. J. Agric. food Chem. 63 (10), 2708e2714. OECD, 1995. In: OECD Guidelines for the Testing of Chemicals. Test Guideline 407. Repeated Dose 28-day Oral Toxicity Study in Rodent. OECD, 2001. In: Procedures for Toxicological Assessment on Food Safety, pp. 1e94. GBI5193. OECD., 1997a. In: OECD Guidelines for the Testing of Chemicals. Test Guideline 471. Bacterial Reverse Mutation Test. OECD., 1997b. In: OECD Guidelines for the Testing of Chemicals. Test Guideline 475. Mammalian Bone Marrow Chromosome Aberration Test. Petry, N., Egli, I., Gahutu, J.B., Tugirimana, P.L., Boy, E., Hurrell, R., 2014. Phytic acid concentration influences iron bioavailability from biofortified beans in Rwandese women with low iron status. J. Nutr. 144 (11), 1681e1687. Ramm, G.A., Ruddell, R.G., 2005. Hepatotoxicity of iron overload: mechanisms of iron-induced hepatic fibrogenesis. 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Toxicological evaluation of ferrous N-carbamylglycinate chelate: Acute, Sub-acute toxicity and mutagenicity Dan Wan a, Xihong Zhou a, Chunyan Xie a, Xugang Shu b, Xin Wu a, *, Yulong Yin a, ** a
Hunan Provincial Engineering Research Center for Healthy Livestock and Poultry Production, Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, 410125, China b College of Chemistry and Chemical Engineering, Zhongkai University of Agriculture and Engineering, Guangzhou, 510225, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 August 2015 Received in revised form 7 September 2015 Accepted 8 September 2015 Available online 11 September 2015
Iron is an essential trace element that is vital important in various biological process. A deficiency in iron could induce public health problem e.g. anaemia, while an overload could induce ROS production, lipid peroxidation and DNA bases modifications. In the present study, a new iron fortifier was synthesized, and its acute/sub-acute toxicity was investigated. According to the improved Karber's method, the median lethal dose (LD50) of the ferrous N-carbamylglycinate in SD rat was 3.02 g/kg and the 95% confidence intervals were between 2.78 and 3.31 g/kg. No biologically significant or test substance-related differences were observed in body weights, feed consumption, clinical signs, organ weights, histopathology, ophthalmology, hematology, and clinical chemistry parameters in any of the treatment groups of ferrous N-carbamylglycinate at target concentrations corresponding to 150, 300, and 600 mg/kg/day for 28 days. The no observed adverse effect level (NOAEL) for ferrous N-carbamylglycinate was at least 600 mg/kg b.w. day in rats. In addition, no evidence of mutagenicity was found, either in vitro in bacterial reverse mutation assay or in vivo in mice bone marrow micronucleus assay and sperm shape abnormality assay. On the basis of our findings, we conclude that ferrous N-carbamylglycinate is a low-toxic substance with no genotoxicity. © 2015 Elsevier Inc. All rights reserved.
Keywords: Iron amino acid chelates Ferrous N-carbamylglycinate Acute toxicity Sub-acute toxicity Mutagenicity
1. Introduction Iron is an essential trace element in both animal and human nutrition. It plays vital roles in electron transfer reactions, gene regulation, oxygen transport and storage, proteins and enzymes function, energy metabolism as well as in redox reactions. A deficiency in iron in the diet could induce anaemia, which concerns
Abbrevations: MMS, Methyl methyl sulfonate; CP, cyclophosphamide; SD, SpragueeDawley; BMM, bone marrow micronucleus; LD50, median lethal dose; ANOVA, one-way analysis of variance; NOAEL, no observed adverse effect level; PCE, polychromatic erythrocytes; NCE, normochromatic erythrocytes; OECD, Organization for Economic Co-operation and Development; WBC, blood cell count; RBC, red blood cell count; HGB, hemoglobin concentration; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PLT, platelet counts; TP, total protein; ALB, total albumin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TCHO, total cholesterol; TG, triglyceride; Cr, creatinine; BUN, blood urea nitrogen; GLU, glucose; A/G, total albumin/globulin ratios; HE, hematoxylin and eosin. * Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (X. Wu), [email protected] (Y. Yin). http://dx.doi.org/10.1016/j.yrtph.2015.09.013 0273-2300/© 2015 Elsevier Inc. All rights reserved.
