Child Nutrition and Health

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40 Terezie Tolar Mosby and Ronald D. Barr clinical trials in chronic graft-versus-host disease: I. Diagnosis and Staging Working Group report. Biology of Blood and Marrow Transplantation, 11, 945-956. [104] Aquino V. M., Harvey A. R., Garvin J. H., Godder K. T., Nieder M. L., Adams R. H., et al. (2005). A double-blind randomized placebo-controlled study of oral glutamine in the prevention of mucositis in children undergoing hematopoietic stem cell transplantation: A pediatric blood and marrow transplant consortium study. Bone Marrow Transplant 36, 611-616. [105] Thompson, J. L. and Duffy J. (2008). Nutrition support challenges in hematopoietic stem cell transplant patients. Nutrition in Clinical Practice, 23,533-46. [106] Food and Nutrition Technical Report Series. (2001). Human energy requirements. Report of a joint FAO/WHO/UNU expert consultation. Rome, Italy. [107] AARC. (2004). Metabolic measurement using indirect calorimetry during mechanical ventilation: 2004 revision and update. Respiratory Care, 49, 1073-1079. [108] Duggan, C., Bechard, L., Donovan, K., Vangel, M., O'Leary, A., Holmes, C., et al. (2003). Changes in resting energy expenditure among children undergoing allogeneic stem cell transplantation. American Journal of Clinical Nutrition, 78, 104-109. [109] Duncan, C. N, Vrooman, L., Apfelbaum, E. M., Whitley, K., Bechard, L. and Lehmann, L. E. (2011) 25-hydroxy vitamin D deficiency following pediatric hematopoietic stem cell transplant. Biology of Blood and Marrow Transplantation. 17, 749-753. [110] McClune, B. L., Polgreen, L. E., Burmeister, L.A., Blaes, A. H., Mulrooney, D. A., Burns, L. J. et al. (2011). Screening, prevention and management of osteoporosis and bone loss in adult and pediatric hematopoietic cell transplant recipients. Bone Marrow Transplantation, 46, 1-9. [111] IOM - Institute of Medicine. (2011). Dietary Reference Intakes for Calcium and Vitamin D. Washington, DC: The National Academies Press. [112] Sefcick, A., Anderton, D., Byrne, J. L., Teahon, K. and Russell, N. H. (2001). Naso- jejunal feeding in allogeneic bone marrow transplant recipients: Results of a pilot study. Bone Marrow Transplantatio, 28,1135-1139. [113] Szeluga, D. J., Stuart, R. K., Brookmeyer, R., Utermohlen, V. and Santos, G. W. (1987). Nutritional support of bone marrow transplant recipients: A prospective, randomized clinical trial comparing total parenteral nutrition to an enteral feeding program. Cancer Research, 47, 3309-3316. [114] Papadopoulou, A., MacDonald, A., Williams, M. D., Darbyshire, P. J. and Booth, I. W. (1997). Enteral nutrition after bone marrow transplantation. Archives of Disease in Childhood, 77, 131-136. [115] Langdana, A., Tully, N., Molloy, E., Bourke, B. and O'Meara, A. (2001). Intensive enteral nutrition support in paediatric bone marrow transplantation. Bone Marrow Transplantation, 27, 741-746. [116] Weisdorf, S., Hofland, C., Sharp, H. L., Teasley, K., Schissel, K., McGlave, P. B, et al. (1984). Total parenteral nutrition in bone marrow transplantation: A clinical evaluation. Journal of Pediatric Gastroenterology and Nutrition, 3, 95-100. [117] Hastings, Y., White, M., and Young, J. (2006). Enteral nutrition and bone marrow transplantation. Journal of Pediatric Oncology Nursing, 23, 103-110. [118] Stratton, J. F., Thompson, D., Bobrow, L., Dalal, N., Gore, M., Bishop, et al. (1999). The genetic epidemiology of early-onset epithelial ovarian cancer: a population-based study. American Journal of Human Genetics, 65, 1725-1732.

Nutrition in Children and Adolescents with Cancer 41 [119] Charuhas, P. M., Fosberg, K. L., Bruemmer, B., Aker, S. N., Leisenring, W., Seidel, K. et al. (1997). A double-blind randomized trial comparing outpatient parenteral nutrition with intravenous hydration: effect on resumption of oral intake after marrow transplantation. Journal of Parenteral and Enteral Nutrition. 21:157-161. [120] Heubi, J. E. (1999). Whenever possible, use the gut! Journal of Pediatric Hematology/Oncology, 21,88-90. [121] Moore, F. A., Moore, E. E., Jones, T. N., McCroskey, B. L. and Peterson, V. M. (1989). TEN versus TPN following major abdominal trauma-reduced septic morbidity. Journal of Trauma and Acute Care Surgery, 29, 916-922. [122] Moore, F. A., Moore, E. E., Poggetti, R. S. and Read, R. A. (1992). Postinjury shock and early bacteremia. A lethal combination. Archives of Surgery. 127, 893-897; discussion 897-898. [123] Bechard L. (2000). Oncology and bone marrow transplantation. In: Hendricks K, Duggan C, Walker W, (Eds.). Manual of Pediatric Nutrition. (3rd ed, 490-502) Hamilton, Ontario. [124] Ziegler, T. R., Young, L. S., Benfell, K., Scheltinga, M., Hortos, K., Bye, R., et al. (1992). Clinical and metabolic efficacy of glutamine-supplemented parenteral nutrition after bone marrow transplantation. A randomized, double-blind, controlled study. Annals of Internal Medicine. 116, 821-828. [125] Schloerb, P.R. and Amare, M. (1993). Total parenteral nutrition with glutamine in bone marrow transplantation and other clinical applications (a randomized, double-blind study).Journal of Parenteral and Enteral Nutrition ,17, 407-413. [126] Piccirillo, N., De Matteis, S., Laurenti, L., Chiusolo, P., Sora, F., Pittiruti, M., et al. (2003). Glutamine-enriched parenteral nutrition after autologous peripheral blood stem cell transplantation: Effects on immune reconstitution and mucositis. Haematologica, 88, 192-200. [127] Pytlik R, Benes P, Patorkova M, Chocenska E, Gregora E, Prochazka B et al: Standardized parenteral alanyl-glutamine dipeptide supplementation is not beneficial in autologous transplant patients: A randomized, double-blind, placebo controlled study. Bone Marrow Transplant 2002; 30:953-961. [128] Schloerb, P. R. and Skikne, B. S. (1999). Oral and parenteral glutamine in bone marrow transplantation: A randomized, double-blind study. Journal of Parenteral and Enteral Nutrition, 23, 117-122. [129] Coghlin Dickson, T. M., Wong, R. M., Offrin, R. S., Shizuru, J. A., Johnston, L. J., Hu, W. W., et al. (2000). Effect of oral glutamine supplementation during bone marrow transplantation. Journal of Parenteral and Enteral Nutrition, 24,61-66. [130] Anderson P. M., Schroeder G. and Skubitz K. M. (1998). Oral glutamine reduces the duration and severity of stomatitis after cytotoxic cancer chemotherapy. Cancer. 83,1433-1439. [131] Hearing, S. D. (2004). Refeeding syndrome. British Medical Journal, 328, 908-909. [132] Nutrition Care Manual (2009). Hematopoietic Stem Cell Transplant. Chicago, IL: AmericanDieteticAssociation,.http://www.nutrition care manual.org/auth.cfm?p=%2 Findex%2Ecfm%3Fanderr=NotLoggedIn [133] Howlader, N., Noone, A. M., Krapcho, M., Neyman, N., Aminou, R., Waldron, W., et al. (eds). SEER Cancer Statistics Review, 1975-2008, National Cancer Institute.

