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Toxicological evaluation of pomegranate seed oil
Food and Chemical Toxicology 47 (2009) 1085–1092
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Toxicological evaluation of pomegranate seed oil I.A.T.M. Meerts a,*, C.M. Verspeek-Rip a, C.A.F. Buskens a, H.G. Keizer b, J. Bassaganya-Riera c, Z.E. Jouni d, A.H.B.M. van Huygevoort e, F.M. van Otterdijk e, E.J. van de Waart a a
Department of In vitro and Environmental Toxicology, NOTOX B.V., Hambakenwetering 7, 5231 DD ‘s-Hertogenbosch, The Netherlands Lipid Nutrition B.V., Hogeweg 1, 1520 AZ Wormerveer, The Netherlands c Nutrition Therapeutics Inc., Blacksburg, VA 24060, USA d Mead Johnson Nutritionals, Evansville, IN, USA e Department of Toxicology, NOTOX B.V., ’s-Hertogenbosch, The Netherlands b
a r t i c l e
i n f o
Article history: Received 8 August 2008 Accepted 27 January 2009
Keywords: Pomegranate seed oil Acute oral toxicity Mutagenicity 28-day toxicity
a b s t r a c t In this manuscript, the toxicology and safety of pomegranate seed oil (PSO) was evaluated by in vitro (Ames, chromosomal aberration), and in vivo toxicity tests (acute toxicity and 28-day toxicity in Wistar rats). No mutagenicity of PSO was observed in the absence and presence of metabolic activation up to precipitating concentrations of 5000 lg/plate (Ames test) or 333 lg/ml (chromosome aberration test). The acute oral toxicity study revealed no significant findings at 2000 mg PSO/kg body weight. In the 28day dietary toxicity study PSO was dosed at concentrations of 0, 10,000, 50,000 and 150,000 ppm, which resulted in a mean intake of 0–0, 825–847, 4269–4330 and 13,710–14,214 mg PSO/kg body weight per day in males–females, respectively. At 150,000 ppm dietary exposure to PSO, a much higher dose than the level of PSO that elicits antidiabetic and anti-inflammatory efficacy, increased hepatic enzyme activities determined in plasma (aspartate, alanine aminotransferase and alkaline phosphatase) and increased liver-to-body weight ratios were observed. However, these effects might be the result of a physiological response to exposure to a very high level of a fatty acid which is not part of the normal diet, and are most likely not toxicologically relevant. The no observable adverse effect level (NOAEL) was 50,000 ppm PSO (=4.3 g PSO/kg body weight/day). Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction The pomegranate (Punica granatum) is an ancient fruit which has been widely consumed in many different cultures for thousands of years, largely without incidents, and thus is considered generally safe by the general public. It belongs to the family Punicaceae which grows only in Socotra, an island off the Yemeni coast (Lansky and Newman, 2007). The pomegranate is native from Iran to the Himalayas in northern India, and has been cultivated and naturalized over the whole Mediterranean region since ancient times. Over 1000 cultivars of P. granatum exist (Levin, 1994). It is widely cultivated throughout Iran, India, the drier parts of Southeast Asia, Malaysia, the East Indies, and tropical Africa. Pomegranate is now cultivated mainly in the drier parts of California and Abbreviations: 2-AA, 2-aminoanthracene; CLNA, conjugated linolenic acids; DMSO, dimethyl sulfoxide; EEC, European Economic Committee; GLP, Good Laboratory Practice; MMS, methylmethanesulfonate; 4-NQO, 4-nitroquinoline N-oxide; OECD, Organisation for Economic Co-operation and Development; PSO, pomegranate seed oil. * Corresponding author. Tel.: +31 (0)73 6406700; fax: +31 (0)73 6406799. E-mail address: [email protected] (I.A.T.M. Meerts). 0278-6915/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2009.01.031
Arizona for its fruits exploited commercially as juice products gaining in popularity since 2001, especially because of the appearance of several reports presenting the antioxidant activities of pomegranate fractions in vitro (Schubert et al., 1999; Gil et al., 2000; Singh et al., 2002). Pomegranate seed oil (PSO) comprises 12–20% of total seed weight. The oil is characterized by a high content of conjugated linolenic acids (CLNA, about 31.8–86.6%), followed by linoleic acid (0.7–24.4%), oleic acid (0.4–17.7%), stearic acid (2.8–16.7%) and palmitic acid (0.3–9.9%) (El-Shaarawy and Nahapetian, 1983; Ozgul-Yucel, 2005; Fadavi et al., 2006). Variability in concentration is due to differences between cultivars. The CLNA found in PSO are all 9, 11, 13 isomers, with punicic acid (9 cis, 11 trans, 13 cis) as the predominant conjugated triene (Ozgul-Yucel, 2005). Coldpressed PSO has been commercially available during the last 5– 6 years. The pomegranate seeds are cleaned and pressed giving virgin oil which is further purified by centrifugation, sedimentation or filtration. The cold-pressed oil obtained can then be used for skin care recipes or food use as a dietary supplement. Although pomegranate has been widely consumed for thousands of years, little is known about the possible toxicity and safety
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of the pomegranate or the PSO. Several studies indicate that coldpressed PSO is able to reduce tumor occurrence, tumor incidence and multiplicity in both ex vivo models and in vivo in mice and rats. In an ex vivo model using mouse mammary organ culture (Mehta and Lansky, 2004), cold-pressed PSO reduced the tumor occurrence up to 87%. In an in vivo study with female CD-1 mice with skin tumors induced by topical application of 7,12-dimetylbenz[a]anthracene and promoted by 12-O-tetradecanoylphorbol 13-acetate, PSO applied topically (5%) resulted in significant decreases in tumor incidence and multiplicity (Hora et al., 2003). In male F344 rats treated with the carcinogen azoxymethane, dietary PSO at concentrations of 0.1% already reduced the incidence and multiplicity of colon tumors (Kohno et al., 2004). However, besides several very intriguing and promising studies on the possible beneficial effects of PSO, little is known about the possible toxicity and safety of pomegranate fruits or PSO, and standard in vitro and in vivo toxicology studies have not been performed with PSO. Therefore, in the current investigations we studied the possible mutagenicity of PSO using two in vitro mutagenicity studies (the Ames test and chromosomal aberration test), and the possible in vivo toxicity of PSO using Wistar rats (acute oral toxicity study and 28-day toxicity test). 2. Materials and methods
and a relative humidity of 30–70%. The rooms were illuminated with 12 h artificial fluorescent light and 12 h darkness per day. Unless otherwise stated, the animals were provided a standard pelleted laboratory animal diet (SM R/M-Z from SSNIFFÒ Spezialdiäten GmbH, Soest, Germany) that met or exceeded all nutritional needs and tap water ad libitum. The animals were allowed to acclimatize for at least 5 days before the start of the treatment. Results of analysis for ingredients and/or contaminants of diet, bedding, paper and water were assessed and did not reveal any findings that were considered to have affected the study integrity. 2.4. Preparation of rat liver S9-fraction for in vitro mutagenicity studies Rat liver S9 was prepared from young adult male Wistar rats (8 weeks of age), which were obtained from Charles River (Sulzfeld, Germany). Five male rats per S9batch were group-housed in labeled polycarbonate cages (type MIV height: 18 cm) containing Woody Clean bedding (Woody-Clean type 3/4; Technilab-BMI BV, Someren, The Netherlands) and paper as cage-enrichment (Technilab-BMI BV). The rats were orally dosed for three consecutive days with a suspension of phenobarbital (80 mg/kg body weight) and b-naphthoflavone (100 mg/kg body weight) in corn oil. The rats were denied access to food for 3–4 h preceding each dosing. One day after the final exposure, the rats were sedated using oxygen/carbon dioxide and sacrificed by decapitation. The rats received a limited quantity of food during the night before sacrifice. The livers of the rats were removed aseptically, and washed in cold (0 °C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA (Merck, Germany). Subsequently the livers were minced in a blender and homogenized in 3 volumes of 0.1 M sodium phosphate buffer (pH 7.4) with a Potter-Elvehjem tube and Teflon pestle. The homogenate was centrifuged for 15 min at 9000g (4 °C). The resulting supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen ( 196 °C) for a maximum of 1 year. All batches were checked for sterility and the concentration of cytochrome P450 was calculated from the CO-reduced difference spectrum according to Omura and Sato (1964).
2.1. Pomegranate seed oil (PSO) Cold-pressed PSO was obtained from Lipid Nutrition, Wormerveer, The Netherlands. The composition of the PSO was determined using GC analysis and is presented in Table 1. 2.2. Chemicals and bacterial strains The Salmonella typhimurium strains TA1535, TA1537 and TA98 were obtained from Dr. B.N. Ames (University of California, Berkeley, USA) and the strain TA100 was obtained from Xenometric (Boulder, Co, USA). The Escherichia coli strain was obtained from Prof. Dr. B.A. Bridges (University of Sussex, Brighton, UK). Positive controls used in the Ames test without metabolic activation were sodium azide (TA1535), 9-aminoacridine (TA1537), 2-nitrofluorene (TA98), methylmethanesulfonate (TA100) and 4-nitroquinoline N-oxide (WP2uvrA). In the presence of 5 and 10% S9-mix as metabolic activation, 2-aminoanthracene (2AA) was used as positive control. Sodium azide, methylmethanesulfonate, 4-nitroquinoline N-oxide, 2-aminoanthracene, b-naphthoflavone were obtained from Sigma, Zwijndrecht, The Netherlands. 9-Aminoacridine and colchicine were from Acros Organics, Geel, Belgium. Dimethyl sulfoxide, 2-nitrofluorene and Giemsa were obtained from Merck, Darmstadt, Germany.
2.5. In vitro mutagenicity tests 2.5.1. Ames test PSO was tested in the S. typhimurium reverse mutation assay with four histidine-requiring strains of S. typhimurium (TA1535, TA1537, TA98 and TA100) and in the E. coli reverse mutation assay with a tryptophan-requiring strain of E. coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of metabolic activation (phenobarbital- and b-naphthoflavone-induced rat liver S9-mix), and was performed under GLP conditions according to OECD and EEC guidelines (OECD 471 and 2000/32/EC). PSO was dissolved in dimethyl sulfoxide and tested in TA100 and WP2uvrA at concentrations of 0 (solvent control), 3, 10, 33, 100, 333, 1000, 3330 and 5000 lg/ plate in the absence and presence of 5% (v:v) S9-mix (first experiment). Since PSO precipitated on the plates at concentrations of 3330 lg/plate and 5000 lg/ plate, in the strains TA1535, TA1537 and TA98, PSO was tested at concentrations of 33, 100, 333, 1000 and 3330 lg/plate. An independent repeat assay was performed, in which PSO was tested at concentrations of 33, 100, 333, 1000 and 3330 lg/plate in all bacterial strains in the absence and presence of 10% (v:v) S9mix (second experiment). Appropriate negative and strain-specific positive controls were added.
2.3. Animal husbandry All procedures concerning the use of animals were approved by the Animal Experimental Committee of NOTOX. The animals were housed in a controlled environment, with approximately 15 air changes per hour, a temperature of 21 ± 3 °C Table 1 Chemical composition of the two PSO batches used. Batch Aa (%)
Batch Bb (%)
Triglycerides Diglycerides Monoglycerides Free fatty acids Polymers Total composition (%)
94 2.3 0.4 0.9 2.1 99.7
91 3.2 1.0 2.2 2.8 100.2
Fatty acid analysis (%) C16:0 stearic acid C18:0 palmitic acid C18:1 oleic acid C18:2 linoleic acid CLNA Total fatty acid analysis (%)
2.7 2.6 5.9 5.8 82.8 99.8
2.9 2.2 7 6.5 79.6 98.2
a Batch A is used for the two in vitro mutagenicity assays and the in vivo acute oral toxicity study. b Batch B is used for preparation of the diets for the 28-day dietary toxicity study.