about 30% of the world population (especially female and young) and decrease in immunity (Recalcati et al., 2012; Nairz et al., 2013; Wu et al., 2013; Ganz and Nemeth, 2015). Among the different strategies available today to fight against this public health problem, the development of iron fortifiers appears as the most costeffective and the most appropriate long-term approach (Fox et al., € f et al., 2002; Berhanu et al., 2014). 1998; Domello Up to date, increasing results revealed that ferrous sulfate are usually not optimum as iron complementary product. The solubility of ferrous sulfate in the presence of water has been shown to decrease linearly with time (Lee and Clydesdale, 1980), and various processing methods are known to change the chemical nature of added iron compounds (Lee et al., 1979). It easily changed by oxidation or chelated with macromolecular, e.g. proteins, phytic acid, etc, and therefore its bioavailability was reduced (Aluru et al., 2011; Torres-Fuentes et al., 2012; Petry et al., 2014). Thus, less reactive compounds such as reduced iron and ferric orthophosphate or chelates are used to fortify food with low moisture content. However, some dietary compounds, such as reducing components, stearic acid, certain amino acids (His, Glu, Asp, and Cys) and peptides enhance iron absorption and improving iron
D. Wan et al. / Regulatory Toxicology and Pharmacology 73 (2015) 644e651
bioavailability (Storcksdieck et al., 2007). In recent years, new iron complementary products, such as ferrous EDTANa, iron chelated amino acids (Gly, Met, Lys, etc.) were commercially available in the market. A lot of studies have shown peptides chelate irons that with low molecular weights (below 500 Da) had a better bioavailability than ferrous sulfate. For instance, iron chelating peptides generated from chickpea protein (Torres-Fuentes et al., 2012) and iron chelating peptide Ser-Cys-His identified by Guo et al. (2013) exhibited high chelating activities. It was found that the total iron solubility was improved by 72% in the presence of the 1 kDa-Protein fraction following simulated gastrointestinal digestion compared to control FeSO4$7H2O solutions (O'Loughlin et al., 2015). In addition, the improved bioactivity of iron amino acid complex supplementation was also found in animals, e.g. piglets, etc. It was revealed that increases of iron amino acid complex supplementation was significantly better in the total iron content, heme-iron concentration in the muscle and skin color than ferrous sulfate supplementation in weaning piglets (Yu et al., 2000). Hence, it would be considerable to design a structural determined amino acid/amino acid derivative chelated iron to be used in the prevention of iron deficiency in both human and animals in medicinal industry and farming. Overall results of various studies indicated ferrous glycinate to be well suited for use as an iron fortificant and beneficial in the treatment of iron deficient anaemia at lower doses than those associated with ferrous sulphate preparations. When comparing the bioavailability of ferrous sulfate to ferrous glycinate in different meals, the absorption of ferrous glycinate, which partially prevented the inhibitory effect of phytates, was about twice the absorption from ferrous sulfate in human (Layrisse et al., 2000). While in broilers, it was found that the administration of Fe-Gly significantly reduced the fecal Fe concentrations and improved growth performance, iron tissue storage and antioxidant status compared to the ferrous sulfate (Ma et al., 2012). Moreover, it was reported 6 months after supplementation of bis-glycinate chelate iron and ferrous sulfate, the ferritin concentration was significantly higher in the group that received bisglycinate chelate iron in Mexican Children (Duque et al., 2014). Based on the structures of ferrous glycinate and N-carbamylglutamate (Wu et al., 2015), here we designed a new iron chelate ferrous N-carbamylglycinate as an iron fortifier, which aimed to improve the iron bioavailability (compared to inorganic irons) and reduce extra digestion (compared to macromolecule, e.g. proteins chelated irons) (Fig. 1). The safety of this compound, including acute, sub-acute and mutagenicity toxicities were evaluated in the present study. In detail, the present study was conducted to assess the potential genotoxicity and acute and sub-acute (28-day) oral toxicity of ferrous N-carbamylglycinate. All studies here were conducted in accordance with Good Laboratory Practice guidelines
645
(OECD, 1995, 1997a; 1997b, 2001; FDA, 2010). 2. Materials and methods 2.1. Chemicals 2-aminofluorene (C13H11N, CAS 153-78-6), 4-Nitroquinoline Noxide (C9H6N2O3, CAS 56-57-5), cyclophosphamide (CP, C7H17Cl2N2O3P, CAS 50-18-0) were purchased from Aladdin Reagent Co., LTD (Shanghai, China). Fenaminosulf (C8H10N3NaO3S, CAS 140-56-7), Methyl methyl sulfonate (MMS) (C2H6O3S, CAS 6627-3) were purchased from J&K Scientific LTD (Shanghai, China). Phenobarbital/benzoflavone (10%)-induced rat liver S9 was purchased from Platt Bio-Pharmaceutical Co., Ltd. (Beijing, China). New-born calf serum was produced by Hangzhou Sijiqing Biological Materials Limited Company (Hangzhou, China). The rat and mice diet were procured by the Comparative Medicine Centre of Yangzhou University (Yangzhou, China). All other reagents used were of analytical grade or better, as indicated in the certificate of analysis supplied by the manufacturer. 2.2. Ferrous N-carbamylglycinate Ferrous N-carbamylglycinate (Fe, 13%) was produced by a modified method according to Li et al. (2013), as illustrated in Fig. 2. Briefly, the mixture of glycine (C2H5NO2, CAS 56-40-6) (1) and potassium cyanate (KCNO, CAS 590-28-3) in KOH/water (1/100, w/ w) was reacted for 20 h, then crystallized (0 C) and dried to give the product of N-carbamylglycinate (C3H6N2O3, CAS 462-60-2) (2). After then, the N-carbamylglycinate (2) was mixed with FeSO4 and reacted at water in 60 C for 6 h. The crude product was filtrated twice, and the filtrate was washed by ethyl alcohol/water (v/v, 95/ 100) and then dried, finally given a light green crystallized Ferrous N-carbamylglycinate (3). 2.3. Animals Specific pathogen-free (SPF) SpragueeDawley (SD) rats (8 weeks, 180e220 g) and Kunming mice (6 weeks, 18e22 g) were purchased from the Comparative Medicine Centre of Yangzhou University (Yangzhou, China) (license No.: SCXK (Su) 2007-0001). The basic feed without any drugs was produced according to the Chinese standard “Laboratory animal rats and mice feed” (GB14924.3, 2010). Rats and mice were kept in a barrier-maintained animal room conditioned at a temperature of 25 ± 3 C, a relative humidity of 50 ± 10% and a 12-h light/dark cycle. Before treatment, animals were individually handled and carefully examined for abnormal
Fig. 1. Chemical structures of ferrous glycinate (A) and ferrous N-carbamlglycinate (B).