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In: Child Nutrition and Health ISBN: 978-1-62257-981-5 Editors: G. Cvercko and L. Predovnik © 2013 Nova Science Publishers, Inc. Chapter II Calcium Supplementation in Young Children in Asia: Prevalence, Benefits and Risks Shu Che, Colin Binns and Bruce Maycock School of Public Health and Curtin Health Innovation Research Institute, Curtin University, Perth, Western Australia Abstract Calcium is essential for maintaining bone health in infants and young children. The calcium intakes of weaning infants and children in Asia are relatively low in comparison to their Western counterparts. This is an increasing concern for Asian parents and is one reason the Asia Pacific region is becoming a large market for vitamins and dietary supplements. However, there is a lack of data on the long-term benefits to early calcium supplementation of healthy infants and young children. The objective of this chapter is to discuss the appropriate calcium intakes for infants and young children, the risks and benefits of calcium supplementation and to review the proportion of children in Asia who are taking calcium supplements. To achieve our objective a literature review was undertaken of the English language databases PubMed and Web of Knowledge. Studies were selected that reported outcomes of calcium intake in infants and young children, as well as systematic reviews of such studies. Studies were undertaken of children in China and a comparison group of Chinese children living in Australia to document the use of calcium supplements. The prevalence of dietary supplementation among children under five years old in China (30.0%) was higher than in Australia (21.6%). In supplement users in China, 60.3% of them took calcium supplementation while only a small number in Australia (8%) took calcium supplements. Age and feeding method of the child (ever breastfed or not) were associated with nutritional supplementation in Australia, while household income and mother’s educational status were significantly related to the use of dietary supplements including calcium supplements in China. More than half of the children took supplemental calcium

44 Shu Che, Colin Binns and Bruce Maycock in the form of calcium gluconate (51.8%) and the average intake from supplements was 131 mg per day. There is little evidence to support the general use of calcium supplements in infants who were exclusively breastfed or formula fed. Evidence from recent studies does not support the use of calcium supplementation in healthy children as a public health intervention. However, for weaning infants and children with low calcium intakes, increased intake of calcium-rich foods should be encouraged. If adequate calcium cannot be achieved through food sources, supplementation may be an effective alternative. More studies are required in infants and young children with low calcium intakes, particularly those living in Asian countries or children of Asian ethnic origin. Abbreviations EAR Estimated Average Requirement RDA Recommended Dietary Allowance AI Adequate Intake UL Tolerable Upper Intake Level PRI Population Reference Intakes RI Recommended Intakes BMC Bone Mineral Content BMD Bone Mineral Density DRI Dietary Reference Values RDI Recommended Dietary Intakes NRV Nutrient Reference Values CV Coefficient of Variation SD Standard Deviation WHO World Health Organization NHMRC The Australia National Health and Medical Research Council FAO The Food and Agriculture Organization of the United Nations IOM Institute of Medicine SCF Scientific Committee on Food US United States Introduction Adequate calcium intake is important for bone health throughout the lifespan [1]. It is required for the normal development and maintenance of the skeleton as well as for the proper functioning of neuromuscular and cardiac function [2]. Providing adequate dietary intakes of calcium during infancy and early childhood may not only prevent diseases influence immediate health but may also delay or prevent osteoporosis in the elderly [3-7]. Although calcium is essential for maintaining bone health in infants and young children, the calcium intakes of weaning infants and children in Asia are relatively low in comparison to their Western counterparts. This can be partly attributable to the high incidence of lactase deficiency in Asia children, non-milk based diets, poor dietary habits in some families,