2.5.2. In vitro chromosomal aberration test using cultured peripheral human lymphocytes A chromosomal aberration test using cultured peripheral human lymphocytes was performed under GLP conditions according to OECD and EEC guidelines (OECD 473 and 2000/32/EC). Two independent experiments were performed. In the first experiment, PSO was tested at concentrations of 33, 100 and 333 lg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v:v) S9-fraction. PSO precipitated in the culture medium at a concentration of 333 lg/ ml. In the second experiment, PSO was again tested at concentrations of 33, 100 and 333 lg/ml, with a change in incubation time and fixation time. In the absence of S9-mix, 24 h and 48 h continuous exposure times were used with a 24 and 48 h fixation time. In the presence of S9-mix, a 3 h exposure time and 48 h fixation time was used. As positive controls, mitomycin (Sigma, Zwijndrecht, The Netherlands) was used for incubations without metabolic activation, and cyclophosphamide (Endoxan-Asta, Asta-Werke, Germany) for incubations with metabolic activation. As negative control, dimethyl sulfoxide was used since this was the solvent used to dissolve PSO. During the last 2.5–3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 lg/ml medium). Thereafter, the cell cultures were centrifuged and the remaining pellet was treated for 5 min with hypotonic 0.56% (w:v) potassium chloride solution at 37 °C. Subsequently, the cells were fixed with 3 changes of methanol:acetic acid 3:1 (v:v). Fixed cells were dropped onto clean slides. Slides were allowed to dry and stained for 10–30 min with 5% (v:v) Giemsa solution in tap water. Thereafter, slides were rinsed in tap water, allowed to dry, embedded in MicroMount and mounted with a coverslip. All slides were randomly coded before examination of chromosome aberrations and scoring was performed in a blind fashion. Per culture, 100 metaphase chromo-
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10,000 ppm PSO
50,000 ppm PSO
150,000 ppm PSO
High fat control diet
Casein Corn starch mod. Maltodextrine Sucrose Cellulose L-Cystine AIN 93G-Vitamin premixa AIN 93G-Min./trace elem. premix Choline chloride Safflower oil PSO
22.00 28.59 13.20 10.00 5.60 0.33 1.10 3.90 0.28 14.00 1.00
22.00 28.59 13.20 10.00 5.60 0.33 1.10 3.90 0.28 10.00 5.00
22.00 28.59 13.20 10.00 5.60 0.33 1.10 3.90 0.28 – 15.00
22.00 28.59 13.20 10.00 5.60 0.33 1.10 3.90 0.28 15.00 –
Dry matter (%) Crude Protein (%) Crude Fat (%) Crude Fiber (%) Crude Ash (%) Starch (%) Sugar and dextrines (%) N free extracts (%) Calcium (%) Phosphorus (%) Sodium (%) Magnesium (%) Lysine (%) Methionine (%) Met + Cys (%) Vitamin A (IU/kg) Vitamin D3 (IU/kg) Vitamin E (IU/kg) Vitamin K3 (IU/kg) GE (MJ/kg) ME (pig) (MJ/kg) ME (Atwater) (MJ/kg) Protein (%) Fat (%) Carbohydrates (%) C14:0 (% in diet) C16:0 (% in diet) C18:0 (% in diet) C18:1 (% in diet) C18:2 (% in diet) C18:3 (% in diet) linolenic (n3) C18:3 (% in diet) punicic (n5) P C18:3 (% in diet) conjug.b C20:0 (% in diet) C20:1 (% in diet)
97.0 19.1 15.1 5.6 3.3 27.5 24.6 53.9 0.57 0.36 0.16 0.09 1.57 0.58 0.99 4,400 1,100 83 4.5 21.2 16.5 17.9 18 32 50 0.01 0.95 0.36 1.55 10.58 0.07 0.40 0.39 0.07 0.07
97.0 19.1 15.1 5.6 3.3 27.5 24.6 53.9 0.57 0.36 0.16 0.09 1.57 0.58 0.99 4,400 1,100 83 4.5 21.2 16.5 17.9 18 32 50 0.01 0.80 0.36 1.42 7.84 0.05 2.02 1.95 0.07 0.08
97.0 19.1 15.1 5.6 3.3 27.5 24.6 53.9 0.57 0.36 0.16 0.09 1.57 0.58 0.99 4,400 1,100 83 4.5 21.2 16.5 17.9 18 32 50 0.02 0.46 0.34 1.08 0.97 0.01 6.06 5.86 0.06 0.09
97.0 19.1 15.1 5.6 3.3 27.5 24.6 53.9 0.57 0.36 0.16 0.09 1.57 0.58 0.99 4,400 1,100 83 4.5 21.2 16.5 17.9 18 32 50 0.01 0.98 0.36 1.58 11.27 0.07 – – 0.07 0.07
PSO: pomegranate seed oil, GE = gross energy, ME = metabolizable energy. Fatty acid (FA) composition is characterized by the FA concentration of safflower oil and PSO. a Provides 150 mg butylhydroxytoluol (BHT)/kg diet and 1000 mg ascorbic acid/kg diet. b Without punicic acid.
some spreads were examined by light microscopy for chromosomal aberrations. Only metaphases containing 46 ± 2 chromosomes were analyzed. Prior to analysis of chromosomal aberrations, toxicity was determined using the mitotic index. The following aberrations were scored: chromatid gaps and breaks, chromosome gaps and breaks, chromatid deletion, minutes, double minutes, dicentric chromosomes, tricentric chromosomes, ring chromosomes, exchange figures, chromosomal intrachanges, pulverised chromosomes, multiple aberrations in one metaphase, polyploidy and endoreduplications. The dose levels of 33, 100 and 333 lg/ml from each incubation and fixation time were selected for scoring of chromosome aberrations. 2.6. In vivo toxicology tests 2.6.1. Acute oral toxicity in rats The acute oral toxicity study in rats was performed as a GLP study according to the guidelines mentioned in OECD 420 (2001) and EC council directive 67/548/EEC. PSO was administered as a single oral dose (by gavage) of 2000 mg/kg body weight to 9–11 weeks old female Wistar Crl:WI rats (n = 5). The animals were group-housed (4 animals per cage) in labeled Macrolon cages (MIII type, height 18 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son, Surrey, UK). A pilot study with one animal (individually housed) was performed to deter-
mine if 2000 mg/kg body weight was a tolerable dose. The test system and procedures of this pilot study with one animal were identical to the main study with 4 animals. Based on the pilot results, a fixed dose level was selected for the main study and four female rats were exposed to 2000 mg/kg body weight. PSO was dosed undiluted, resulting in a dose volume of 2.105 ml/kg body weight when corrected for the density (0.95 g/ml). Food was withheld overnight prior to dosing until approximately 3–4 h after administration. The body weights of the females at day 1 was 193–234 g. Body weights were determined on days 1, 8 and 15. Possible clinical signs occurring were recorded at periodic intervals on the day of dosing (day 1) and once daily thereafter until day 15. On day 15, the animals were sacrificed by oxygen/carbon dioxide procedure and subjected to necropsy. Macroscopic examination was performed after terminal sacrifice. The following organs were examined: lungs, heart, thyroid, thymus, spleen, pancreas, stomach, trachea, duodenum, jejunum, ileum, cecum, colon, liver, kidneys, adrenal glands, urinary bladder, urogenital tract, ovaria, and uterus. 2.6.2. 28-Day dietary toxicity study The 28-day dietary toxicity study in rats was performed as a GLP study based on the guidelines mentioned in OECD 407 and EC council directive 67/548/EEC. Three male and three female Wistar Crl:WI rats (Charles River, Sulzfeld, Germany) of approximately 6 weeks old per sex per group were exposed to 0, 10,000, 50,000 and 150,000 ppm PSO via dietary administration. The diet (semi-synthetic pelleted
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laboratory animal diet using AIN 93G as basal diet) containing 10,000, 50,000 and 150,000 ppm PSO was prepared by SSNIFF Spezialdiäten GmbH, Soest, Germany. Table 2 presents a detailed composition of the delivered diets. The composition of the diets was such that imbalances in nutritional or energy status due to the high fat content of the diets were accounted for. The highest dose of 150,000 ppm was selected since it was anticipated that higher doses would result in non-specific intestinal discomfort due to the high fat content of the diet. The control group received a diet with 150,000 ppm safflower oil as control oil. In the other dose groups, the safflower oil was (partly) replaced by PSO at the required concentration. The crude fat content of the diets was kept constant across all dose groups. The diet was provided ad libitum. Diets in the food hoppers were replaced with fresh diet twice weekly. Prepared diets were delivered to our laboratory once for the whole study in sealed and labeled vacuum-bags containing approximately 650 g of diet. Upon receipt, the bags were stored in the refrigerator (6 15 °C). Each diet bag contained an oxygen absorber. Diets were administered as delivered and were allowed to acclimatize by storing the required bags at room temperature for at least 1 h before use. The stability of the diet preparations was determined after storage of the diet at room temperature for 5, 8 and 12 days and after storage of the diet in the refrigerator for 14 and 28 days, and the ingredients in the diet were determined to be stable. The general physical condition of each animal was monitored at least twice daily during the 28-day study. Detailed clinical observations were performed for each individual animal once daily. The body weight of the animals was measured weekly, and the food consumption twice per week. On day 28, blood samples were collected from the retro-orbital sinus under isoflurane anaesthesia (minimally 3%). The following hematological parameters were determined: leukocytes, erythrocytes, differential leukocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), reticulocytes, hemoglobin, hematocrit, platelets, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), prothrombin time (PT), and activated partial thromboplastin time (APTT). The plasma from each animal was analyzed for the following clinical biochemistry parameters: alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium and inorganic phosphate. On day 28, the animals were anaesthetized using isoflurane inhalation (Abott Laboratories Ltd., Zwolle) and subsequently exsanguinated. The following organ weights (and terminal body weight) were recorded: adrenal glands, heart, kidneys, liver, spleen, testes and thymus.
3. Results
The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant increase in the number of revertants was observed. PSO did not induce an increase in the number of revertant colonies in all strains tested both in the absence and presence of metabolic activation (Table 3). These results were confirmed in an independently repeated experiment (Table 4). Therefore, it was concluded that PSO is not mutagenic in the Ames test. 3.1.2. In vitro chromosomal aberration test PSO precipitated in the culture medium at a concentration of 333 lg/ml, therefore concentrations above 333 lg/ml were not tested for chromosome aberrations. The concentrations tested for chromosome aberrations (33, 100 and 333 lg/ml) showed no significant toxicity. PSO did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in two independently repeated experiments (Tables 5 and 6). In addition, no effects of PSO on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of metabolic activation. It can therefore be concluded that PSO is not clastogenic and also does not induce numerical chromosomal aberrations. 3.2. In vivo toxicology tests 3.2.1. Acute oral toxicity in rats No mortality occurred at the dose level of 2000 mg/kg body weight. Between days 1 (dosing day) and 2 all animals showed a hunched posture. The body weight gain shown by the animals over the study period was considered to be normal. In addition, no abnormalities were found at macroscopic post mortem examination of the animals. Based on these results, the minimum oral lethal dose of PSO in rats was established to exceed 2000 mg/kg body weight.
3.1. In vitro mutagenicity tests 3.1.1. Ames test PSO precipitated on the plates at concentrations of 3330 and 5000 lg/plate.
3.2.2. 28-Day dietary toxicity study Dietary administration of PSO at 0, 10,000, 50,000 and 150,000 ppm produced no clinical signs, no adverse effects or mortality. Slightly lower body weights and a slightly lower body
Table 3 Response of PSO in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (first experiment). Results represent mean number of revertant colonies/3 replicate plates ± standard deviation. Concentration (lg/plate)
TA1535
TA1537
Without metabolic activation Solvent control 3 10 33 100 333 1000 3330SP 5000SP Positive control
21 ± 4 –a – 16 ± 4 18 ± 4 15 ± 1 13 ± 3 13 ± 3 – 1092 ± 15
5±3 – – 8±5 5±2 5±2 4±1 6±1 – 249 ± 33
13 ± 4 – – 15 ± 5 11 ± 1 12 ± 5 16 ± 2 12 ± 2 – 325 ± 12
6±1 – – 5±2 7±2 6±4 6±1 6±2 – 364 ± 55
With metabolic activation (5% S9-mix) Solvent control 3 10 33 100 333 1000 3330SP 5000SP Positive control SP
Slight precipitate. a Not determined.