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Fig. 2. Synthesis of ferrous N-carbamlglycinate: (i) 0.5 equiv KCNO, KOH/water (1/100, w/w), room temperature, stirred, 20 h (ii) 0.5 equiv FeSO4, water (pH 6.0), 60 C, stirred, 6 h.
behavior and appearance (Ruan et al., 2014; Tang et al., 2014). All animals had a one-week acclimatization period before experiment started and received basic feed and fresh water freely during the experiment period (Ren et al., 2014). Use of animals was in accordance with “Guide for the Care and Use of Laboratory Animals” NIH Publication (NRC, 1996). All the studies were approved by the Ethical Committee of the Faculty of Veterinary Medicine at Yangzhou University. 2.4. Experimental design 2.4.1. Acute oral toxicity study Feed of SpragueeDawley (SD) rats (10 males and 10 females) was withdrawn overnight before administration of starting doses at 12 h. An acute, one-day oral toxicity study was conducted in accordance with the Organization for Economic Co-operation and Development (OECD) Guideline 423 (OECD, 2001). Oral acute study for calculating median lethal dose (LD50) was performed according to modified Karber's method. In this method, the Ferrous N-carbamylglycinate was suspended in 0.5% carboxymethyl-cellulose solution and exposed to a single dose of the Ferrous N-carbamylglycinate 2020, 2420, 2900, 3480, 4170 and 5000 mg/kg b.w. by oral gavage, respectively, which were determined through respective pretesting. Control groups were administered 0.5% carboxymethylcellulose solution equally. Then rats that survived were observed for another 14 days. Observations included evaluation of skin and fur, eyes and mucous membranes, respiratory, autonomic effects (e.g. salivation), central nervous system effects (tremors and convulsions), changes in the level of motor activity, gait and posture, reactivity to handling and stereotypes or bizarre behavior (e.g. selfmutilation, walking backwards). The time of death was recorded as precisely as possible. At the end of the test, surviving animals were sacrificed (Ruan et al., 2013). All studies were conducted in accordance with FDA Good Laboratory Practice guidelines (FDA, 2010). 2.4.2. Sub-acute toxicity study SD rats (8 weeks old, 110e140 g, 40 male and 40 female) were randomly assigned to four groups (0, 150, 300, and 600 mg/kg b.w./ day, 28 days) by a computer-generated (weight-ordered) randomization procedure (Ren et al., 2013a). Each group consisted of 10 female rats and 10 male rats. Dietary Ferrous N-carbamylglycinate concentrations were adjusted weekly according to the average male body weights for each dosage group from the previous week. All rats were observed at least once a day for mortality or morbidity, changes in posture, changes in skin, fur, eyes, mucous membranes and behaviors (Ren et al., 2013b). Changes in gait were assessed weekly by allowing the animal to walk freely (Chen et al., 2014). All rats were anesthetized with pentobarbital sodium and killed at the 28th day after an overnight fast. The endpoints included clinical
and ophthalmological observations, body weight and feed consumption values, feed efficiency (g weight gain per 100 g of food consumed), a functional observational battery, motor activity, hematology and coagulation assessment, serum chemistry levels, and clinical pathology (Zeng et al., 2008). During necropsies, all the organs/tissues were carefully examined, macroscopically and gross lesions were recorded. The organs of each animal including the heart, lung, kidneys, liver, spleen, testicles (epididymis), ovary and adrenal glands were weighed separately (Kong et al., 2009). Organ/body weight ratios were calculated based on the fasted animal's body weight. The organs from each animal, with the exception of testes, were preserved in 10% neutral-buffered formalin and slides were prepared for histopathological examination using the routine paraffin embedding technique. Sections of 5 mm thickness stained with hematoxylin and eosin (HE) were examined under light microscopy for morphological alterations by a board-certified veterinary pathologist. Tissues from other groups were examined as necessary to determine no observed adverse effect level (NOAEL) in target organs. 2.4.3. Mutagenicity studies Bacterial reverse mutation assay was performed to evaluate the mutagenicity of Ferrous N-carbamylglycinate, with and without S9, using the following four Salmonella strains as prescribed in the OECD guideline No.471 (OECD, 1997a): TA97a, TA98, TA100, and TA102. All strains were provided by Chinese (Jiangsu) Center for Disease Control and Prevention for type culture collection and checked for maintenance of genetic markers prior to the study. Test solutions were prepared in water as serial dilutions to deliver the required concentration in a constant volume. 2-aminofluorene (10 mg per plate) was used as a positive control for all strains tested with S9. Fenaminosulf (50 mg per plate) was used as a positive control for TA97 and TA98 strains tested without S9. 4Nitroquinoline N-oxide (0.5 mg per plate) was used as a positive control for TA100 strain tested without S9. Methyl methyl sulfonate (MMS) (1 mL per plate) was used as a positive control for TA102 strain tested without S9. The assay tubes were pre-incubated at 37 C for 20 min before plating onto minimal agar. Three test plates per concentration were incubated at 37 C for 48 h and then counted. The criteria for a positive response were a more than two-fold increase in the average plate count compared with the solvent control for at least one concentration level and a dose response over the range of tested concentrations in at least one strain with or without S9. Mice bone marrow erythrocyte micronucleus assay was performed in accordance with the OECD Guideline No. 474 (OECD, 1997b) for principles of Good Laboratory Practices (GLP). Fifty 810 week-old SPF Kunming mice (18e22 g) were randomly divided
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2.5. Statistical analyses
Table 1 The results acute test of ferrous N-carbamylglycinate in rats. Groups
Number of rats
Dosage (mg/kg b.w.)