Calcium Supplementation in Young Children in Asia 45 inadequate information and knowledge on calcium rich foods of parents [1, 8, 9]. The prevalence of primary lactase deficiency is almost 100% in Asian adults and approximately 20% of Asian children younger than 5 years of age have evidence of lactase deficiency and lactose malabsorption [10-13]. Dietary lactose enhances calcium absorption and, conversely, lactose-free diets generally have lower overall calcium content and also lower calcium absorption [14]. Thus, lactose intolerance (and lactose-free diets) theoretically may predispose to inadequate bone mineralization [15, 16]. It is reported that about 20% of 1-2 years old and 45% of 3-5 years old children fail to achieve the recommended intake of calcium in the USA [17]. Considering the high prevalence of lactose intolerance and low consumption of calcium-rich food in Asia, it may be even more difficult to achieve recommended intakes without calcium supplementation for Asian children. The pattern of complementary feeding in many Chinese infants and young children, especially in rural areas, does not conform to current WHO recommendations for complementary feeding, including in achieving calcium intakes [18]. Dietary supplements enriched with vitamins, minerals, and other substances have received increasing attention worldwide. The North America and the Asia Pacific regions are the dominant markets for vitamins and dietary supplements [19]. A cross-sectional study of infants aged 6-12 months (n=251) in Beijing found that calcium supplements and cod liver oil were prescribed by health care providers for prevention of rickets in 71.6% and 49% of infants, respectively [20]. However, evidence for the association between calcium supplementation and bone changes are insufficient at present to make general recommendations for widespread use. There is also a lack of data proving long-term benefits to early calcium supplementation of healthy infants and young children [6]. A recent meta- analysis showed that although there is a small benefit of giving calcium supplements to children, it is unlikely to substantially reduce fracture risk in later life or even result in a clinically significant decrease in fracture risk in children [21]. In this chapter, we consider normal and abnormal patterns of bone mineralization in infants and sources of calcium in infants and pre-school children aged from 0 to 60 months. We further consider long-term effects of potential interventions related to calcium. To achieve our objective a literature review was undertaken of the English language databases PubMed and Web of Knowledge. Studies were selected that reported outcomes of calcium intake in infants and young children, as well as systematic reviews of such studies. All abstracts were read and relevant full text publications were then retrieved to include in this review. Review of Calcium Recommendations in Infants and Young Children Calcium requirements vary in different ethnic groups for dietary, genetic, body size, physical activity, lifestyle, and geographical reasons [22, 23]. Different bone mass and its accretion rate are evident among Asian, African, Caucasian and Hispanic adolescents [24-26]. Ethnic differences in fractional calcium absorption were also found in studies. Comparing to Caucasian counterparts, Chinese children and adolescents have higher fractional calcium absorption [27-30].

46 Shu Che, Colin Binns and Bruce Maycock A recent study compared calcium and bone accretion between Chinese adolescents and American Caucasian populations. Although habitual calcium intakes and vitamin D status were found to be lower in Chinese adolescents, bone mineralisation from age 10-15 years was similar in the two groups [31]. It further suggested more efficient calcium utilization, calcium absorption, excretion, and retention among the Chinese and calcium absorption efficiency decreased with increasing calcium intakes for the Chinese girls, but not the Caucasian girls. This suggested that bone accretion could be matched between the different ethnic groups at higher calcium intakes [31]. However after considering the ethnic differences in calcium metabolism and bone accretion in skeletal development, different calcium allowance recommendations were developed for different populations. Definitions for the terms used including Dietary Reference Values (DRIs)/ Recommended Dietary Intakes (RDIs)/Nutrient Reference Values (NRVs) for calcium requirements are given below [32-34]: Box 1. Definitions given to DRIs/RDIs/NRVs Estimated Average Requirement (EAR) – Reflects the estimated median requirement and is particularly appropriate for applications related to planning and assessing intakes for groups of persons. Recommended Dietary Allowance (RDA) – Derived from the EAR and meets or exceeds the requirement for 97.5 percent of the population. Adequate Intake (AI) – Used when an EAR/RDA cannot be developed; average intake level based on observed or experimental intakes. Tolerable Upper Intake Level (UL) – As intake increases above the UL, the potential risk of adverse effects may increase. The UL is the highest average daily intake that is likely to pose no risk of adverse effects to almost all individuals in the general population. Recommended Nutrient Intake (RNI) – It is the daily intake, which meets the nutrient requirements of almost all (97.5 percent) apparently healthy individuals in an age and sex-specific population group. Population Reference Intakes (PRI) – the level of (nutrient) intake that is adequate for virtually all people in a population group. Calcium as a nutrient is most commonly associated with the formation and metabolism of bone [32]. At full-term birth, the human infant has accrued about 26 to 30g of calcium, most of which is in the skeleton [32]. When calcium transfer from the placenta ceases at birth, the newborn infant is dependent on dietary calcium [32]. Human milk is recognized as the optimal source of nourishment for infants and the optimum source of calcium [35, 36]. The 2011 Institute of Medicine committee stated that there were no reports of any full-term, vitamin D–replete infants developing calcium deficiency when exclusively fed human milk [32]. The breastfed full-term infant is assumed to have a sufficient calcium intake regardless of the actual intake [6]. Therefore, AIs for calcium for infants up to 6 months old are based on average intake of breastmilk during the first half year of life and the studies that have determined average concentration of calcium in breastmilk [32, 37]. Reasonable estimations of calcium absorption, accretion and excretion are also taken into account [32]. In formula- fed infants, it is assumed that calcium is less bioavailable from infant formula compared to

Calcium Supplementation in Young Children in Asia 47 breastmilk [6]. Statutory guidance in the United States, and common practice throughout the world, is to provide 30% to 100% more calcium in infant formula than in breastmilk [6]. Therefore, the AIs for infants 7–12 months were set by not only considering the calcium intake from breastmilk or infant formula at this age but also an estimate of intake from supplementary foods [2]. The EARs for young children under 5 years old were set by estimating calcium requirements from data on daily rates of calcium accretion from a typical diet and then evaluating, on the basis of available balance data of the target population, the amount of calcium needed in the diet to achieve the requirements [6]. The RDAs were set by additional considering the variability of this intake to achieve the accretion in nearly all children [6]. The UL for calcium is not a recommended intake. Rather, it is intended to specify the level above which the risk for harm begins to increase. It is defined as the highest average daily intake of a nutrient that is likely to pose no risk of adverse health effects for nearly all persons in the general population [32]. As intake increases above the UL, the potential risk for adverse effects increases. Only a limited number of studies have reported the toxicity doses of calcium of children 1 to 5 years. Values for the UL were not set for infants or young children for all the populations. In the USA the ULs were set on the basis of limited available safety data and suggested that calcium toxicity was extremely unlikely to occur in healthy infants unless high-dose supplementation would be provided [32]. Australia and New Zealand The Australia National Health and Medical Research Council (NHMRC) and the New Zealand Ministry of Health developed the Australian/New Zealand RDIs in 2006, which were based on an update of the 2001 USA values. The AI of calcium for Australian and New Zealand for 0–6 months was set based on the estimated average intake of breastmilk (780 ml/day) and the average concentration of calcium in breastmilk (264 mg/L). After considering the lower bioavailability of calcium in infant formula, 350 mg/day was recommended for formula-fed babies (Table 1). In the 2006 Australian/New Zealand RDIs, the a mean intake of breastmilk was considered to be 600 ml/day at 7–12 months with an average concentration of 210 mg/L [2]. Based on this data an intake of 140 mg/day calcium from complementary foods is required during the second six months of life. This resulted in a calculated calcium intake of 266 mg/day, which was rounded up to 270 mg/day [2] (Table 1). For children 1–8 years, the daily net absorbed calcium need was estimated to be 220 mg/day. By assuming absorption rates of one standard deviation (SD) above those of adults and considering an approximate body weight for this age group, a figure of 360 mg/day was given as the EARs for 1–3 year-olds and 520 mg/day to the older group to provide this level of absorbed calcium [2]. The RDI was set assuming a coefficient of variation (CV) of 15% for the EAR and after rounding, giving an RDI of 500 mg/day for 1–3 year-olds and 700 mg/day for 4–8 year-olds [2] (Table 1). As there is little evidence of toxicity in children, the UL was set at 2,500 mg/day by considering the calcium dosage when adverse effects are found in adults with renal stones and considering the need to prevent interference with zinc and iron absorption [2] (Table 1).