TA98
TA100
WP2uvrA
15 ± 5 – – 15 ± 2 16 ± 2 16 ± 3 15 ± 2 19 ± 4 – 979 ± 85
100 ± 3 106 ± 8 107 ± 15 113 ± 4 100 ± 8 109 ± 3 115 ± 2 110 ± 4 118 ± 7 1161 ± 26
11 ± 1 7±2 8±2 9±5 8±2 9±2 10 ± 2 9±2 11 ± 6 916 ± 49
24 ± 2 – – 22 ± 4 17 ± 5 21 ± 5 23 ± 4 23 ± 2 – 1506 ± 159
135 ± 21 131 ± 31 123 ± 9 119 ± 8 123 ± 14 125 ± 5 128 ± 18 116 ± 6 124 ± 6 1832 ± 45
13 ± 6 13 ± 6 9±2 10 ± 2 10 ± 3 12 ± 1 12 ± 4 13 ± 3 11 ± 3 378 ± 37
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Table 4 Response of PSO in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (second experiment). Results represent mean number of revertant colonies/3 replicate plates ± standard deviation. Concentration (lg/plate)
TA1535
TA1537
TA98
TA100
WP2uvrA
Without metabolic activation Solvent control 33 100 333 1000 3330SP Positive control
16 ± 4 21 ± 7 20 ± 6 18 ± 4 20 ± 1 20 ± 5 1067 ± 7
5±1 5±0 7±3 5±2 4±1 3±1 287 ± 55
18 ± 6 15 ± 2 20 ± 2 14 ± 6 17 ± 5 15 ± 3 1099 ± 60
125 ± 15 117 ± 15 128 ± 7 122 ± 10 126 ± 17 98 ± 9 1277 ± 24
30 ± 7 29 ± 1 27 ± 9 27 ± 8 29 ± 6 28 ± 2 1159 ± 15
120 ± 8 123 ± 6 124 ± 21 120 ± 4 135 ± 6 116 ± 8 1342 ± 53
26 ± 8 24 ± 5 26 ± 3 25 ± 4 30 ± 5 27 ± 3 324 ± 29
With metabolic activation (10% S9-mix) Solvent control 33 100 333 1000 3330SP Positive control
19 ± 4 18 ± 4 21 ± 1 18 ± 4 19 ± 6 18 ± 5 224 ± 25
6±2 6±0 6±3 5±2 5±3 8±1 407 ± 6
20 ± 5 26 ± 7 24 ± 2 18 ± 2 28 ± 2 26 ± 5 779 ± 136
SP
Slight precipitate.
Table 5 Chromosome aberrations in donor cultures treated with PSO in the absence of metabolic activation at different exposure and fixation times. Concentration (lg/ml)
Mitotic index (%)
No of cells with structural chromosome aberrations per 200 metaphasesa Gap
Chromatid type
Chromosome type
g
ctb
cte
csb
cse
Others
Total +g
g
Without metabolic activation, 3 h exposure time, 24 h fixation timeb Solvent controlc 100 3 33 86 0 100 84 1 d 89 0 333 MMC 81 2
0 0 0 0 6
0 0 0 0 0
2 0 0 0 15
0 0 0 0 29
0 0 0 0 0
5 0 1 0 50***
2 0 0 0 49***
Without metabolic activation, 24 h exposure time, 24 h fixation time Solvent control 100 0 33 104 0 100 95 1 70 2 333d MMC 41 2
0 3 2 0 17
0 0 0 0 0
0 0 0 0 2
0 0 0 0 21
0 0 0 0 0
0 3 3 2 37***
0 3 2 0 35***
Without metabolic activation, 48 h exposure time, 48 h fixation time Solvent control 100 0 33 90 0 100 76 0 70 0 333d MMC 47 2
0 1 1 3 29
0 0 0 0 0
0 0 0 0 11
0 0 0 0 25
0 0 0 0 2
0 1 1 3 60***
0 1 1 3 60***
Abbreviations: ctb, chromatid break; cte, chromatid exchange; csb, chromosome break; cse chromosome exchange, +g, including gaps; g, excluding gaps; MMC, mytomycin C. a In total 200 metaphases scored for each treatment, i.e. 100 metaphases scored from each duplicate culture. b Human lymphocytes treated with PSO or vehicle for 3 h without metabolic activation and then cultured in fresh medium for further 21 h. c DMSO, dimethyl sulfoxide. d PSO precipitated in the culture medium. *** Significantly different from solvent control (P < 0.001).
weight gain was observed in males at 10,000 and 150,000 ppm PSO in the diet. Since this difference occurred in the absence of a treatment-related distribution, these changes were considered to be of no toxicological relevance. Body weights and body weight gain of other treated groups remained in the same range as the controls. The total food consumption (in g/animal/day) and the relative food consumption (in g/kg body weight/day) showed no significant differences between animals from the different groups (data not shown). The mean intake of PSO, estimated based on the body weight and food consumption values, in the different animal groups was 0–0, 825–847, 4269–4330 and 13710–14214 mg PSO/kg body weight per day in males–females, respectively. No difference was observed in food intake of PSO between the two sexes.
The results of the hematological and clinical biochemistry are presented in Tables 7 and 8. A slightly increased white blood cell count was noted in males exposed to 150,000 ppm PSO and in one female exposed to 15% PSO. Increased relative neutrophil counts with concurrently reduced relative lymphocyte counts were noted in one control male and in all males exposed to 150,000 ppm PSO. Based on historical data this shift in type of white blood cells was considered to be a secondary non-specific response to stress and to be of no toxicological significance. The weight of the different organs and the organ to body weight ratios are presented in Table 9. The liver weight of females exposed to 150,000 ppm PSO and liver-to-body weight ratios of males and females exposed to 150,000 ppm PSO were slightly increased. Other organ weights and organ to body weight ratios were similar to control levels.
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Table 6 Chromosome aberrations in donor cultures treated with PSO in the presence of metabolic activation at different fixation times. Concentration (lg/ml)
Mitotic index (%)
No of cells with structural chromosome aberrations per 200 metaphasesa Gap
Chromatid type
Chromosome type
g
ctb
cte
csb
cse
Others
Total
With metabolic activation, 3 h exposure time, 24 h fixation timeb Solvent controlc 100 0 33 96 0 100 104 0 109 0 333d CP 23 6
0 0 0 0 27
0 0 0 0 0
0 0 0 0 12
0 0 0 0 11
0 0 0 0 1
0 0 0 0 50***
0 0 0 0 47***
With metabolic activation, 3 h exposure time, 48 h fixation timeb Solvent control 100 0 33 91 0 100 84 1 80 1 333d 2 CP –e
1 0 1 3 24
0 0 0 0 0
0 0 0 0 4
0 0 0 0 13
0 0 1 0 1
1 0 2 4 40***
1 0 1 3 38***
+g
g
Abbreviations: ctb, chromatid break; cte, chromatid exchange; csb, chromosome break; cse chromosome exchange, +g, including gaps; g, excluding gaps; CP, cyclophosphamide. a In total 200 metaphases scored for each treatment, i.e. 100 metaphases scored from each duplicate culture. b Human lymphocytes treated with PSO or vehicle for 3 h with metabolic activation and then cultured in fresh medium for further 21 or 45 h. c DMSO, dimethyl sulfoxide. d PSO precipitated in the culture medium. e CP was fixed after 24 h, therefore the mitotic index could not be calculated as percentage of control. *** Significantly different from solvent control (P < 0.001).
Table 7 Haematological findings in rats after 28-day dietary exposure to PSO (mean ± SD). Sex Males
WBC (109/l) Neutrophils (%WBC) Lymphocytes (%WBC) Monocytes (%WBC) Eosinophils (%WBC) Basophils (%WBC) RBC (1012/l) Reticulocytes (%RBC) RDW (%) Haemoglobin (mmol/l) Haematocrit (%) MCV (fL) MCH (fmol) MCHC (mmol/l) Platelets (109/l) PT (s) APTT (s)
Females
Control
10,000 ppm PSO
50,000 ppm PSO
150,000 ppm PSO
Control
10,000 ppm PSO
50,000 ppm PSO
150,000 ppm PSO
10.9 ± 1.6 16.2 ± 4.2 79.0 ± 4.5 3.0 ± 0.5 1.4 ± 0.2 0.5 ± 0.1 7.90 ± 0.51 3.1 ± 0.7 12.5 ± 0.3 9.4 ± 0.4 45.0 ± 2.0 57.0 ± 1.5 1.20 ± 0.03 21.01 ± 0.27 1035 ± 150 17.6 ± 0.6 19.1 ± 0.6
11.3 ± 2.1 12.4 ± 1.1 83.8 ± 1.3 2.3 ± 0.5 1.1 ± 0.5 0.4 ± 0.2 8.27 ± 0.28 2.5 ± 0.4 12.8 ± 0.4 9.4 ± 0.2 44.3 ± 1.0 53.6 ± 0.8 1.14 ± 0.01 21.24 ± 0.13 1155 ± 242 17.4 ± 1.0 16.0 ± 3.2
12.2 ± 1.1 12.3 ± 1.2 82.8 ± 0.6 3.2 ± 0.7 1.2 ± 0.1 0.5 ± 0.1 7.92 ± 0.47 3.1 ± 0.4 13.0 ± 0.4 9.2 ± 0.3 44.1 ± 1.0 55.8 ± 2.7 1.17 ± 0.06 20.88 ± 0.19 1167 ± 43 16.8 ± 0.8 17.2 ± 3.4
15.0 ± 1.4 24.2 ± 4.3 70.5 ± 3.3 3.7 ± 1.2 1.2 ± 0.5 0.5 ± 0.1 8.49 ± 0.48 2.9 ± 0.5 12.7 ± 0.4 9.6 ± 0.2 46.2 ± 1.5 54.5 ± 1.5 1.14 ± 0.05 20.85 ± 0.29 1013 ± 174 16.5 ± 0.5 17.6 ± 3.0
5.4 ± 0.7 12.7 ± 5.1 83.8 ± 6.0 2.0 ± 0.4 1.3 ± 0.4 0.4 ± 0.1 7.77 ± 0.07 2.7 ± 0.3 12.1 ± 0.2 9.1 ± 0.4 42.6 ± 1.6 54.8 ± 2.6 1.18 ± 0.06 21.42 ± 0.06 1136 ± 114 16.5 ± 0.1 18.0 ± 3.5
6.3 ± 2.3 11.2 ± 5.3 84.6 ± 6.9 2.3 ± 1.5 1.6 ± 0.4 0.3 ± 0.3 7.65 ± 0.27 3.0 ± 0.5 12.0 ± 0.8 9.2 ± 0.6 42.3 ± 1.9 55.4 ± 2.9 1.21 ± 0.07 21.86 ± 0.44 907 ± 395 17.0 ± 1.0 14.9 ± 2.6
4.8 ± 1.5 12.8 ± 3.4 83.6 ± 3.7 1.9 ± 0.3 1.4 ± 0.4 0.3 ± 0.1 8.05 ± 0.20 2.4 ± 0.1 12.2 ± 0.6 9.2 ± 0.3 42.6 ± 2.2 52.8 ± 1.4 1.15 ± 0.02 21.69 ± 0.31 1203 ± 80 16.7 ± 0.6 15.4 ± 2.1
9.1 ± 6.5 10.8 ± 5.4 85.3 ± 6.6 2.4 ± 0.9 1.2 ± 0.6 0.4 ± 0.1 7.73 ± 0.11 1.8 ± 0.3 11.6 ± 0.5 9.2 ± 0.3 41.8 ± 1.9 54.1 ± 1.8 1.18 ± 0.03 21.92 ± 0.19 1016 ± 165 16.3 ± 0.7 17.8 ± 3.2
WBC, white blood cells; RBC, red blood cells; red blood cell distribution width; MCV, mean corpuscular volume; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration; PT, prothrombin time; APTT, activated partial thromboplastin time.