Deaths
Death rate (%)
1 2 3 4 5 6
10 10 10 10 10 10
5000 4170 3480 2900 2420 2020
10 9 7 3 1 0
100 90 70 30 10 0
into five groups each consisting of 10 mice (five males and five females). Cyclophosphamide (CP, i.p. 40 mg/kg BW) was administered 6 h before sampling as a positive control and water was used as a negative control. In this doseeresponse study, Ferrous N-carbamylglycinate was administered twice in 30 h with a 24-h interval at a dose level of 375, 750, and 1500 mg/kg BW through oral gavage. Six hours after the last treatment, all the animals were euthanized to obtain cell suspensions from the femur bone marrow. Bone marrows were flushed with 1 mL of newborn calf serum to obtain cell suspensions. One drop of the mixture was smeared on a clean slide, air dried, fixed with 95% methanol for 10 min and stained with Giemsa stain. All slides were coded to ensure that the evaluation was blinded. Micronucleus frequencies were determined for each animal by counting 1000 of polychromatic erythrocytes (PCE) and the micronucleus occurrence rate per one thousand PCE was recorded. The ratio of PCE to normochromatic erythrocytes (NCE) was determined for each animal by counting a total of 1000 erythrocytes. The micronucleus occurrence rate and PCE/NCE ratio of each group were compared using SPSS 17.0 software. Sperm shape abnormality assay was performed in accordance with the principles of Good Laboratory Practices (FDA, 2010) and Chinese standard (GB15193.7-2003). Fifty mice were randomly divided into five groups of 10 each. Ferrous N-carbamylglycinate (oral. 375, 750, and 1500 mg/kg BW) and cyclophosphamide (CP, oral. 40 mg/kg BW), as a positive control were administered for five days with a 24-h interval at a dose level of through oral gavage. Water was used as a negative control. Mice were sacrificed after the last treatment by cervical dislocation. Both of the cauda epididymises were dissected out in a plate with 1.5 mL normal saline then cut twice into pieces. After stirred for 3 min, it was filtered, and smears were made according to the standard protocol for sperm morphology assay. A total of 1000 sperms per animal were scored under a microscope with 40 10 magnification. Sperm head abnormalities were determined as having either normal or abnormal morphology. According to the Chinese standard (GB15193.7-2003), a “hookless head” does not have a spherical spot at the tip of the sperm head; a “banana” has a banana-like form; an “amorphous head” lacks the usual hook and is deformed; and a “folded sperm” is folded on itself.
Statistical analyzes were conducted by comparing the treatment groups with the control group using the SPSS 17.0 program (SPSS Inc., Chicago, USA). Levene's test was used to examine the homogeneity of variances. If the variance was homogeneous, the data was analyzed with one-way analysis of variance (ANOVA); otherwise they were analyzed by the KruskaleWallis non-parametric ANOVA. If the variance was significant (<0.05), Dunnett's test was used to identify the statistical significance of the individual groups. For histopathological findings and abnormal sperm morphology, they were subjected to Fisher's exact probability test. The pre-set two-sided P-value of <0.05 or 0.01 was taken as statistically significant. Values are expressed as mean ± SD. 3. Results 3.1. Acute toxicity During the acute experiment period, the rats were found motionless and breathless, suddenly jumped and squealed. Their furs were messy and gray loose stool was around the anus. Except for the 2020 mg/kg b.w. group, some rats were found dead in other groups (Table 1). Particularly, the rats in the highest dosed group (5000 mg/kg b.w.) were found dead within 4e16 h after administration. All rats died were dissected and submitted to the pathoanatomical analysis. At post-mortem, no obvious changes were noted except the gastrointestinal tracts which were distended and filled with reddish-brown liquid and gray contents. For the 14-day observation, most rats got a recovery. LD50 values and its 95% confidence intervals of the Ferrous N-carbamylglycinate in SD rats were calculated according to improved Karber's method. The results indicated that the LD50 values of the Ferrous N-carbamylglycinate in SD rats were 3.02 g/kg and the 95% confidence intervals were between 2.78 and 3.31 g/kg. 3.2. Sub-acute toxicity No deaths or obvious clinical signs were found in any groups throughout the sub-acute experimental period. The body weight changes, feed intakes and efficiency were summarized in Table 2, but no significant difference (p > 0.05) was found in each group. Except for GLU concentration in serum, no significant difference (p > 0.05) was observed in any other hematology and serum biochemical parameters (Table 3). No significant difference in organ weight of the investigated tissues, including heart, liver, lung, spleen, kidney, ovary, testis and adrenals in both male and females in the 150 mg/kg b.w., 300 mg/kg b.w. and 600 mg/kg b.w. diet groups was found (Table 4). And no treatment related
Table 2 Effect of the 28-day ferrous N-carbamylglycinate repeated dosage on body weight and feed consumption in rats. Groups (n ¼ 5) Control Initial body weight (g) Final body weight (g) Body weight gain (g) Total feed consumption (g) Feed consumption rate
Male Female Male Female Male Female Male Female Male Female
132.50 117.20 351.70 230.80 219.20 113.60 6623 4277 33.10 26.56
FeN-150 ± ± ± ± ± ±
7.09 5.14 28.94 7.42 30.52 6.88
133.00 129.80 354.10 226.30 221.10 105.70 6012 3550 36.78 29.77
± ± ± ± ± ±
FeN-300 5.85 8.47 21.49 14.68 21.06 12.50
131.90 134.40 333.80 222.00 201.90 106.00 5890 3779 30.88 28.05
FeN, ferrous N-carbamylglycinate; FeN-150, 150 mg/kg BW/day; FeN-300, 300 mg/kg BW/day; FeN-600, 600 mg/kg BW/day.