Table 1. Current Dietary Reference Intake values for calcium for infants and young children (mg/day) Australia and New WHO 2002 United States and Canada 2011 European Japan 2010 China 2010 Zealand 2006 AI EAR RDA UL Union AI UL 1993 AI EAR RDA AI UL RNI PRI Males Females Males Females 0–6 months Breastmilk Breastmilk 200 1000 200 300 210 300 Formula 350 Formula 400 6–12 months 270 400 260 1500 400 250 400 1-2 years 360 2500 500 500 700 2500 400 350 350 400 400 600 2000 3 years 360 2500 500 500 700 2500 400 500 450 600 550 600 2000 4-5 years 520 2500 600 800 1000 2500 450 500 450 600 550 800 2000 AI, adequate intake; UL, tolerable upper intake level; RNI, recommended nutrient intake; EAR, estimated average requirement; RDA, recommended dietary allowance; and PRI,Population Reference Intakes.

Calcium Supplementation in Young Children in Asia 49 World Health Organization The Food and Agriculture Organization (FAO) of the United Nations and the World Health Organization (WHO) expert consultation had produced a publication on defining standards for micronutrient requirements in 1998 [34]. The recommendations for calcium allowances were based on Western European, American and Canadian data [34]. For infants, the concentration of calcium in breastmilk formed the basis of recommendations of calcium intakes in infants [22]. The FAO/WHO determined the daily calcium increment in the skeleton is about 100 mg in the first two years of life using data from American Academy of Pediatrics Committee on Nutrition [22, 38]. Together with information on the urinary calcium of infants and insensible losses of 20 mg/day, infants calcium absorption need was calculated as 120 mg daily to allow for normal growth [22]. For breastfed babies, a mean intake of 240 mg was assumed to meet the need of 120 mg net absorption. Based on the average daily breastmilk production of 750 ml, the calcium recommended intake of 300 mg for breastfed babies can be achieved. With cow milk, calcium intake needs to be set at about 300 mg to meet the requirement of 120 mg net calcium absorption and 400 mg of calcium intake was the recommended (Table 1). From age 2 to 9 years, as whole-body calcium increases with skeletal growth, the daily rate of calcium accumulation rises to 120 mg and urinary calcium increases to 60 mg. A dermal loss of 40 mg is added to these figures leading to an average daily net absorbed calcium requirement of 220 mg during this period [22]. Assuming that the net absorption of calcium by children is one SD above that of adults, the average daily requirement during this period is about 440 mg and the average recommended intake is 600 mg [22] (Table 1). United States and Canada The National Academy of Sciences, Institute of Medicine (IOM) released their new DRIs for calcium and vitamin D intake for the United States and Canada in 2011 [32]. Using estimates of a mean calcium concentration in breastmilk (259 mg/ml) and the amount of milk consumed per day (780 ml), the AI for calcium for infants 0 to 6 months of age is 200 mg/day (Table 1). It is a value reflective of the calcium provided to exclusively breastfed infants and to be considered as sufficient amounts of calcium to meet most infant’s growth needs by 2011 IOM committee [32]. From 6 to 12 months of age, the intake of calcium from solid foods becomes more significant. The IOM 2011 committee determined that the mean calcium intake from solid foods was about 140 mg/day for formula fed infants based on the limited data and assumed that breastfed babies had similar intakes of solid food to those of formula fed infants of the same age. Based on the mean breastmilk intake during the second 6 months of life (600 ml/day) and a calcium concentration (200 mg/L) in breastmilk during this age span, the calcium intake from breastmilk would be approximately 120 mg/day. Adding those figures together gives a total intake of 260 mg/day [32] (Table 1). From the results of studies that used children as subjects, the 2011 IOM committee used the average bone calcium accretion to set an EAR rather than an AI for young children older than one year [32]. An estimated EAR is established as 500 mg of calcium per day, rounded from 474 mg/day by the 2011 DRI panel for children 1 through 3 years of age. An additional

50 Shu Che, Colin Binns and Bruce Maycock 30% calcium retention would meet the needs of 97.5% of age group 4 to 8 years. This results in an estimated RDA for calcium of 700 mg/day calcium, with rounding [32] (Table 1). Studies of Abrams et al. and Ames et al., indicate a calcium intake of 800 mg/day could be expected to achieve the levels of calcium needed for bone accretion for Children 4 through 8 years of age [39, 40]. Again, the assumption that another approximately 30% is needed to cover about 97.5% of the population results in a calculated and rounded RDA value for calcium of 1,000 mg/day [32] (Table 1). Within the confines of the limitations of the data, a ‘no observed adverse effect’ level of 1750 mg/day was established for infants [32]. To reduce the uncertainty factor, the UL for the life stage group of 0 to 6 months was adjusted for weight difference and rounded to 1,000 mg/day [32]. Given the limitation of data, a slight uncertainty correction is warranted, and the UL is set at 1,500 mg/day for infants 7 to 12 months of age [32] (Table 1). A UL of 2,500 mg of calcium per day was continually used for children between the age of 1 and 8 years as it was established in 1997 [41] (Table 1). New data on adverse outcomes due to over-dose of calcium intake among children have not emerged since then. Given the expected body weight and metabolic capacities increases for old ages, the level of 2500 mg/day is a reasonable compared with the new UL set for infants [32]. European Union The population reference intakes defined by the Scientific Committee on Food (SCF) in 1993 are based on a factorial approach without considering measurements of bone mineral accretion under different calcium intakes [33]. The 1993 SCF estimated the mean calcium retention needed per day for skeletal growth was 150 mg/day. The PRI is 400 mg/day for infants in the second half of the first year and for children up to age 3 years, 450 mg/day for children between 4 and 6 years [33]. Those figures were based on the assumption that the net absorption of dietary calcium is 35% and 30% were added to the calculated amount to allow for individual variation [33]. Because of the absence of reliable data, the PRI for 6-11 months old infants was taken as the same as for 1-3 years olds [33]. Possible adverse health effects of individual micronutrients at intakes in excess of dietary requirements have been evaluated in European population groups. It was reported that European infants and young had a high intake of calcium [42]. Although there are no data to set a numerical UL for children and adolescents and no appreciable risk has been identified even with the extreme levels of calcium intake in this age group [42]. China At present insufficient evidence is available from Chinese studies to establish EARs for calcium intake from which RDAs would be determined. Therefore, the AIs were established, based on maximal calcium retention for different age groups. The AI for calcium is 300 mg/day for infants before 6 months and 400 mg/day for infants 6 to 11 months. A calcium intake of 600 mg/day is recommended to children aged 1 to 3 years. An AI of 800 mg/day was given for 4-10 year-olds [43] (Table 1).