4. Discussion The current paper is the first paper focusing on safety assessment of pomegranate seed oil (PSO). The possible mutagenicity and toxicity of PSO was evaluated using the in vitro Ames and in vitro chromosomal aberration test (in cultured peripheral human lymphocytes), an in vivo acute oral toxicity and an in vivo 28-day dietary toxicity study. The results of the mutagenicity studies reveal that PSO is neither mutagenic nor clastogenic, both in the absence or presence of metabolic activation. In the oral acute toxicity study, no abnormalities were observed at macroscopic post mortem examination of the animals, and no effects on body weight or body weight gain were observed at a concentration of 2000 mg/kg body weight. According to the OECD 423 test guideline, the LD50 cut-off value can then be considered to
exceed 5000 mg/kg body weight, and no classification or labeling for oral toxicity is required for PSO. In the 28-day dietary toxicity study no effects were observed at 10,000 ppm and 50,000 ppm PSO in the diet. The mean PSO intake over the treatment period at these dietary levels was 825–847 and 4269–4330 (male–female animals) mg/kg body weight per day. Thus it can be concluded that 4.3 g of PSO per kg body weight per day is the no observed adverse effect level in this study. At higher concentrations, e.g. 150,000 ppm PSO in the diet (corresponding to 13,710 and 14,214 mg/kg body weight/day in males and female rats, respectively), mainly effects on hepatic enzyme activities are observed and a higher liver weight and liver-to-body weight ratio, indicating that the liver might be a target organ. However, the effects observed on liver weight and liver-to-body weight ratio at the highest exposure group (150,000 ppm PSO) might be the result of a physiological response to exposure to a very high le-
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ALAT (U/l) ASAT (U/l) ALP (U/l) Total protein (g/l) Albumin (g/l) Total bilirubin (lmol/l) Urea (mmol/l) Creatinine (lmol/l) Glucose (mmol/l) Cholesterol (mmol/l) Sodium (mmol/l) Potassium (mmol/l) Chloride (mmol/l) Calcium (mmol/l) Inorganic phosphate (mmol/ l)
Females
Control
10,000 ppm PSO
50,000 ppm PSO
150,000 ppm PSO
Control
10,000 ppm PSO
50,000 ppm PSO
150,000 ppm PSO
27.1 ± 7.2 72.0 ± 13.3 251 ± 140 68.0 ± 4.6 34.4 ± 1.8 2.7 ± 0.3 6.1 ± 0.8 37.6 ± 1.6 7.76 ± 1.30 2.36 ± 0.23 142.3 ± 1.3 3.70 ± 0.14 102 ± 1 2.91 ± 0.08 2.59 ± 0.17
33.0 ± 4.4 73.6 ± 11.8 231 ± 61 68.0 ± 0.8 34.5 ± 0.9 2.4 ± 0.2 5.2 ± 0.6 38.0 ± 1.1 7.72 ± 0.91 1.62 ± 0.16 142.8 ± 0.7 3.62 ± 0.24 105 ± 1 2.92 ± 0.05 2.58 ± 0.17
34.6 ± 7.5 92.0 ± 15.2 241 ± 55 66.1 ± 3.1 33.0 ± 0.4 2.4 ± 0.2 5.5 ± 1.3 39.0 ± 1.1 7.77 ± 0.70 1.77 ± 0.44 141.9 ± 1.2 3.62 ± 0.07 103 ± 0 2.86 ± 0.07 2.75 ± 0.20
106.1 ± 14.0 430.7 ± 118.7 651 ± 92 69.1 ± 2.9 36.3 ± 0.5 2.7 ± 0.1 7.2 ± 0.1 41.4 ± 2.2 9.37 ± 2.78 1.58 ± 0.22 141.5 ± 0.1 3.75 ± 0.19 101 ± 1 2.91 ± 0.07 2.97 ± 0.10
32.4 ± 7.9 71.4 ± 1.0 94 ± 20 69.4 ± 1.5 35.9 ± 1.9 3.4 ± 0.4 5.0 ± 0.1 41.1 ± 1.8 6.74 ± 1.00 1.44 ± 0.33 140.4 ± 1.1 3.25 ± 0.17 103 ± 2 2.83 ± 0.07 1.98 ± 0.19
29.1 ± 2.1 99.2 ± 31.2 122 ± 13 65.2 ± 3.5 35.3 ± 0.7 3.2 ± 0.6 6.1 ± 1.0 45.3 ± 1.8 7.96 ± 0.87 1.28 ± 0.31 142.4 ± 1.3 3.33 ± 0.14 105 ± 3 2.76 ± 0.06 2.19 ± 0.03
29.7 ± 4.5 80.0 ± 6.3 123 ± 35 67.5 ± 3.7 34.8 ± 2.1 2.5 ± 0.5 6.9 ± 0.8 42.1 ± 4.6 8.27 ± 0.95 1.48 ± 0.11 141.8 ± 0.4 3.53 ± 0.48 105 ± 2 2.78 ± 0.04 2.32 ± 0.21
42.7 ± 7.1 96.3 ± 36.6 291 ± 70 71.5 ± 2.8 38.5 ± 1.5 3.0 ± 0.2 7.0 ± 1.9 44.2 ± 2.8 9.12 ± 1.37 1.31 ± 0.44 141.0 ± 0.6 3.37 ± 0.15 104 ± 1 2.78 ± 0.07 2.19 ± 0.31
ALAT, alanine aminotransferase; ASAT, aspartate aminotransferase; ALP, alkaline phosphatase.
Table 9 Organ weights of male and female rats after 28-day dietary exposure to PSO (mean ± SD). Sex Males Control
Females 10,000 ppm PSO
50,000 ppm PSO
150,000 ppm PSO
Control
10,000 ppm PSO
50,000 ppm PSO
150,000 ppm PSO
Organ weights (g) Body weight 377 ± 23 Heart 1.330 ± 0.136 Liver 11.28 ± 0.96 Thymus 0.638 ± 0.238 Kidneys 3.05 ± 0.16 Adrenals 0.077 ± 0.018 Spleen 0.925 ± 0.186 Testes 3.65 ± 0.20
332 ± 11 1.104 ± 0.064 9.51 ± 0.48 0.547 ± 0.072 2.43 ± 0.31 0.068 ± 0.004 0.907 ± 0.082 3.43 ± 0.09
370 ± 29 1.175 ± 0.132 10.66 ± 0.92 0.625 ± 0.273 2.57 ± 0.24 0.072 ± 0.006 0.960 ± 0.170 3.66 ± 0.24
324 ± 33 1.263 ± 0.180 11.58 ± 1.66 0.493 ± 0.120 2.52 ± 0.37 0.063 ± 0.012 0.954 ± 0.127 3.35 ± 0.29
236 ± 5 0.835 ± 0.026 6.82 ± 0.39 0.410 ± 0.025 1.71 ± 0.08 0.079 ± 0.009 0.589 ± 0.047 n.a.
232 ± 13 0.926 ± 0.119 6.46 ± 0.09 0.424 ± 0.110 1.68 ± 0.07 0.084 ± 0.002 0.673 ± 0.034 n.a.
232 ± 14 0.826 ± 0.011 6.92 ± 0.02 0.432 ± 0.039 1.77 ± 0.11 0.087 ± 0.004 0.585 ± 0.052 n.a.
219 ± 18 0.885 ± 0.047 8.11 ± 1.01 0.378 ± 0.119 1.79 ± 0.11 0.076 ± 0.004 0.675 ± 0.028 n.a.
Organ to body weight ratio (%) Heart 0.352 ± 0.023 Liver 2.99 ± 0.08 Thymus 0.167 ± 0.056 Kidneys 0.81 ± 0.04 Adrenals 0.020 ± 0.004 Spleen 0.245 ± 0.044 Testes 0.97 ± 0.07
0.333 ± 0.022 2.87 ± 0.14 0.165 ± 0.022 0.73 ± 0.07 0.020 ± 0.002 0.274 ± 0.034 1.03 ± 0.04
0.319 ± 0.044 2.88 ± 0.06 0.166 ± 0.059 0.69 ± 0.01 0.020 ± 0.001 0.258 ± 0.027 0.99 ± 0.05
0.390 ± 0.035 3.57 ± 0.30 0.153 ± 0.039 0.78 ± 0.07 0.019 ± 0.002 0.294 ± 0.022 1.04 ± 0.02
0.354 ± 0.004 2.89 ± 0.12 0.174 ± 0.008 0.72 ± 0.03 0.033 ± 0.003 0.250 ± 0.017 n.a.
0.401 ± 0.067 2.78 ± 0.14 0.183 ± 0.046 0.72 ± 0.04 0.036 ± 0.002 0.291 ± 0.031 n.a.
0.356 ± 0.017 2.99 ± 0.17 0.186 ± 0.019 0.76 ± 0.06 0.038 ± 0.004 0.252 ± 0.011 n.a.
0.405 ± 0.030 3.69 ± 0.16 0.171 ± 0.046 0.82 ± 0.09 0.035 ± 0.004 0.308 ± 0.018 n.a.
vel of a fatty acid which is not part of the normal diet, and are most likely adaptive and nature and not toxicologically relevant. Conflict of interest statement I.A.T.M. Meerts, C.M. Verspeek-Rip, A.H.B.M. van Huygevoort, F.M. van Otterdijk declare that there are no conflicts of interest. Funding source: nil. C.A.F. Buskens declare that there are no conflicts of interest. Funding source: The study sponsor (Lipid Nutrition) was involved in the design of the protocol. However they had no influence on data collection, analysis and interpretation of the data. Lipid Nutrition asked NOTOX to write the manuscript and submit it for publication. E.J. van de Waart declares that there are no conflicts of interest. Funding source: nil. Z.E. Jouni, a co-author, is employed by Mead Johnson Nutritionals. Mead Johnson is collaborating with Lipid Nutrition in its evaluation of the safety and efficacy of pomegranate seed oil. Funding source: Mead Johnson had no influence on design of study, data collection, analyses and interpretation of the data.
H.G. Keizer, a co-author, is employed by Lipid Nutrition, the sponsor of this study. This company investigates the safety and efficacy of pomegranate seed oil in various indications. If safety is proven and efficacy is shown, pomegranate seed oil may become a market product. Funding source: The study sponsor (Lipid Nutrition) was involved in the design of the protocol. However they had no influence on data collection, analyses and interpretation of the data. The study sponsors asked NOTOX to write the manuscript and submit it for publication. J. Bassaganya-Riera is an inventor in a patent that covers the use of pomegranate seed oil as a nutritional supplement and functional food ingredient for the prevention and treatment of chronic diseases. Funding source: nil. References El-Shaarawy, M.I., Nahapetian, A., 1983. Studies on pomegranate seed oil. Fette. Seife. Anstrichmittel 85 (3), 123–126. European Community (EC), Council Directive 67/548/EEC, Annex V, Part B, 2004. Methods for Determination of Toxicity, as last amended by Commission Directive 2004/73/EC, B1 bis: Acute toxicity-oral, Fixed Dose Method.
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European Community (EC), Council Directive 67/548/EEC, Annex V, Part B, 1996. Methods for Determination of Toxicity and other Health Effects; B7, ‘‘Repeated Dose (28 days) Toxicity (oral)”. Official Journal of the European Communities No. L248. Fadavi, A., Barzegar, M., Azizi, M.H., 2006. Determination of fatty acids and total lipid content in oilseed of 25 pomegranates varieties grown in Iran. J. Food Comp. Anal. 19, 676–680. Gil, M.I., Tomas-Barberan, F.A., Hess-Pierce, B., Holcroft, D.M., Kader, A.A., 2000. Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing. J. Agr. Food Chem. 48, 4581–4589. Hora, J.J., Maydew, E.R., Lansky, E.P., Dwivedi, C., 2003. Chemopreventive effects of pomegranate seed oil on skin tumor development in CD1 mice. J. Med. Food 6 (3), 157–161. Kohno, H., Suzuki, R., Yasui, Y., Hosokawa, M., Miyashita, K., Tanaka, T., 2004. Pomegranate seed oil rich in conjugated linolenic acid suppresses chemically induced colon carcinogenesis in rats. Cancer Sci. 95, 481–486. Lansky, E.P., Newman, R.A., 2007. Punica granatum (pomegranate) and its potential for prevention and treatment of inflammation and cancer. J. Ethnopharmacol. 109, 177–206. Levin, G.M., 1994. Pomegranate (Punica granatum) plant genetic resources in Turkmenistan. Plant Gen. Resour. Newslett. 97, 31–37.