± ± ± ± ± ±
FeN-600 5.74 8.26 39.41 12.09 37.87 9.96
132.70 116.30 344.50 222.30 211.80 106.00 6290 4280 33.67 23.36
± ± ± ± ± ±
7.20 3.74 15.72 10.76 18.81 12.17
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Table 3 Effects of ferrous N-carbamylglycinate on hematology and serum clinical chemistry parameters of rats in the 28-day sub-acute test. Groups (n ¼ 5) Control 9
WBC (10 /L)
Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female
12
RBC(10 /L) HGB (g/L) HCT (%) MCV (fL) MCH (pg) MCHC (g/L) PLT (109/L) ALT (U/L) AST (U/L) CHO (mmol/L) TG (mmol/L) CRE (mmol/L) GLu (mmol/L) TP (g/L) ALB (g/L)
7.53 4.69 7.50 7.11 145.90 135.00 42.69 40.55 55.20 55.01 18.22 18.17 340.00 332.90 579.40 515.70 58.80 55.20 144.80 117.10 1.23 1.13 1.84 0.83 33.81 38.50 8.15 8.15 60.75 56.61 17.31 18.07
FeN-150 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.75 1.09 0.23 0.28 4.63 7.13 1.72 1.74 2.32 4.03 0.97 0.68 12.27 7.45 134.95 74.75 8.07 11.61 26.97 16.82 0.28 0.37 0.83 0.42 11.39 7.81 0.51 0.78 6.39 1.60 1.02 0.94
8.26 5.73 7.41 7.27 135.30 136.70 40.11 40.10 54.21 55.39 18.28 18.86 337.30 340.80 614.60 545.10 62.50 56.00 151.20 138.50 1.29 1.17 1.63 1.10 35.59 37.91 9.62 9.73 63.43 59.49 19.35 18.41
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
FeN-300 3.84 1.82 0.38 0.57 8.03 5.10 1.92 1.00 1.76 3.32 0.77 1.06 5.46 6.96 64.98 54.34 8.14 7.26 26.25 18.76 0.23 0.16 0.85 0.29 10.98 6.35 1.50* 1.12* 10.62 7.29 3.78 3.74
8.18 4.99 7.78 7.11 150.80 136.00 43.95 40.38 56.56 56.75 18.62 18.73 343.10 337.30 652.10 481.10 60.50 61.70 140.50 133.60 1.31 1.24 1.68 1.11 34.18 41.63 8.73 9.75 63.65 60.57 17.31 18.84
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
FeN-600 1.10 1.71 0.43 0.31 8.01 7.63 2.20 2.75 1.56 2.25 0.84 0.49 9.42 10.73 191.99 149.44 9.07 7.97 14.69 20.91 0.18 0.16 0.71 0.43 8.24 7.70 0.81 2.57* 4.54 6.20 1.37 1.68
8.11 4.66 7.63 7.02 146.40 135.00 42.66 41.20 55.95 57.81 18.70 18.75 343.30 328.10 546.80 515.70 53.10 48.70 140.70 110.80 1.13 1.17 1.93 0.96 27.17 43.50 8.60 9.22 60.28 56.29 18.07 18.89
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.17 0.99 0.22 0.25 5.08 3.43 1.35 1.65 2.09 1.73 0.75 0.64 10.01 11.73 102.66 86.79 9.04 13.80 17.19 33.30 0.09 0.37 0.45 0.25 3.51 7.39 1.11 1.12* 2.15 2.48 0.59 1.15
* Significantly different from the control group at p < 0.05. FeN, Ferrous N-carbamylglycinate; FeN-150, 150 mg/kg BW/day; FeN-300, 300 mg/kg BW/day; FeN-600, 600 mg/kg BW/day; WBC, white blood cell count; RBC, red blood cell count; HGB, hemoglobin concentration; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PLT, platelet counts; TP, total protein; ALB, total albumin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TCHO, total cholesterol; TG, triglyceride; Cr, creatinine; BUN, blood urea nitrogen; GLU, glucose; A/G, total albumin/globulin ratio.
macroscopical changes in rats or histopathological changes in liver, spleen, heart, lung, ovary and testis were found in each group.