Calcium Supplementation in Young Children in Asia 51 The UL for calcium was set at 2000mg/day for Children aged 1-6 years old by the Chinese Nutrition Society [43] (Table 1). Japan The AI for calcium is 200 mg/day for infants of 0-5 months and 250 for infants of 6-11 months. The EAR and RDA for calcium for 1-2 years old children is 350 mg/day and 400 mg/day respectively. The EAR and RDA of calcium for boys in 3-5 years are 50 mg higher than that for girls in the same age group [44] (Table 1). Calcium Intakes of Children in Asia Calcium intakes vary between countries, generally following the different dietary habits and depending largely on dairy product consumption [22]. The FAO/WHO reported the daily protein and calcium intakes in different regions of the world during 1987 and 1989, the lowest calcium intakes occur in Asia, and the highest in North America and Europe [22]. Some study results further indicate variation in calcium intake among child and adolescent ethnic groups. Asians as the ethnic group were found with relatively low calcium intakes in comparison to the Western counterparts [45-48]. Milk or milk products are a good source of many nutrients and the best known food source for calcium. Dairy products provide calcium in a readily absorbable and convenient form. Human milk is the best source of calcium for infants, averagely providing the infant with 202 mg of calcium per day in the first half year and 120 mg per day in the second half [32]. The calcium concentration in milk is 120 mg per 100 g and up to 1100 mg/100 g milk products, from which about 32% is absorbable [49]. The average calcium contribution from milk differed among ethnic groups. Lactose restriction reduces milk consumption in Asians who have the highest prevalence of lactase deficiency in the world, close to 100% [12, 13]. The incidence of of some degree of lactase deficiency in Shanghai children aged 0～6 years was reported to be 47.4% and lactase intolerance was 16.5%, with a trend of increasing with the age [50]. Only a small number of studies have examined sources of calcium from foods and beverages as consumed by Asian populations especially in the child age group. A study in the US on ethnic diversity and calcium intakes found that vegetables and legumes were a major source of non-dairy calcium for Asian Americans, who generally had lower dairy consumption when compared to other population groups. The Asian Americans were reported to consume only 10% to 11% calcium from dairy products [46]. Similar results were found in China, where the main sources of calcium were vegetables (35.2%), bean and bean products (13.9%), wheat (11.2%) and rice (9.1%) and less than 5% of calcium came from dairy foods [51]. Those figures were extracted from 2002 China National Nutrition and Health Survey where it was reported that the deficiency of calcium was a common problem in Chinese residents [51]. In children aged 2 and 3 years, only 3.7% males and 5.1% females met the AI for calcium intake (600 mg/day), which were the highest in all age groups of each gender. In 4-6 years age group, the percentage of meeting the AI (800 mg/day) dropped to 1.8% for males and 1.1% for females [51].

52 Shu Che, Colin Binns and Bruce Maycock Prevalence of Use of Calcium Supplements in Australia and China Although calcium intake can be increased by dietary means, long-term adherence to high- calcium diets is difficult to achieve for Asians, as they often report having a low dairy consumption [18, 46, 51, 52]. Calcium supplements may be a useful way of helping Asians to obtain sufficient calcium and enhance health and wellbeing [53]. Most studies on calcium supplements have focused on adults or older persons and little is known regarding the intake of calcium supplements by infants and young children. A recent study from Taiwan reported that 34.9% of the infants had been given a dietary supplement and 15.5% took calcium supplement between birth and 6 months of age [54]. A survey of infant feeding practices (n=251) in Beijing, China found that 71.6% infants aged 6-12 months were taking calcium supplementation [20]. In Hubei, PR. China, a survey reported a prevalence of 90.2% (1523/1688) of calcium supplementation in pre-school children in four kindergartens and more than half of them were taking calcium supplements without medical prescriptions [55]. Australians have a high prevalence of taking dietary supplements. A representative population survey conducted in 2004 in South Australia reported the use of vitamin supplements by 39.2% respondents and mineral supplementations by13.6% of the population [56]. No recent data is available on the use of calcium supplements by infants or young children in Australia. Until recently, few studies have investigated the intake of calcium dietary supplement by infants and young children under five years old. And there have been no studies of calcium supplementation among Chinese children under five years in mainland China or overseas published in English. To document the prevalence of use of calcium supplements in these populations, a survey was carried out of Chinese mothers living in Perth, Australia and Chengdu and Wuhan, PR China. The China Australia Supplements Study (CASS Study) A survey was undertaken of 231 Chinese mothers living in Perth Australia, 360 mothers living in Wuhan and 1335 in Chengdu, PR China. The participants in Perth were mothers with children under 5 years old who were recruited from the Perth Chinese community, including Chinese schools and community organizations. A total of 238 mothers agreed to participate with a response rate of 96.0% and 231 mothers completed the dietary supplementation questionnaire, a final response rate of 93.1%. Participants in China were recruited from kindergartens in Wuhan and Chengdu. A total of 2800 questionnaires were distributed by kindergarten teachers and 1702 and 556 were returned by the mothers in Chengdu and Wuhan respectively. The dietary supplementation questionnaire was completed by 1335 mothers in Chengdu and 360 mothers in Wuhan, a total response rate of 60.5% in China. The study was approved by the Curtin University Human Research Ethics Committee. Demographic and breastfeeding information was collected using a validated and reliable questionnaire previously used in Chinese population studies [57]. Mothers were classified