Mehta, R., Lansky, E.P., 2004. Breast cancer chemopreventive properties of pomegranate (Punica granatum) fruit extracts in a mouse mammary organ culture. Eur. J. Cancer Prevent. 13, 345–348. Organisation for Economic Co-operation and Development (OECD), 1995. OECD Guidelines for Testing of Chemicals, Section 4, Health Effects, No. 407, ‘‘Repeated Dose 28-day Oral Toxicity Study in Rodents”, Paris Cedex. Organisation for Economic Co-operation and Development (OECD), 2001. OECD Guidelines for Testing of Chemicals, Section 4, Health Effects, No. 420, ‘‘Acute Oral Toxicity – Fixed Dose Method”, Paris, Cedex. Omura, T., Sato, R., 1964. The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem. 239, 2370– 2378. Ozgul-Yucel, S., 2005. Determination of conjugated linolenic acid content of selected oil seeds grown in Turkey. JAOCS 82 (12), 893–897. Schubert, S.Y., Lansky, E.P., Neeman, I., 1999. Antioxidant and eicosanoid enzyme inhibition properties of pomegranate seed oil and fermented juice flavonoids. J. Ethnopharmacol. 66, 11–17. Singh, R.P., Chidambara Murthy, K.N., Jayaprakasha, G.K., 2002. Studies on the antioxidant activity of pomegranate (Punica granatum) peel and seed extracts using in vitro models. J. Agr. Food Chem. 50, 81–86.
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Toxicological evaluation of pomegranate seed oil I.A.T.M. Meerts a,*, C.M. Verspeek-Rip a, C.A.F. Buskens a, H.G. Keizer b, J. Bassaganya-Riera c, Z.E. Jouni d, A.H.B.M. van Huygevoort e, F.M. van Otterdijk e, E.J. van de Waart a a
Department of In vitro and Environmental Toxicology, NOTOX B.V., Hambakenwetering 7, 5231 DD ‘s-Hertogenbosch, The Netherlands Lipid Nutrition B.V., Hogeweg 1, 1520 AZ Wormerveer, The Netherlands c Nutrition Therapeutics Inc., Blacksburg, VA 24060, USA d Mead Johnson Nutritionals, Evansville, IN, USA e Department of Toxicology, NOTOX B.V., ’s-Hertogenbosch, The Netherlands b
a r t i c l e
i n f o
Article history: Received 8 August 2008 Accepted 27 January 2009
Keywords: Pomegranate seed oil Acute oral toxicity Mutagenicity 28-day toxicity
a b s t r a c t In this manuscript, the toxicology and safety of pomegranate seed oil (PSO) was evaluated by in vitro (Ames, chromosomal aberration), and in vivo toxicity tests (acute toxicity and 28-day toxicity in Wistar rats). No mutagenicity of PSO was observed in the absence and presence of metabolic activation up to precipitating concentrations of 5000 lg/plate (Ames test) or 333 lg/ml (chromosome aberration test). The acute oral toxicity study revealed no significant findings at 2000 mg PSO/kg body weight. In the 28day dietary toxicity study PSO was dosed at concentrations of 0, 10,000, 50,000 and 150,000 ppm, which resulted in a mean intake of 0–0, 825–847, 4269–4330 and 13,710–14,214 mg PSO/kg body weight per day in males–females, respectively. At 150,000 ppm dietary exposure to PSO, a much higher dose than the level of PSO that elicits antidiabetic and anti-inflammatory efficacy, increased hepatic enzyme activities determined in plasma (aspartate, alanine aminotransferase and alkaline phosphatase) and increased liver-to-body weight ratios were observed. However, these effects might be the result of a physiological response to exposure to a very high level of a fatty acid which is not part of the normal diet, and are most likely not toxicologically relevant. The no observable adverse effect level (NOAEL) was 50,000 ppm PSO (=4.3 g PSO/kg body weight/day). Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction The pomegranate (Punica granatum) is an ancient fruit which has been widely consumed in many different cultures for thousands of years, largely without incidents, and thus is considered generally safe by the general public. It belongs to the family Punicaceae which grows only in Socotra, an island off the Yemeni coast (Lansky and Newman, 2007). The pomegranate is native from Iran to the Himalayas in northern India, and has been cultivated and naturalized over the whole Mediterranean region since ancient times. Over 1000 cultivars of P. granatum exist (Levin, 1994). It is widely cultivated throughout Iran, India, the drier parts of Southeast Asia, Malaysia, the East Indies, and tropical Africa. Pomegranate is now cultivated mainly in the drier parts of California and Abbreviations: 2-AA, 2-aminoanthracene; CLNA, conjugated linolenic acids; DMSO, dimethyl sulfoxide; EEC, European Economic Committee; GLP, Good Laboratory Practice; MMS, methylmethanesulfonate; 4-NQO, 4-nitroquinoline N-oxide; OECD, Organisation for Economic Co-operation and Development; PSO, pomegranate seed oil. * Corresponding author. Tel.: +31 (0)73 6406700; fax: +31 (0)73 6406799. E-mail address: [email protected] (I.A.T.M. Meerts). 0278-6915/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2009.01.031
Arizona for its fruits exploited commercially as juice products gaining in popularity since 2001, especially because of the appearance of several reports presenting the antioxidant activities of pomegranate fractions in vitro (Schubert et al., 1999; Gil et al., 2000; Singh et al., 2002). Pomegranate seed oil (PSO) comprises 12–20% of total seed weight. The oil is characterized by a high content of conjugated linolenic acids (CLNA, about 31.8–86.6%), followed by linoleic acid (0.7–24.4%), oleic acid (0.4–17.7%), stearic acid (2.8–16.7%) and palmitic acid (0.3–9.9%) (El-Shaarawy and Nahapetian, 1983; Ozgul-Yucel, 2005; Fadavi et al., 2006). Variability in concentration is due to differences between cultivars. The CLNA found in PSO are all 9, 11, 13 isomers, with punicic acid (9 cis, 11 trans, 13 cis) as the predominant conjugated triene (Ozgul-Yucel, 2005). Coldpressed PSO has been commercially available during the last 5– 6 years. The pomegranate seeds are cleaned and pressed giving virgin oil which is further purified by centrifugation, sedimentation or filtration. The cold-pressed oil obtained can then be used for skin care recipes or food use as a dietary supplement. Although pomegranate has been widely consumed for thousands of years, little is known about the possible toxicity and safety
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of the pomegranate or the PSO. Several studies indicate that coldpressed PSO is able to reduce tumor occurrence, tumor incidence and multiplicity in both ex vivo models and in vivo in mice and rats. In an ex vivo model using mouse mammary organ culture (Mehta and Lansky, 2004), cold-pressed PSO reduced the tumor occurrence up to 87%. In an in vivo study with female CD-1 mice with skin tumors induced by topical application of 7,12-dimetylbenz[a]anthracene and promoted by 12-O-tetradecanoylphorbol 13-acetate, PSO applied topically (5%) resulted in significant decreases in tumor incidence and multiplicity (Hora et al., 2003). In male F344 rats treated with the carcinogen azoxymethane, dietary PSO at concentrations of 0.1% already reduced the incidence and multiplicity of colon tumors (Kohno et al., 2004). However, besides several very intriguing and promising studies on the possible beneficial effects of PSO, little is known about the possible toxicity and safety of pomegranate fruits or PSO, and standard in vitro and in vivo toxicology studies have not been performed with PSO. Therefore, in the current investigations we studied the possible mutagenicity of PSO using two in vitro mutagenicity studies (the Ames test and chromosomal aberration test), and the possible in vivo toxicity of PSO using Wistar rats (acute oral toxicity study and 28-day toxicity test). 2. Materials and methods
and a relative humidity of 30–70%. The rooms were illuminated with 12 h artificial fluorescent light and 12 h darkness per day. Unless otherwise stated, the animals were provided a standard pelleted laboratory animal diet (SM R/M-Z from SSNIFFÒ Spezialdiäten GmbH, Soest, Germany) that met or exceeded all nutritional needs and tap water ad libitum. The animals were allowed to acclimatize for at least 5 days before the start of the treatment. Results of analysis for ingredients and/or contaminants of diet, bedding, paper and water were assessed and did not reveal any findings that were considered to have affected the study integrity. 2.4. Preparation of rat liver S9-fraction for in vitro mutagenicity studies Rat liver S9 was prepared from young adult male Wistar rats (8 weeks of age), which were obtained from Charles River (Sulzfeld, Germany). Five male rats per S9batch were group-housed in labeled polycarbonate cages (type MIV height: 18 cm) containing Woody Clean bedding (Woody-Clean type 3/4; Technilab-BMI BV, Someren, The Netherlands) and paper as cage-enrichment (Technilab-BMI BV). The rats were orally dosed for three consecutive days with a suspension of phenobarbital (80 mg/kg body weight) and b-naphthoflavone (100 mg/kg body weight) in corn oil. The rats were denied access to food for 3–4 h preceding each dosing. One day after the final exposure, the rats were sedated using oxygen/carbon dioxide and sacrificed by decapitation. The rats received a limited quantity of food during the night before sacrifice. The livers of the rats were removed aseptically, and washed in cold (0 °C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA (Merck, Germany). Subsequently the livers were minced in a blender and homogenized in 3 volumes of 0.1 M sodium phosphate buffer (pH 7.4) with a Potter-Elvehjem tube and Teflon pestle. The homogenate was centrifuged for 15 min at 9000g (4 °C). The resulting supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen ( 196 °C) for a maximum of 1 year. All batches were checked for sterility and the concentration of cytochrome P450 was calculated from the CO-reduced difference spectrum according to Omura and Sato (1964).
2.1. Pomegranate seed oil (PSO) Cold-pressed PSO was obtained from Lipid Nutrition, Wormerveer, The Netherlands. The composition of the PSO was determined using GC analysis and is presented in Table 1. 2.2. Chemicals and bacterial strains The Salmonella typhimurium strains TA1535, TA1537 and TA98 were obtained from Dr. B.N. Ames (University of California, Berkeley, USA) and the strain TA100 was obtained from Xenometric (Boulder, Co, USA). The Escherichia coli strain was obtained from Prof. Dr. B.A. Bridges (University of Sussex, Brighton, UK). Positive controls used in the Ames test without metabolic activation were sodium azide (TA1535), 9-aminoacridine (TA1537), 2-nitrofluorene (TA98), methylmethanesulfonate (TA100) and 4-nitroquinoline N-oxide (WP2uvrA). In the presence of 5 and 10% S9-mix as metabolic activation, 2-aminoanthracene (2AA) was used as positive control. Sodium azide, methylmethanesulfonate, 4-nitroquinoline N-oxide, 2-aminoanthracene, b-naphthoflavone were obtained from Sigma, Zwijndrecht, The Netherlands. 9-Aminoacridine and colchicine were from Acros Organics, Geel, Belgium. Dimethyl sulfoxide, 2-nitrofluorene and Giemsa were obtained from Merck, Darmstadt, Germany.
2.5. In vitro mutagenicity tests 2.5.1. Ames test PSO was tested in the S. typhimurium reverse mutation assay with four histidine-requiring strains of S. typhimurium (TA1535, TA1537, TA98 and TA100) and in the E. coli reverse mutation assay with a tryptophan-requiring strain of E. coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of metabolic activation (phenobarbital- and b-naphthoflavone-induced rat liver S9-mix), and was performed under GLP conditions according to OECD and EEC guidelines (OECD 471 and 2000/32/EC). PSO was dissolved in dimethyl sulfoxide and tested in TA100 and WP2uvrA at concentrations of 0 (solvent control), 3, 10, 33, 100, 333, 1000, 3330 and 5000 lg/ plate in the absence and presence of 5% (v:v) S9-mix (first experiment). Since PSO precipitated on the plates at concentrations of 3330 lg/plate and 5000 lg/ plate, in the strains TA1535, TA1537 and TA98, PSO was tested at concentrations of 33, 100, 333, 1000 and 3330 lg/plate. An independent repeat assay was performed, in which PSO was tested at concentrations of 33, 100, 333, 1000 and 3330 lg/plate in all bacterial strains in the absence and presence of 10% (v:v) S9mix (second experiment). Appropriate negative and strain-specific positive controls were added.