3.3. Mutagenicity No increase > two-fold in the number of revertants was observed with the S. typhimurium TA97a, TA98, TA100, TA102 and
Table 4 Effects of the 28-day ferrous N-carbamylglycinate treatment on weight of rat organ. Groups (n ¼ 5) Control Heart Liver Spleen Lung Kidney Adrenals Testis Ovary
Male Female Male Female Male Female Male Female Male Female Male Female
0.34 0.36 4.40 3.53 0.22 0.26 0.40 0.47 0.78 0.75 0.16 0.17 0.98 0.06
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.02 0.02 0.29 0.27 0.03 0.04 0.03 0.06 0.05 0.03 0.02 0.04 0.10 0.01
4. Disscussion
FeN-150 0.35 0.37 4.16 3.58 0.23 0.25 0.38 0.52 0.79 0.72 0.15 0.18 1.01 0.06
TA1535 strains after treatment with Ferrous N-carbamylglycinate at 0.005, 0.05, 0.5 and 5 mg/plate compared with the negative control (Table 5). No statistically significant changes (p > 0.05) in the ratios of MN-PCE to PCE was found in the Ferrous N-carbamylglycinate (370, 750, or 1500 mg/kg b.w./day) treatment groups (Table 6). No significant difference (p > 0.05) in the percentage of abnormal sperm was found in Ferrous N-carbamylglycinate treated groups (Table 7). These results above indicated that the Ferrous N-carbamylglycinate can be classified as a low-toxic substance without genotoxicity.
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.03 0.03 0.36 0.35 0.03 0.04 0.10 0.06 0.05 0.05 0.03 0.04 0.07 0.02
FeN-300 0.37 0.37 4.09 3.67 0.20 0.25 0.50 0.44 0.78 0.72 0.15 0.15 1.05 0.06
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.03 0.02 0.48 0.23 0.02 0.03 0.22 0.15 0.05 0.11 0.03 0.03 0.23 0.01
FeN-600 0.30 0.36 4.26 3.71 0.21 0.26 0.39 0.51 0.76 0.79 0.16 0.22 0.94 0.06
± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.08 0.04 0.28 0.27 0.02 0.03 0.04 0.04 0.04 0.04 0.04 0.16 0.20 0.01
No statistically significant differences were found. FeN, ferrous N-carbamylglycinate; FeN-150, 150 mg/kg BW/day; FeN-300, 300 mg/kg BW/day; FeN-600, 600 mg/ kg BW/day.
In the present study, a series of preclinical trials were performed to evaluate the safety of ferrous N-carbamylglycinate, including acute toxicity, sub-acute (28-day oral) toxicity and mutagenicity tests. All the tests provided a comprehensive safety assessment for Ferrous N-carbamylglycinate and could contribute to the development of Ferrous N-carbamylglycinate to be used in future studies. In the earlier studies, the acute oral LD50 of ferrous glycinate were 560 mg iron/kg body weight of rat (Jeppsen and Borzelleca, 1999; Jeppsen, 2001). However, the present study showed that the LD50 of the ferrous N-carbamlglycinate was 3.02 g/kg, equaled to 392 mg iron/kg body weight of rat. The results of the present study on acute oral toxicity were similar but slightly lower than ferrous glycinate in the former research. It indicated the N-carbamlglycinate chelates may have a better bioavailability than glycinate chelate in rats. The results of acute oral toxicity in this study
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Table 5 Mean number of revertants ±SD in the presence or absence of metabolic activation (n ¼ 3, mean ± SD). Treatment
TA97
TA98
(S9) FeN 0.03 FeN 0.3 FeN 3 FeN 30 Water DMSO Fenaminosulf (500 mg/mL) 2-AF (1 mg/mL) 4-nitroquinoline N-oxide (50 mg/mL) MMS (10 mL/mL)
þ (S9)
130.00 135.20 121.68 131.00 132.00 128.10 708.00
± ± ± ± ± ± ±
5.18 3.96 2.36 5.55 4.90 5.42 90.51a
114.55 125.66 119.00 125.33 120.33 130.32 e
± ± ± ± ± ±
5.73 6.21 4.96 5.25 6.23 5.12
TA100
(S9)
þ (S9)
35.00 ± 2.16 33.96 ± 3.08 38.68 ± 3.25 34.33 ± 2.88 39.55 ± 1.96 32.88 ± 0.92 1145.33 ± 48.45a
32.33 34.00 34.67 40.00 35.67 35.67 e
TA102
(S9)
± ± ± ± ± ±
0.94 2.16 2.05 4.97 1.25 2.46
151.67 155.00 150.66 145.25 162.32 151.34 e
þ (S9) ± ± ± ± ± ±
9.69 8.49 8.89 7.38 7.24 4.27
178.67 166.67 171.00 169.67 173.67 177.00 e
(S9) ± ± ± ± ± ±
11.08 11.61 9.04 17.23 6.31 9.42
275.33 281.33 279.67 274.00 277.38 278.67 e
e e
1348.33 ± 88.24a e e e
1245.67 ± 68.37a e 1698.33 ± 162.45a e e 688.67 ± 98.87a e e
e
e
e
e
e
e
þ (S9) ± ± ± ± ± ±
11.27 6.80 6.94 11.66 18.00 8.99
289.26 296.67 284.00 280.00 263.33 272.67 e
± ± ± ± ± ±
8.90 12.04 16.00 13.07 14.08 8.38
1520.00 ± 108.24a e
1428.00 ± 98.43a e
a Number of revertant colonies induced was double or more in the sample/number of spontaneous in the negative control. 2-AF, 2-aminofluorene; MMS, methyl methyl sulfonate; FeN, ferrous N-carbamylglycinate; FeN 0.03, 100 mL of FeN at the concentration of 0.03 mg/mL was added to the plate; FeN 0.3, 100 mL of FeN at the concentration of 0.3 mg/mL was added to the plate; FeN 3, 100 mL of FeN at the concentration of 3 mg/mL was added to the plate; FeN 30, 100 mL of FeN at the concentration of 30 mg/mL was added to the plate.