Calcium Supplementation in Young Children in Asia 53 into three groups to compare their economic status based on the local annual household income [58, 59]. Data were analysed using the IBM Statistical Package for Social Sciences (SPSS) Version 20.0. Independent samples t-test was used to compare means between groups. Chi-square (χ2) test was used to test associations between basic characteristics and factors potentially related to the use of supplements among young children. P values <0.05 were considered statistically significant. Results of the CASS Study A total of 231 Chinese mothers living in Perth Australia and 1355 mothers living in Chengdu, Sichuan Province and 360 mothers living in Wuhan, Hubei Province, PR China completed the supplement questionnaire. The distribution analysis shows no difference in age, education attainment, marital status, working status, family income status, breastfeeding initiation and duration, between mothers who completed the supplement questionnaire and mothers who did not. There was also no difference in education attainment, marital status, family income status, breastfeeding initiation and duration, between mothers in Chengdu and Wuhan. The only two statistically significant differences between mothers in Wuhan and Chengdu were the average age (31.2 years in Chengdu and 30.8 years in Wuhan, p<0.001) and working status (68.7%Wuhan mothers have full-time work compared to 60.1% in Chengdu, χ2=8.1, df=2, p<0.05). Because the differences are so small in Wuhan and Chengdu mothers, their data have been combined into one group. A total of 21.6% of the Chinese children living in Perth were taking dietary supplements, including multivitamins/minerals, fish oil, probiotics, calcium and vitamin D. In Chengdu and Wuhan, China, 30.0% of young children were having dietary supplements and 60.3% of those supplement users were taking calcium supplements. Compared to Chinese Australians, Chinese parents living in China were more likely to give their children dietary supplements (χ2=6.9, df=1, p<0.001) and especially calcium supplements (χ2=40.3, df=1, p<0.001). About half of the Chinese children taking calcium supplements were also taking Vitamin D (including the use of multi-vitamins) (Table 2). Table 2. Prevalence of supplements use by type in Chinese children under 5 years living in Australia and China Supplement type Australia % Supplement n China % Supplement Any supplement users users Calcium n % (n=231) 100 509 % (n=1695) 100 Calcium + Vitamin D 307 50 21.6 8 160 30.0 60.3 4 1.7 18.1 2 0.9 4 9.7 31.4 In Australia, only four children were given specific calcium supplements. One was taking calcium carbonate tablets, the other calcium lactate, and two were unknown. The most common forms of supplemental calcium used in Chinese children up to five years old are

54 Shu Che, Colin Binns and Bruce Maycock gluconate (51.8%) and carbonate (37.5%). The dosage range of calcium supplements for Chinese children is 54 to 725 mg/day. The average intake for carbonate users (307.4 mg/day) is higher than gluconate calcium users (81 mg/day) (Table 3). Table 3. Calcium supplement form and dosage used by Chinese children under five years in China Supplements Number % Calcium Average intake Intake range form 115 supplement users (mg/day) (mg/day) Carbonate 159 37.5 307.4 (n=106) 85-725 Gluconate 11 51.8 81 (n=154) 54-360 Lactate 9 3.6 - - Others 13 2.9 116.7 (n=3) 100-150 Unknown 307 4.2 - - Total 100 131.4 (n=264) 54-725 Table 4. Calcium supplement use by maternal and child characteristic variables Age (year) Australia China Calcium p (%) ≥31 Any supplement Any supplement n 0.776 <31 N p (%) n p (%) 0.351 Education 121 (19.1) 0.001 ≥University 0.221 0.675 122 (18.3) ≤High school 39 (24.5) 194 (30.6) 0.374 Household income 11 (16.2) 212 (31.8) 109 (19.4) 0.687 High 122 (17.4) Middle 0.268 0.035 0.083 Low 41 (23.6) 190 (33.7) 60 (22.9) Gender of the child 9 (15.8) 216 (28.3) 139 (19.9) Male 22 (10.3) Female 0.477 0.000 Child's age (year) 15 (26.3) 93 (35.5) 173 (19.4) <1 year 27 (21.8) 242 (34.6) 133 (17.6) 1-2 6 (15.8) 37 (17.3) 2-3 5 (18.5) 3-4 0.635 0.549 6 (26.1) 4-5 28 (23.0) 285 (31.9) 46 (22.0) Infant feeding 22 (20.2) 221 (29.3) 119 (19.2) Ever breastfed 97 (18.0) Never breastfed 0.016 0.724 0 8 (29.6) 267 (18.9) 13 (14.8) 8 (34.8) 33 (14.0) 11 (21.6) 72 (34.4) 10 (26.3) 201 (32.5) 11 (39.3) 160 (29.7) 0.038 0.192 44 (20.2) 436 (30.8) 6 (46.2) 62 (26.4)