2.3. Animal husbandry All procedures concerning the use of animals were approved by the Animal Experimental Committee of NOTOX. The animals were housed in a controlled environment, with approximately 15 air changes per hour, a temperature of 21 ± 3 °C Table 1 Chemical composition of the two PSO batches used. Batch Aa (%)
Batch Bb (%)
Triglycerides Diglycerides Monoglycerides Free fatty acids Polymers Total composition (%)
94 2.3 0.4 0.9 2.1 99.7
91 3.2 1.0 2.2 2.8 100.2
Fatty acid analysis (%) C16:0 stearic acid C18:0 palmitic acid C18:1 oleic acid C18:2 linoleic acid CLNA Total fatty acid analysis (%)
2.7 2.6 5.9 5.8 82.8 99.8
2.9 2.2 7 6.5 79.6 98.2
a Batch A is used for the two in vitro mutagenicity assays and the in vivo acute oral toxicity study. b Batch B is used for preparation of the diets for the 28-day dietary toxicity study.
2.5.2. In vitro chromosomal aberration test using cultured peripheral human lymphocytes A chromosomal aberration test using cultured peripheral human lymphocytes was performed under GLP conditions according to OECD and EEC guidelines (OECD 473 and 2000/32/EC). Two independent experiments were performed. In the first experiment, PSO was tested at concentrations of 33, 100 and 333 lg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v:v) S9-fraction. PSO precipitated in the culture medium at a concentration of 333 lg/ ml. In the second experiment, PSO was again tested at concentrations of 33, 100 and 333 lg/ml, with a change in incubation time and fixation time. In the absence of S9-mix, 24 h and 48 h continuous exposure times were used with a 24 and 48 h fixation time. In the presence of S9-mix, a 3 h exposure time and 48 h fixation time was used. As positive controls, mitomycin (Sigma, Zwijndrecht, The Netherlands) was used for incubations without metabolic activation, and cyclophosphamide (Endoxan-Asta, Asta-Werke, Germany) for incubations with metabolic activation. As negative control, dimethyl sulfoxide was used since this was the solvent used to dissolve PSO. During the last 2.5–3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 lg/ml medium). Thereafter, the cell cultures were centrifuged and the remaining pellet was treated for 5 min with hypotonic 0.56% (w:v) potassium chloride solution at 37 °C. Subsequently, the cells were fixed with 3 changes of methanol:acetic acid 3:1 (v:v). Fixed cells were dropped onto clean slides. Slides were allowed to dry and stained for 10–30 min with 5% (v:v) Giemsa solution in tap water. Thereafter, slides were rinsed in tap water, allowed to dry, embedded in MicroMount and mounted with a coverslip. All slides were randomly coded before examination of chromosome aberrations and scoring was performed in a blind fashion. Per culture, 100 metaphase chromo-
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10,000 ppm PSO
50,000 ppm PSO
150,000 ppm PSO
High fat control diet
Casein Corn starch mod. Maltodextrine Sucrose Cellulose L-Cystine AIN 93G-Vitamin premixa AIN 93G-Min./trace elem. premix Choline chloride Safflower oil PSO
22.00 28.59 13.20 10.00 5.60 0.33 1.10 3.90 0.28 14.00 1.00
22.00 28.59 13.20 10.00 5.60 0.33 1.10 3.90 0.28 10.00 5.00
22.00 28.59 13.20 10.00 5.60 0.33 1.10 3.90 0.28 – 15.00
22.00 28.59 13.20 10.00 5.60 0.33 1.10 3.90 0.28 15.00 –
Dry matter (%) Crude Protein (%) Crude Fat (%) Crude Fiber (%) Crude Ash (%) Starch (%) Sugar and dextrines (%) N free extracts (%) Calcium (%) Phosphorus (%) Sodium (%) Magnesium (%) Lysine (%) Methionine (%) Met + Cys (%) Vitamin A (IU/kg) Vitamin D3 (IU/kg) Vitamin E (IU/kg) Vitamin K3 (IU/kg) GE (MJ/kg) ME (pig) (MJ/kg) ME (Atwater) (MJ/kg) Protein (%) Fat (%) Carbohydrates (%) C14:0 (% in diet) C16:0 (% in diet) C18:0 (% in diet) C18:1 (% in diet) C18:2 (% in diet) C18:3 (% in diet) linolenic (n3) C18:3 (% in diet) punicic (n5) P C18:3 (% in diet) conjug.b C20:0 (% in diet) C20:1 (% in diet)
97.0 19.1 15.1 5.6 3.3 27.5 24.6 53.9 0.57 0.36 0.16 0.09 1.57 0.58 0.99 4,400 1,100 83 4.5 21.2 16.5 17.9 18 32 50 0.01 0.95 0.36 1.55 10.58 0.07 0.40 0.39 0.07 0.07
97.0 19.1 15.1 5.6 3.3 27.5 24.6 53.9 0.57 0.36 0.16 0.09 1.57 0.58 0.99 4,400 1,100 83 4.5 21.2 16.5 17.9 18 32 50 0.01 0.80 0.36 1.42 7.84 0.05 2.02 1.95 0.07 0.08
97.0 19.1 15.1 5.6 3.3 27.5 24.6 53.9 0.57 0.36 0.16 0.09 1.57 0.58 0.99 4,400 1,100 83 4.5 21.2 16.5 17.9 18 32 50 0.02 0.46 0.34 1.08 0.97 0.01 6.06 5.86 0.06 0.09
97.0 19.1 15.1 5.6 3.3 27.5 24.6 53.9 0.57 0.36 0.16 0.09 1.57 0.58 0.99 4,400 1,100 83 4.5 21.2 16.5 17.9 18 32 50 0.01 0.98 0.36 1.58 11.27 0.07 – – 0.07 0.07
PSO: pomegranate seed oil, GE = gross energy, ME = metabolizable energy. Fatty acid (FA) composition is characterized by the FA concentration of safflower oil and PSO. a Provides 150 mg butylhydroxytoluol (BHT)/kg diet and 1000 mg ascorbic acid/kg diet. b Without punicic acid.
some spreads were examined by light microscopy for chromosomal aberrations. Only metaphases containing 46 ± 2 chromosomes were analyzed. Prior to analysis of chromosomal aberrations, toxicity was determined using the mitotic index. The following aberrations were scored: chromatid gaps and breaks, chromosome gaps and breaks, chromatid deletion, minutes, double minutes, dicentric chromosomes, tricentric chromosomes, ring chromosomes, exchange figures, chromosomal intrachanges, pulverised chromosomes, multiple aberrations in one metaphase, polyploidy and endoreduplications. The dose levels of 33, 100 and 333 lg/ml from each incubation and fixation time were selected for scoring of chromosome aberrations. 2.6. In vivo toxicology tests 2.6.1. Acute oral toxicity in rats The acute oral toxicity study in rats was performed as a GLP study according to the guidelines mentioned in OECD 420 (2001) and EC council directive 67/548/EEC. PSO was administered as a single oral dose (by gavage) of 2000 mg/kg body weight to 9–11 weeks old female Wistar Crl:WI rats (n = 5). The animals were group-housed (4 animals per cage) in labeled Macrolon cages (MIII type, height 18 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son, Surrey, UK). A pilot study with one animal (individually housed) was performed to deter-
mine if 2000 mg/kg body weight was a tolerable dose. The test system and procedures of this pilot study with one animal were identical to the main study with 4 animals. Based on the pilot results, a fixed dose level was selected for the main study and four female rats were exposed to 2000 mg/kg body weight. PSO was dosed undiluted, resulting in a dose volume of 2.105 ml/kg body weight when corrected for the density (0.95 g/ml). Food was withheld overnight prior to dosing until approximately 3–4 h after administration. The body weights of the females at day 1 was 193–234 g. Body weights were determined on days 1, 8 and 15. Possible clinical signs occurring were recorded at periodic intervals on the day of dosing (day 1) and once daily thereafter until day 15. On day 15, the animals were sacrificed by oxygen/carbon dioxide procedure and subjected to necropsy. Macroscopic examination was performed after terminal sacrifice. The following organs were examined: lungs, heart, thyroid, thymus, spleen, pancreas, stomach, trachea, duodenum, jejunum, ileum, cecum, colon, liver, kidneys, adrenal glands, urinary bladder, urogenital tract, ovaria, and uterus. 2.6.2. 28-Day dietary toxicity study The 28-day dietary toxicity study in rats was performed as a GLP study based on the guidelines mentioned in OECD 407 and EC council directive 67/548/EEC. Three male and three female Wistar Crl:WI rats (Charles River, Sulzfeld, Germany) of approximately 6 weeks old per sex per group were exposed to 0, 10,000, 50,000 and 150,000 ppm PSO via dietary administration. The diet (semi-synthetic pelleted
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laboratory animal diet using AIN 93G as basal diet) containing 10,000, 50,000 and 150,000 ppm PSO was prepared by SSNIFF Spezialdiäten GmbH, Soest, Germany. Table 2 presents a detailed composition of the delivered diets. The composition of the diets was such that imbalances in nutritional or energy status due to the high fat content of the diets were accounted for. The highest dose of 150,000 ppm was selected since it was anticipated that higher doses would result in non-specific intestinal discomfort due to the high fat content of the diet. The control group received a diet with 150,000 ppm safflower oil as control oil. In the other dose groups, the safflower oil was (partly) replaced by PSO at the required concentration. The crude fat content of the diets was kept constant across all dose groups. The diet was provided ad libitum. Diets in the food hoppers were replaced with fresh diet twice weekly. Prepared diets were delivered to our laboratory once for the whole study in sealed and labeled vacuum-bags containing approximately 650 g of diet. Upon receipt, the bags were stored in the refrigerator (6 15 °C). Each diet bag contained an oxygen absorber. Diets were administered as delivered and were allowed to acclimatize by storing the required bags at room temperature for at least 1 h before use. The stability of the diet preparations was determined after storage of the diet at room temperature for 5, 8 and 12 days and after storage of the diet in the refrigerator for 14 and 28 days, and the ingredients in the diet were determined to be stable. The general physical condition of each animal was monitored at least twice daily during the 28-day study. Detailed clinical observations were performed for each individual animal once daily. The body weight of the animals was measured weekly, and the food consumption twice per week. On day 28, blood samples were collected from the retro-orbital sinus under isoflurane anaesthesia (minimally 3%). The following hematological parameters were determined: leukocytes, erythrocytes, differential leukocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), reticulocytes, hemoglobin, hematocrit, platelets, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), prothrombin time (PT), and activated partial thromboplastin time (APTT). The plasma from each animal was analyzed for the following clinical biochemistry parameters: alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium and inorganic phosphate. On day 28, the animals were anaesthetized using isoflurane inhalation (Abott Laboratories Ltd., Zwolle) and subsequently exsanguinated. The following organ weights (and terminal body weight) were recorded: adrenal glands, heart, kidneys, liver, spleen, testes and thymus.
3. Results
The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant increase in the number of revertants was observed. PSO did not induce an increase in the number of revertant colonies in all strains tested both in the absence and presence of metabolic activation (Table 3). These results were confirmed in an independently repeated experiment (Table 4). Therefore, it was concluded that PSO is not mutagenic in the Ames test. 3.1.2. In vitro chromosomal aberration test PSO precipitated in the culture medium at a concentration of 333 lg/ml, therefore concentrations above 333 lg/ml were not tested for chromosome aberrations. The concentrations tested for chromosome aberrations (33, 100 and 333 lg/ml) showed no significant toxicity. PSO did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in two independently repeated experiments (Tables 5 and 6). In addition, no effects of PSO on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of metabolic activation. It can therefore be concluded that PSO is not clastogenic and also does not induce numerical chromosomal aberrations. 3.2. In vivo toxicology tests 3.2.1. Acute oral toxicity in rats No mortality occurred at the dose level of 2000 mg/kg body weight. Between days 1 (dosing day) and 2 all animals showed a hunched posture. The body weight gain shown by the animals over the study period was considered to be normal. In addition, no abnormalities were found at macroscopic post mortem examination of the animals. Based on these results, the minimum oral lethal dose of PSO in rats was established to exceed 2000 mg/kg body weight.