microscopic findings were found. The NOAEL of ferrous glycinate was at least 500 mg ferrous glycinate chelate/kg body weight/day, the highest dose tested (Jeppsen and Borzelleca, 1999). Moreover, an in vivo toxicity study of Iron N-(2-hydroxy acetophenone) glycinate (FeNG) did not reveal any mortality or symptoms of toxicity, including pathological changes, body weight gain/loss pattern, organ weight, haematological or biochemical parameters in treated groups as compared to the control group of animals (Ganguly et al., 2012). Here in the present 28-day sub-acute study, no significant difference in organ weight and treatment related macroscopical or histopathological changes were found in liver or any other tissues in Ferrous N-carbamylglycinate treated rats. Moreover, no significant difference was found in hepatic function related serum AST, ALT, CHO and TG concentration. Therefore, the NOAEL was at least 600 mg for ferrous N-carbamylglycinate chelate/kg body weight/ day in rats, the highest dose tested in the present study. Iron-mediated formation of free radicals, caused various modifications to DNA bases, enhanced lipid peroxidation, and altered calcium and sulfhydryl homeostasis (Valko et al., 2005; Guo et al., 2014). Lipid peroxides, formed by the attack of radicals on polyunsaturated fatty acid residues of phospholipids, can further react with redox irons and finally produce mutagenic and carcinogenic malondialdehyde, 4-hydroxynonenal and other exocyclic DNA adducts. Therefore, the mutagenicity of the ferrous N-carbamylglycinate was investigated both in vivo and in vitro. Fortunately, the results indicated that the ferrous N-carbamylglycinate was not mutagenic in the bacterial reverse mutation assay, mice bone marrow erythrocyte micronucleus assay and sperm shape abnormality assay. Since previous studies did not investigate the potential genotoxicity of iron amino acid chelates, the results of the present study indicated the class of these compounds probably be not genotoxic. In summary, the results of the acute oral toxicity study (LD50
Table 6 Effect of ferrous N-carbamylglycinate on bone marrow polychromatic erythroblast micronuclei (MN) in mice (n ¼ 10, mean ± SD). Treatment (mg/kg BW/day, 5 days)
PCE
FeN
10 10 10 10 10
CP Water
375 750 1500 40
PCE/NCE
MN/PCE
P value
MN MN (%, mean ± SD) 000 000 000 000 000
1.18 1.21 1.09 0.88 1.20
± ± ± ± ±
0.10 0.06 0.09 0.11 0.07
11 12 14 168 10
1.02 1.19 1.20 4.79 1.00
± ± ± ± ±
0.57 1.98 2.89 4.87 0.55
<0.01
FeN, ferrous N-carbamylglycinate; CP, cyclophosphamide; NCE, normochromatic erythrocyte; PCE, polychromatic erythrocyte.
indicated that ferrous N-carbamlglycinate could be classified as category 5 (2000e5000 mg/kg), based on the Globally Harmonized System of Classification and Labelling of Chemicals system (GHS, 2005). According to the provision of a low-toxic substance (501e5000 mg/kg) of an acute toxicity test in Chinese Procedures for Toxicological Assessment of Food (MOH, 2003), ferrous N-carbamlglycinate was considered to be a low-toxic substance. Iron is an essential element required for growth and survival of almost every organism. Chronic toxicity of iron consumption can be associated with a high dietary iron intake. Cases of acute iron toxicity are relatively rare and mostly related to hepatoxicity (Tenenbein, 2001). The excessive intestinal absorption of iron resulted in lipid peroxidation and hepatocellular organelles damage, such as mitochondria and lysosomes, which was thought to contribute to hepatocyte necrosis and apoptosis, and ultimately lead to the development of hepatic fibrogenesis (Ramm and Ruddell, 2005). In an early subchronic 90-day toxicity study of ferrous glycinate, there were no biologically or statistically significant compound-related and dose-dependent macroscopic or
Table 7 Effect of ferrous N-carbamylglycinate on spermhead morphology in mice (n ¼ 10, mean ± SD). Treatment (mg/kg BW/day, 5 days)
Number of animals
Number of sperms
FeN
10 10 10 10 10
10 10 10 10 10
Water CP
375 750 1500 40
1000 1000 1000 1000 1000
Total abnormalities Hookless
Banana
Amorphous
Folded
Double-headed
Double-tailed
55 46 46 52 125
20 22 22 21 68
106 102 104 100 302
7 8 9 6 18
5 4 2 0 15
4 2 3 2 16
FeN, ferrous N-carbamylglycinate; CP, cyclophosphamide.