Calcium Supplementation in Young Children in Asia 55 In Australia, older children (χ2=12.24, df=4, p<0.05) and children who were never be breastfed (χ2=4.88, df=1, p<0.05) were more likely to take dietary supplements (Table 4). In China, no specific child characteristics where associated with taking supplements. However higher household income and higher education of the mother were significantly related to the use of all types of child supplements as well as calcium supplements (Table 4). The CASS study assessed the intake of calcium supplements used by Chinese young children in China and in Australia. It appears to be the first study reporting on the use of calcium supplements in Chinese young children up to five years old. In this study, one fifth of Chinese children in Perth were taking at least one nutritional supplement. Older children and formula fed children were more likely to be given nutritional supplements, but relatively few were on specific calcium supplements. This may be due to higher rates of dairy consumption in Australia than in China and less emphasis on calcium supplements in the lay press. Data from the Australian nationally representative 1995 National Nutrition Survey shows that the mean calcium intakes were 833 mg/day in children aged 2-3 years and 769 mg/day in children aged 4-7 years, which is higher than the Australian and New Zealand AI for calcium for those age group and suggest no need for calcium supplementation [60]. In China 30.0% of young children were taking nutritional supplements and 60.3% of these supplements users were on calcium supplementation. It was found that nutritional supplementation including calcium supplementation was more likely to occur among those children from a higher income family and with higher educated mother. However, the average intake of calcium from supplementation was only 131.4 mg per day, which is about 20% of the AI for calcium for Chinese children in this age group. It is less than half of calcium consumption that can be provided from one serve (250 ml) of milk, besides milk can provide other nutrients like protein to support child growth [49]. Studies in children regarding calcium supplementation and bone changes indicate that BMD changes are influenced by baseline calcium intake, stage of development, and the sites evaluated for BMD [61]. When baseline habitual calcium consumption is low, larger increments in BMD occur with increased dietary calcium intake [62]. The average calcium intake from supplements in Chinese young children (131.4 mg) is lower that all randomized controlled trials studies (nineteen studies) included in a meta-analysis assessing effects of calcium supplementation on BMD in healthy children. Calcium supplementation was with a calcium dose of 300-1200 mg per day in those nineteen studies [63]. Thus, it is not likely that the low calcium intake from supplements would result a significant change in BMD in children. Calcium Supplement Form and Absorption The most common forms of supplemental calcium used in Chinese children are calcium gluconate and calcium carbonate. More than half of the supplements users choose the oral solution of calcium gluconate. Elemental calcium is less concentrated in this form, containing only 9% elemental calcium [64]. Because calcium gluconate contains a lower proportion of elemental calcium, it is not considered practical for clinical practice. One popular brand of calcium gluconate contained 54 mg of elemental calcium in a 10 ml bottle. Chinese children taking calcium gluconate only take an average of 81 mg calcium a day, which is less than calcium contained in 100 ml milk [49].

56 Shu Che, Colin Binns and Bruce Maycock Calcium carbonate is the most common and least expensive form of calcium [64]. Generally calcium carbonate provides more elemental calcium with the same number of pills [65]. It contains 40% calcium and well-absorbed and tolerated in most individuals when taken with a meal [66]. The bioavailability of calcium carbonate depends on the dosage and whether they are taken with a meal [44]. It was found to be equivalent to skim milk and orange juice fortified with calcium-citrate malate [67]. Calcium can compete or interfere with the absorption of iron, zinc, and magnesium. Therefore, for persons with known deficiencies of these other minerals who require calcium supplementation, taking calcium supplements between meals is advisable [64]. Benefits of Calcium Supplantation Increased Bone Density and Bone Strength Low bone mineral density is an important risk factor for osteoporotic fractures [68]. Calcium deficiency leads to a reduction in bone mass by increasing bone resorption to preserve the level of ionised calcium in the extracellular fluid [61]. Dietary calcium deficiency may also be a major cause of rickets in children in developing countries [61]. Although nutritional rickets has long been considered a disease caused by vitamin D deficiency, calcium deficiency has also been reported as an important cause of rickets by recent studies in Nigeria, Bangladesh, India and the US [3, 69-71]. Study did in Europe also found that low 25-(OH) D level combined with low calcium intakes and possibly digestive disorders, were associated with an increased risk of genu valgum in children [4]. Optimal calcium intake is especially important during childhood, when most mineral accretion occurs [72]. Evidence has shown that increased calcium intakes, with and without vitamin D, increases BMC/BMD in children [73-75]. Studies did in Asia children also suggest that higher long-term habitual calcium intake and physical activity may lead to higher BMC in children [76, 77]. A review on calcium supplementations in children reported that almost all studies (seventeen out of nineteen) resulted a statistically significant improvement of supplementation on BMD in children [78]. Subjects in eight of the seventeen studies had a baseline daily calcium intake of 800–1300 mg. Those studies (eight of the seventeen) concluded that calcium supplementation was efficient even if baseline calcium intake was adequate [78]. However, evidence for an association between calcium supplementation and bone changes in children is conflicting [63, 75]. A systematic review evaluated the effect of calcium supplementation on BMD and concluded that such supplementation has little effect on BMD in children [63]. The only site with a significant increase in BMD was the upper limb. This effect translated into a 1.7% greater increase in BMD in the supplemented groups compared with non-supplemented groups. The review does not support the use of calcium supplementation in healthy children as a public health intervention [63]. Considering that few Asian children can meet the recommended calcium intakes for their ages and low BMD is a risk factor for fracture in childhood, increasing their calcium intake and optimising age appropriate bone mass may have a immediate beneficial effect [79, 80].

Calcium Supplementation in Young Children in Asia 57 Lower Body Fat Calcium intake has been associated with a reduction of body weight or weight gain in several studies [81-84]. Although the effect of calcium intake on body composition remains unclear, it may due to the reduced consumption of sugar-sweetened drinks and increased resting energy expenditure [82, 84]. A study of a multiethnic sample of children on calcium intakes and body fat suggested that calcium intake may play a role in fat accumulation and energy balance through its effects on resting energy expenditure [84]. However clinical, longitudinal, retrospective and cross-sectional studies in children show inconsistent findings regarding calcium intake and bone changes. Some studies reported no association between calcium and/or dairy intake in children and weight and/or body composition [85-87]. A systematic review of placebo-controlled randomized controlled trials of calcium supplementation found no statistically significant effects of calcium supplementation on weight, body fat or lean mass [88]. Although the results do not exclude an effect of calcium supplementation with dairy products on weight gain or body composition, at the present time there is insufficient evidence to recommend taking dairy products or calcium supplements as a means of population weight control [88]. Decreased Osteoporosis in Later In Life Bone loss in later life is related to the quality of peak bone mass established over the first two decades of life [89]. Considerable studies have been carried out over the past several decades to discuss whether osteoporosis originates in childhood and if providing high dietary intakes of calcium may delay or prevent this disease in the elderly [6]. Calcium is the primary bone-forming mineral that must be supplied to the diet and is the most important during childhood when approximately 200 mg/day is accreted into the skeleton [90]. Postmenopausal BMD is a function of peak bone mass formed during the first two decades of life and the rate of subsequent bone loss index during the aging process, which are equally important risk factors for fracture in later life [91, 92]. One of the recommended primary preventions of osteoporosis is the adequate calcium intake during infancy and childhood to optimize the gain in bone mass [5]. Thus, efforts to maximise peak bone mass through calcium supplementation during childhood have been encouraged. However, there has been no intervention study long enough to test the effect of nutritional factors to maximize peak bone mass [1]. It remains unclear that whether increases of BMD benefited from calcium supplements would persist into later life after supplementation stopped. Risks of Excess Consumption of Calcium There has no report of excess intake of calcium from food sources, however, as the use of calcium supplements increasing, excess consumption of calcium may occur [93]. Calcium plays a major role in the metabolism of virtually every cell in the body and interacts with a large number of other nutrients, like iron, zinc, magnesium and phosphorus，and as a result, disturbances of calcium metabolism may give rise to a variety of adverse effects [42, 94].