3.1. In vitro mutagenicity tests 3.1.1. Ames test PSO precipitated on the plates at concentrations of 3330 and 5000 lg/plate.
3.2.2. 28-Day dietary toxicity study Dietary administration of PSO at 0, 10,000, 50,000 and 150,000 ppm produced no clinical signs, no adverse effects or mortality. Slightly lower body weights and a slightly lower body
Table 3 Response of PSO in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (first experiment). Results represent mean number of revertant colonies/3 replicate plates ± standard deviation. Concentration (lg/plate)
TA1535
TA1537
Without metabolic activation Solvent control 3 10 33 100 333 1000 3330SP 5000SP Positive control
21 ± 4 –a – 16 ± 4 18 ± 4 15 ± 1 13 ± 3 13 ± 3 – 1092 ± 15
5±3 – – 8±5 5±2 5±2 4±1 6±1 – 249 ± 33
13 ± 4 – – 15 ± 5 11 ± 1 12 ± 5 16 ± 2 12 ± 2 – 325 ± 12
6±1 – – 5±2 7±2 6±4 6±1 6±2 – 364 ± 55
With metabolic activation (5% S9-mix) Solvent control 3 10 33 100 333 1000 3330SP 5000SP Positive control SP
Slight precipitate. a Not determined.
TA98
TA100
WP2uvrA
15 ± 5 – – 15 ± 2 16 ± 2 16 ± 3 15 ± 2 19 ± 4 – 979 ± 85
100 ± 3 106 ± 8 107 ± 15 113 ± 4 100 ± 8 109 ± 3 115 ± 2 110 ± 4 118 ± 7 1161 ± 26
11 ± 1 7±2 8±2 9±5 8±2 9±2 10 ± 2 9±2 11 ± 6 916 ± 49
24 ± 2 – – 22 ± 4 17 ± 5 21 ± 5 23 ± 4 23 ± 2 – 1506 ± 159
135 ± 21 131 ± 31 123 ± 9 119 ± 8 123 ± 14 125 ± 5 128 ± 18 116 ± 6 124 ± 6 1832 ± 45
13 ± 6 13 ± 6 9±2 10 ± 2 10 ± 3 12 ± 1 12 ± 4 13 ± 3 11 ± 3 378 ± 37
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Table 4 Response of PSO in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (second experiment). Results represent mean number of revertant colonies/3 replicate plates ± standard deviation. Concentration (lg/plate)
TA1535
TA1537
TA98
TA100
WP2uvrA
Without metabolic activation Solvent control 33 100 333 1000 3330SP Positive control
16 ± 4 21 ± 7 20 ± 6 18 ± 4 20 ± 1 20 ± 5 1067 ± 7
5±1 5±0 7±3 5±2 4±1 3±1 287 ± 55
18 ± 6 15 ± 2 20 ± 2 14 ± 6 17 ± 5 15 ± 3 1099 ± 60
125 ± 15 117 ± 15 128 ± 7 122 ± 10 126 ± 17 98 ± 9 1277 ± 24
30 ± 7 29 ± 1 27 ± 9 27 ± 8 29 ± 6 28 ± 2 1159 ± 15
120 ± 8 123 ± 6 124 ± 21 120 ± 4 135 ± 6 116 ± 8 1342 ± 53
26 ± 8 24 ± 5 26 ± 3 25 ± 4 30 ± 5 27 ± 3 324 ± 29
With metabolic activation (10% S9-mix) Solvent control 33 100 333 1000 3330SP Positive control
19 ± 4 18 ± 4 21 ± 1 18 ± 4 19 ± 6 18 ± 5 224 ± 25
6±2 6±0 6±3 5±2 5±3 8±1 407 ± 6
20 ± 5 26 ± 7 24 ± 2 18 ± 2 28 ± 2 26 ± 5 779 ± 136
SP
Slight precipitate.
Table 5 Chromosome aberrations in donor cultures treated with PSO in the absence of metabolic activation at different exposure and fixation times. Concentration (lg/ml)
Mitotic index (%)
No of cells with structural chromosome aberrations per 200 metaphasesa Gap
Chromatid type
Chromosome type
g
ctb
cte
csb
cse
Others
Total +g
g
Without metabolic activation, 3 h exposure time, 24 h fixation timeb Solvent controlc 100 3 33 86 0 100 84 1 d 89 0 333 MMC 81 2
0 0 0 0 6
0 0 0 0 0
2 0 0 0 15
0 0 0 0 29
0 0 0 0 0
5 0 1 0 50***
2 0 0 0 49***
Without metabolic activation, 24 h exposure time, 24 h fixation time Solvent control 100 0 33 104 0 100 95 1 70 2 333d MMC 41 2
0 3 2 0 17
0 0 0 0 0
0 0 0 0 2
0 0 0 0 21
0 0 0 0 0
0 3 3 2 37***
0 3 2 0 35***
Without metabolic activation, 48 h exposure time, 48 h fixation time Solvent control 100 0 33 90 0 100 76 0 70 0 333d MMC 47 2
0 1 1 3 29
0 0 0 0 0
0 0 0 0 11
0 0 0 0 25
0 0 0 0 2
0 1 1 3 60***
0 1 1 3 60***
Abbreviations: ctb, chromatid break; cte, chromatid exchange; csb, chromosome break; cse chromosome exchange, +g, including gaps; g, excluding gaps; MMC, mytomycin C. a In total 200 metaphases scored for each treatment, i.e. 100 metaphases scored from each duplicate culture. b Human lymphocytes treated with PSO or vehicle for 3 h without metabolic activation and then cultured in fresh medium for further 21 h. c DMSO, dimethyl sulfoxide. d PSO precipitated in the culture medium. *** Significantly different from solvent control (P < 0.001).
weight gain was observed in males at 10,000 and 150,000 ppm PSO in the diet. Since this difference occurred in the absence of a treatment-related distribution, these changes were considered to be of no toxicological relevance. Body weights and body weight gain of other treated groups remained in the same range as the controls. The total food consumption (in g/animal/day) and the relative food consumption (in g/kg body weight/day) showed no significant differences between animals from the different groups (data not shown). The mean intake of PSO, estimated based on the body weight and food consumption values, in the different animal groups was 0–0, 825–847, 4269–4330 and 13710–14214 mg PSO/kg body weight per day in males–females, respectively. No difference was observed in food intake of PSO between the two sexes.
The results of the hematological and clinical biochemistry are presented in Tables 7 and 8. A slightly increased white blood cell count was noted in males exposed to 150,000 ppm PSO and in one female exposed to 15% PSO. Increased relative neutrophil counts with concurrently reduced relative lymphocyte counts were noted in one control male and in all males exposed to 150,000 ppm PSO. Based on historical data this shift in type of white blood cells was considered to be a secondary non-specific response to stress and to be of no toxicological significance. The weight of the different organs and the organ to body weight ratios are presented in Table 9. The liver weight of females exposed to 150,000 ppm PSO and liver-to-body weight ratios of males and females exposed to 150,000 ppm PSO were slightly increased. Other organ weights and organ to body weight ratios were similar to control levels.
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Table 6 Chromosome aberrations in donor cultures treated with PSO in the presence of metabolic activation at different fixation times. Concentration (lg/ml)
Mitotic index (%)
No of cells with structural chromosome aberrations per 200 metaphasesa Gap
Chromatid type
Chromosome type
g
ctb
cte
csb
cse
Others
Total
With metabolic activation, 3 h exposure time, 24 h fixation timeb Solvent controlc 100 0 33 96 0 100 104 0 109 0 333d CP 23 6
0 0 0 0 27
0 0 0 0 0
0 0 0 0 12
0 0 0 0 11
0 0 0 0 1
0 0 0 0 50***
0 0 0 0 47***
With metabolic activation, 3 h exposure time, 48 h fixation timeb Solvent control 100 0 33 91 0 100 84 1 80 1 333d 2 CP –e
1 0 1 3 24
0 0 0 0 0
0 0 0 0 4
0 0 0 0 13
0 0 1 0 1
1 0 2 4 40***
1 0 1 3 38***
+g
g
Abbreviations: ctb, chromatid break; cte, chromatid exchange; csb, chromosome break; cse chromosome exchange, +g, including gaps; g, excluding gaps; CP, cyclophosphamide. a In total 200 metaphases scored for each treatment, i.e. 100 metaphases scored from each duplicate culture. b Human lymphocytes treated with PSO or vehicle for 3 h with metabolic activation and then cultured in fresh medium for further 21 or 45 h. c DMSO, dimethyl sulfoxide. d PSO precipitated in the culture medium. e CP was fixed after 24 h, therefore the mitotic index could not be calculated as percentage of control. *** Significantly different from solvent control (P < 0.001).
Table 7 Haematological findings in rats after 28-day dietary exposure to PSO (mean ± SD). Sex Males
WBC (109/l) Neutrophils (%WBC) Lymphocytes (%WBC) Monocytes (%WBC) Eosinophils (%WBC) Basophils (%WBC) RBC (1012/l) Reticulocytes (%RBC) RDW (%) Haemoglobin (mmol/l) Haematocrit (%) MCV (fL) MCH (fmol) MCHC (mmol/l) Platelets (109/l) PT (s) APTT (s)
Females
Control
10,000 ppm PSO
50,000 ppm PSO
150,000 ppm PSO
Control
10,000 ppm PSO
50,000 ppm PSO
150,000 ppm PSO
10.9 ± 1.6 16.2 ± 4.2 79.0 ± 4.5 3.0 ± 0.5 1.4 ± 0.2 0.5 ± 0.1 7.90 ± 0.51 3.1 ± 0.7 12.5 ± 0.3 9.4 ± 0.4 45.0 ± 2.0 57.0 ± 1.5 1.20 ± 0.03 21.01 ± 0.27 1035 ± 150 17.6 ± 0.6 19.1 ± 0.6
11.3 ± 2.1 12.4 ± 1.1 83.8 ± 1.3 2.3 ± 0.5 1.1 ± 0.5 0.4 ± 0.2 8.27 ± 0.28 2.5 ± 0.4 12.8 ± 0.4 9.4 ± 0.2 44.3 ± 1.0 53.6 ± 0.8 1.14 ± 0.01 21.24 ± 0.13 1155 ± 242 17.4 ± 1.0 16.0 ± 3.2
12.2 ± 1.1 12.3 ± 1.2 82.8 ± 0.6 3.2 ± 0.7 1.2 ± 0.1 0.5 ± 0.1 7.92 ± 0.47 3.1 ± 0.4 13.0 ± 0.4 9.2 ± 0.3 44.1 ± 1.0 55.8 ± 2.7 1.17 ± 0.06 20.88 ± 0.19 1167 ± 43 16.8 ± 0.8 17.2 ± 3.4
15.0 ± 1.4 24.2 ± 4.3 70.5 ± 3.3 3.7 ± 1.2 1.2 ± 0.5 0.5 ± 0.1 8.49 ± 0.48 2.9 ± 0.5 12.7 ± 0.4 9.6 ± 0.2 46.2 ± 1.5 54.5 ± 1.5 1.14 ± 0.05 20.85 ± 0.29 1013 ± 174 16.5 ± 0.5 17.6 ± 3.0
5.4 ± 0.7 12.7 ± 5.1 83.8 ± 6.0 2.0 ± 0.4 1.3 ± 0.4 0.4 ± 0.1 7.77 ± 0.07 2.7 ± 0.3 12.1 ± 0.2 9.1 ± 0.4 42.6 ± 1.6 54.8 ± 2.6 1.18 ± 0.06 21.42 ± 0.06 1136 ± 114 16.5 ± 0.1 18.0 ± 3.5
6.3 ± 2.3 11.2 ± 5.3 84.6 ± 6.9 2.3 ± 1.5 1.6 ± 0.4 0.3 ± 0.3 7.65 ± 0.27 3.0 ± 0.5 12.0 ± 0.8 9.2 ± 0.6 42.3 ± 1.9 55.4 ± 2.9 1.21 ± 0.07 21.86 ± 0.44 907 ± 395 17.0 ± 1.0 14.9 ± 2.6
4.8 ± 1.5 12.8 ± 3.4 83.6 ± 3.7 1.9 ± 0.3 1.4 ± 0.4 0.3 ± 0.1 8.05 ± 0.20 2.4 ± 0.1 12.2 ± 0.6 9.2 ± 0.3 42.6 ± 2.2 52.8 ± 1.4 1.15 ± 0.02 21.69 ± 0.31 1203 ± 80 16.7 ± 0.6 15.4 ± 2.1
9.1 ± 6.5 10.8 ± 5.4 85.3 ± 6.6 2.4 ± 0.9 1.2 ± 0.6 0.4 ± 0.1 7.73 ± 0.11 1.8 ± 0.3 11.6 ± 0.5 9.2 ± 0.3 41.8 ± 1.9 54.1 ± 1.8 1.18 ± 0.03 21.92 ± 0.19 1016 ± 165 16.3 ± 0.7 17.8 ± 3.2
WBC, white blood cells; RBC, red blood cells; red blood cell distribution width; MCV, mean corpuscular volume; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration; PT, prothrombin time; APTT, activated partial thromboplastin time.