Mean abnormalities 20.08 18.20 19.00 18.00 50.80
± ± ± ± ±
5.20 5.36 6.20 2.05 16.42
Abnormalities ratio 1.97 1.84 1.86 1.81 5.44
P value
p＜0.01
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3.02 g/kg) indicated that ferrous N-carbamylglycinate can be classified as category 5 or is a low-toxic substance according to GHS or the Chinese chemical classification system, respectively. Based on the sub-chronic study, the NOAEL of ferrous N-carbamylglycinate was 600 mg/kg b.w. day, which was the highest dose we investigated. In addition, it is not genotoxic. The results here provided comprehensive safety information of ferrous N-carbamylglycinate, which would further help researches in clinical use. Conflicts of interest The authors declared no conflicts of interest. Acknowleagement This research was granted by National Science Foundation of China (31330075, 31110103909), National Key Technology Research and Development Program of the Ministry of Science and Technology of China (2012BAD39B00), the Chinese Academy of Sciences comprehensive strategic cooperation project of Guangdong Province (2013B091500095, 2013B090900007), Science and Technology Planning Project of Guangdong Province (2013B091500095, 2013B090900007, 2013B070704081) and Guangzhou Industry Research Collaborative innovation major special (201508020048). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.yrtph.2015.09.013. References Aluru, M.R., Rodermel, S.R., Reddy, M.B., 2011. Genetic modification of low phytic acid 1-1 maize to enhance iron content and bioavailability. J. Agric. Food Chem. 59 (24), 12954e12962. Berhanu, G., Mesfin, A., Kebebu, A., Whiting, S.J., Henry, C.J., 2014. Household food processing methods to enhance iron and zinc bioavailability in formulated haricot bean and maize complementary food. Afr. J. Food Sci. 8 (4), 190e195. Chen, S., Liu, S., Zhang, F., Ren, W.K., Li, N.Z., Yin, J., Duan, J.L., Peng, Y.Y., Liu, G., Yin, Y.L., Wu, G.Y., 2014. Effects of dietary L-glutamine supplementation on specific and general defense responses in mice immunized with inactivated Pasteurella multocida vaccine. Amino Acids 46, 2365e2375. € f, M., Lo € nnerdal, B., Abrams, S.A., Hernell, O., 2002. Iron absorption in Domello breast-fed infants: effects of age, iron status, iron supplements, and complementary foods. Am. J. Clin. Nutr. 76 (1), 198e204. Duque, X., Martinez, H., Vilchis-Gil, J., Mendoza, E., Flores-Hern andez, S., Mor an, S., Navarro, F., Roque-Evangelista, V., Serrano, A., Mera, R.M., 2014. Effect of supplementation with ferrous sulfate or iron bis-glycinate chelate on ferritin concentration in Mexican schoolchildren: a randomized controlled trial. Nutr. J. 13 (1), 71. FDA., 2010. Good Laboratory Practice Regulations 21 CFR Part 58. Docket No. FDA2010-N-0548. Fox, T.E., Eagles, J., Fairweather-Tait, S.J., 1998. Bioavailability of iron glycine as a fortificant in infant foods. Am. J. Clin. Nutr. 67 (4), 664e668. Ganguly, A., Chakraborty, P., Banerjee, K., Chatterjee, S., Basu, S., Sarkar, A., Chatterjee, M., Choudhuri, S.K., 2012. Iron N-(2-hydroxy acetophenone) glycinate (FeNG), a non-toxic glutathione depletor circumvents doxorubicin resistance in Ehrlich ascites carcinoma cells in vivo. Biometals 25 (1), 149e163. Ganz, T., Nemeth, E., 2015. Iron homeostasis in host defence and inflammation. Nat. Rev. Immunol. 15 (8), 500e510. GHS (Globally Harmonized System of Classification and Lablling of Chemicals), 2005. Chapter 3. Health Hazards. (3-2-1) Acute Toxicity. Guo, L., Harnedy, P.A., Li, B., Hou, H., Zhang, Z., Zhao, X., FitzGerald, R.J., 2014. Food protein-derived chelating peptides: biofunctional ingredients for dietary mineral bioavailability enhancement. Trends Food Sci. Technol. 37 (2), 92e105. Guo, L., Hou, H., Li, B., Zhang, Z., Wang, S., Zhao, X., 2013. Preparation, isolation and identification of iron-chelating peptides derived from Alaska pollock skin. Process Biochem. 48 (5), 988e993. Jeppsen, R.B., 2001. Toxicology and safety of Ferrochel and other iron amino acid chelates. Arch. Latinoam. Nutr. 51 (1 Suppl. 1), 26e34. Jeppsen, R.B., Borzelleca, J.F., 1999. Safety evaluation of ferrous bisglycinate chelate. Food Chem. Toxicol. 37 (7), 723e731. Kong, X.F., Zhang, Y.Z., Yin, Y.L., Wu, G.Y., Zhou, H.J., Tan, Z.L., Yang, F., Bo, M.J.,
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