4. Discussion The current paper is the first paper focusing on safety assessment of pomegranate seed oil (PSO). The possible mutagenicity and toxicity of PSO was evaluated using the in vitro Ames and in vitro chromosomal aberration test (in cultured peripheral human lymphocytes), an in vivo acute oral toxicity and an in vivo 28-day dietary toxicity study. The results of the mutagenicity studies reveal that PSO is neither mutagenic nor clastogenic, both in the absence or presence of metabolic activation. In the oral acute toxicity study, no abnormalities were observed at macroscopic post mortem examination of the animals, and no effects on body weight or body weight gain were observed at a concentration of 2000 mg/kg body weight. According to the OECD 423 test guideline, the LD50 cut-off value can then be considered to
exceed 5000 mg/kg body weight, and no classification or labeling for oral toxicity is required for PSO. In the 28-day dietary toxicity study no effects were observed at 10,000 ppm and 50,000 ppm PSO in the diet. The mean PSO intake over the treatment period at these dietary levels was 825–847 and 4269–4330 (male–female animals) mg/kg body weight per day. Thus it can be concluded that 4.3 g of PSO per kg body weight per day is the no observed adverse effect level in this study. At higher concentrations, e.g. 150,000 ppm PSO in the diet (corresponding to 13,710 and 14,214 mg/kg body weight/day in males and female rats, respectively), mainly effects on hepatic enzyme activities are observed and a higher liver weight and liver-to-body weight ratio, indicating that the liver might be a target organ. However, the effects observed on liver weight and liver-to-body weight ratio at the highest exposure group (150,000 ppm PSO) might be the result of a physiological response to exposure to a very high le-
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ALAT (U/l) ASAT (U/l) ALP (U/l) Total protein (g/l) Albumin (g/l) Total bilirubin (lmol/l) Urea (mmol/l) Creatinine (lmol/l) Glucose (mmol/l) Cholesterol (mmol/l) Sodium (mmol/l) Potassium (mmol/l) Chloride (mmol/l) Calcium (mmol/l) Inorganic phosphate (mmol/ l)
Females
Control
10,000 ppm PSO
50,000 ppm PSO
150,000 ppm PSO
Control
10,000 ppm PSO
50,000 ppm PSO
150,000 ppm PSO
27.1 ± 7.2 72.0 ± 13.3 251 ± 140 68.0 ± 4.6 34.4 ± 1.8 2.7 ± 0.3 6.1 ± 0.8 37.6 ± 1.6 7.76 ± 1.30 2.36 ± 0.23 142.3 ± 1.3 3.70 ± 0.14 102 ± 1 2.91 ± 0.08 2.59 ± 0.17
33.0 ± 4.4 73.6 ± 11.8 231 ± 61 68.0 ± 0.8 34.5 ± 0.9 2.4 ± 0.2 5.2 ± 0.6 38.0 ± 1.1 7.72 ± 0.91 1.62 ± 0.16 142.8 ± 0.7 3.62 ± 0.24 105 ± 1 2.92 ± 0.05 2.58 ± 0.17
34.6 ± 7.5 92.0 ± 15.2 241 ± 55 66.1 ± 3.1 33.0 ± 0.4 2.4 ± 0.2 5.5 ± 1.3 39.0 ± 1.1 7.77 ± 0.70 1.77 ± 0.44 141.9 ± 1.2 3.62 ± 0.07 103 ± 0 2.86 ± 0.07 2.75 ± 0.20
106.1 ± 14.0 430.7 ± 118.7 651 ± 92 69.1 ± 2.9 36.3 ± 0.5 2.7 ± 0.1 7.2 ± 0.1 41.4 ± 2.2 9.37 ± 2.78 1.58 ± 0.22 141.5 ± 0.1 3.75 ± 0.19 101 ± 1 2.91 ± 0.07 2.97 ± 0.10
32.4 ± 7.9 71.4 ± 1.0 94 ± 20 69.4 ± 1.5 35.9 ± 1.9 3.4 ± 0.4 5.0 ± 0.1 41.1 ± 1.8 6.74 ± 1.00 1.44 ± 0.33 140.4 ± 1.1 3.25 ± 0.17 103 ± 2 2.83 ± 0.07 1.98 ± 0.19
29.1 ± 2.1 99.2 ± 31.2 122 ± 13 65.2 ± 3.5 35.3 ± 0.7 3.2 ± 0.6 6.1 ± 1.0 45.3 ± 1.8 7.96 ± 0.87 1.28 ± 0.31 142.4 ± 1.3 3.33 ± 0.14 105 ± 3 2.76 ± 0.06 2.19 ± 0.03
29.7 ± 4.5 80.0 ± 6.3 123 ± 35 67.5 ± 3.7 34.8 ± 2.1 2.5 ± 0.5 6.9 ± 0.8 42.1 ± 4.6 8.27 ± 0.95 1.48 ± 0.11 141.8 ± 0.4 3.53 ± 0.48 105 ± 2 2.78 ± 0.04 2.32 ± 0.21
42.7 ± 7.1 96.3 ± 36.6 291 ± 70 71.5 ± 2.8 38.5 ± 1.5 3.0 ± 0.2 7.0 ± 1.9 44.2 ± 2.8 9.12 ± 1.37 1.31 ± 0.44 141.0 ± 0.6 3.37 ± 0.15 104 ± 1 2.78 ± 0.07 2.19 ± 0.31
ALAT, alanine aminotransferase; ASAT, aspartate aminotransferase; ALP, alkaline phosphatase.
Table 9 Organ weights of male and female rats after 28-day dietary exposure to PSO (mean ± SD). Sex Males Control
Females 10,000 ppm PSO
50,000 ppm PSO
150,000 ppm PSO
Control
10,000 ppm PSO
50,000 ppm PSO
150,000 ppm PSO
Organ weights (g) Body weight 377 ± 23 Heart 1.330 ± 0.136 Liver 11.28 ± 0.96 Thymus 0.638 ± 0.238 Kidneys 3.05 ± 0.16 Adrenals 0.077 ± 0.018 Spleen 0.925 ± 0.186 Testes 3.65 ± 0.20
332 ± 11 1.104 ± 0.064 9.51 ± 0.48 0.547 ± 0.072 2.43 ± 0.31 0.068 ± 0.004 0.907 ± 0.082 3.43 ± 0.09
370 ± 29 1.175 ± 0.132 10.66 ± 0.92 0.625 ± 0.273 2.57 ± 0.24 0.072 ± 0.006 0.960 ± 0.170 3.66 ± 0.24
324 ± 33 1.263 ± 0.180 11.58 ± 1.66 0.493 ± 0.120 2.52 ± 0.37 0.063 ± 0.012 0.954 ± 0.127 3.35 ± 0.29
236 ± 5 0.835 ± 0.026 6.82 ± 0.39 0.410 ± 0.025 1.71 ± 0.08 0.079 ± 0.009 0.589 ± 0.047 n.a.
232 ± 13 0.926 ± 0.119 6.46 ± 0.09 0.424 ± 0.110 1.68 ± 0.07 0.084 ± 0.002 0.673 ± 0.034 n.a.
232 ± 14 0.826 ± 0.011 6.92 ± 0.02 0.432 ± 0.039 1.77 ± 0.11 0.087 ± 0.004 0.585 ± 0.052 n.a.
219 ± 18 0.885 ± 0.047 8.11 ± 1.01 0.378 ± 0.119 1.79 ± 0.11 0.076 ± 0.004 0.675 ± 0.028 n.a.
Organ to body weight ratio (%) Heart 0.352 ± 0.023 Liver 2.99 ± 0.08 Thymus 0.167 ± 0.056 Kidneys 0.81 ± 0.04 Adrenals 0.020 ± 0.004 Spleen 0.245 ± 0.044 Testes 0.97 ± 0.07
0.333 ± 0.022 2.87 ± 0.14 0.165 ± 0.022 0.73 ± 0.07 0.020 ± 0.002 0.274 ± 0.034 1.03 ± 0.04
0.319 ± 0.044 2.88 ± 0.06 0.166 ± 0.059 0.69 ± 0.01 0.020 ± 0.001 0.258 ± 0.027 0.99 ± 0.05
0.390 ± 0.035 3.57 ± 0.30 0.153 ± 0.039 0.78 ± 0.07 0.019 ± 0.002 0.294 ± 0.022 1.04 ± 0.02
0.354 ± 0.004 2.89 ± 0.12 0.174 ± 0.008 0.72 ± 0.03 0.033 ± 0.003 0.250 ± 0.017 n.a.
0.401 ± 0.067 2.78 ± 0.14 0.183 ± 0.046 0.72 ± 0.04 0.036 ± 0.002 0.291 ± 0.031 n.a.
0.356 ± 0.017 2.99 ± 0.17 0.186 ± 0.019 0.76 ± 0.06 0.038 ± 0.004 0.252 ± 0.011 n.a.
0.405 ± 0.030 3.69 ± 0.16 0.171 ± 0.046 0.82 ± 0.09 0.035 ± 0.004 0.308 ± 0.018 n.a.
vel of a fatty acid which is not part of the normal diet, and are most likely adaptive and nature and not toxicologically relevant. Conflict of interest statement I.A.T.M. Meerts, C.M. Verspeek-Rip, A.H.B.M. van Huygevoort, F.M. van Otterdijk declare that there are no conflicts of interest. Funding source: nil. C.A.F. Buskens declare that there are no conflicts of interest. Funding source: The study sponsor (Lipid Nutrition) was involved in the design of the protocol. However they had no influence on data collection, analysis and interpretation of the data. Lipid Nutrition asked NOTOX to write the manuscript and submit it for publication. E.J. van de Waart declares that there are no conflicts of interest. Funding source: nil. Z.E. Jouni, a co-author, is employed by Mead Johnson Nutritionals. Mead Johnson is collaborating with Lipid Nutrition in its evaluation of the safety and efficacy of pomegranate seed oil. Funding source: Mead Johnson had no influence on design of study, data collection, analyses and interpretation of the data.
H.G. Keizer, a co-author, is employed by Lipid Nutrition, the sponsor of this study. This company investigates the safety and efficacy of pomegranate seed oil in various indications. If safety is proven and efficacy is shown, pomegranate seed oil may become a market product. Funding source: The study sponsor (Lipid Nutrition) was involved in the design of the protocol. However they had no influence on data collection, analyses and interpretation of the data. The study sponsors asked NOTOX to write the manuscript and submit it for publication. J. Bassaganya-Riera is an inventor in a patent that covers the use of pomegranate seed oil as a nutritional supplement and functional food ingredient for the prevention and treatment of chronic diseases. Funding source: nil. References El-Shaarawy, M.I., Nahapetian, A., 1983. Studies on pomegranate seed oil. Fette. Seife. Anstrichmittel 85 (3), 123–126. European Community (EC), Council Directive 67/548/EEC, Annex V, Part B, 2004. Methods for Determination of Toxicity, as last amended by Commission Directive 2004/73/EC, B1 bis: Acute toxicity-oral, Fixed Dose Method.
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