58 Shu Che, Colin Binns and Bruce Maycock There is no data on children taking calcium from dietary sources or from the usual level of supplements that provides reliable information on adverse effects. Data from European populations indicate that the intakes of calcium from all sources in infants can be close to the UL in a small percentage of the population [42]. In British infants the 97.5th percentile of calcium intake was 1400 mg/day. In German non-breastfed infants the 90th percentile of calcium intake was 700 to 900 mg/day [42]. And it was reported by European Commission of SCF and the Scientific Panel on Dietetic Products, Nutrition and Allergies that no adverse effects of calcium citrate-malate supplements or extra dairy foods (500 to 1000 mg extra calcium over 1 to 3 years) were reported in 217 children between 6 and 14 years, in comparison to un-supplemented controls [42]. Hypercalciuria, as a secondary outcome to high calcium intake, can occur in children. However, the incidence of kidney stones in children is rare. There is limited evidence concerning high calcium intakes in young children relative to calcium excretion. In a study undertaken in 4 to 9 months infants, three infants (6%) who received a calcium-enriched formula (1700 to 1560 mg calcium per day), developed hypercalciuria [95]. Another study tested the effects of 1,800 mg/day total calcium (supplementation adjusted on the basis of dietary calcium questionnaire) in children ages 1 to 6 years reported no diffrence in urinary calcium/creatinine ratios between children who took 1,800 mg/day calcium and those of placebo controls [96]. A study by Sargent et al. (1999) provides information relevant to infants and calcium excretion. Formula with added calcium glycerophosphate (1800 mg of calcium and 1390 mg of phosphate /L) for 9 months were given to infants aged 3.5-6 months old [97]. Together with calcium from solid foods, those infants had a mean calcium intake of 1,563 ± 703 mg/day at 9 months. Although the focus of the study was lead absorption, the data demonstrated that total calcium intakes of about 1,550 to 1,750 mg/day did not affect urinary calcium excretion. However, these data were insufficient to rule out or conclude that a definate risk exists for calcium supplements use in infants or young children. Food Sources of Calcium Compared to calcium from dietary supplements, calcium from food sources may be preferable for the evidence of better health outcomes. Two recent meta-analysis from same group on the effect of calcium supplementation on myocardial infarction and cardiovascular events suggested that calcium supplements in adults in higher doses with or without vitamin D have been associated with a modest increased risk of cardiovascular events [98, 99]. However, the effect of an equivalent dose of calcium from dairy products has a lower risk than calcium supplements and result in lower peaks of serum calcium levels [100]. Additionally，calcium intake from dairy is often reported as a possible factor that may reduce body weight or weight gain [81-84]. A meta-analysis on twenty-one randomized controlled trials (RCTs) found out increased dietary calcium/dairy products, with and without vitamin D, significantly increases total body and lumbar spine BMC in children with low base-line intakes [73]. However, another systematic review reported no evidence to support the use of calcium supplementation as a public health intervention to reduce weight gain or body fat in healthy children [88].

Calcium Supplementation in Young Children in Asia 59 Calcium is present in many foods, but is most concentrated in dairy products. Although lactose intolerance can be a barrier to milk consumption among Asians, studies have shown that subjects with lactose intolerance can consume milk and dairy foods without developing symptoms, if amounts are divided into smaller doses throughout the day [12, 101]. It was reported that dairy-rich diets up to 1500 mg/day of calcium can be consumed by lactose maldigesters without significant symptoms [102]. A recent study comparing calcium intake and bone mass between children with (n=47) and without (n=29) lactose malabsorption reported no statistically significant difference between the groups with respect to the intake of total calcium, milk calcium, milk, cheese, yogurt, ice cream, and calcium density of the diet [103]. The American Academy of Pediatrics Committee on Nutrition has stated that milk and dairy-product avoidance has a negative effect on calcium and vitamin D intake in infants, children, and adolescents. Other nutrients such as protein make dairy products an important source of nutrition for growing children [104]. Therefore, it is important to encourage all Asian children, have lactose intolerance or not, to take dairy products as a primary determinant of calcium intake. For those who avoid cow milk protein or lactose or low-lactose milks, other available calcium sources should be considered [6]. Some common Asian foods have been identified as containing an appreciable amount of calcium. Non-dairy foods such as tofu, tempeh, sea weeds, nuts and seeds dishes and green leafy vegetables etc. have been tested for calcium bioavailability in human studies. Low-oxalate greens (eg, bok choy, broccoli, Chinese cabbage, collards, and kale) and fruit juices fortified with calcium citrate or malate are good sources of highly bioavailable calcium, while calcium-set tofu have good bioavailability of calcium, and foods rich in oxalic acid (eg spinach, rhubarb, beans) or phytic acid (seeds, nuts, grains, certain raw beans and soy isolates) have a lower bioavailability [105]. Compared to milk, calcium absorption from dried beans is about 50% and from spinach, 10% [2]. The calcium absorption is equivalent for soymilk and cow’s milk at similar calcium loads, if the soymilk is fortified with calcium carbonate, not tricalcium phosphate which have lower calcium bioavailability [106]. Conclusion Breastmilk provides adequate calcium to meet the needs of all full-term infants. There is no need to recommend giving calcium supplements to infants who are exclusively breastfed or formula fed. Achieving adequate calcium is important in maximizing bone accretion during growth, preventing child rickets, and perhaps preventing fragility fractures in childhood or even preventing future osteoporosis. For all weaning infants and young children, calcium intake from calcium-rich foods especially from dietary sources should be encouraged at home, schools, and by parents, paediatricians, dietitians and by other health professionals. Current evidence from recent studies does not support the general use of calcium supplementation in healthy young children as a public health intervention. However, given that infancy and childhood are critical periods for the acquisition of bone mass, if adequate calcium cannot be achieved through food sources, supplementation is a useful alternative. More studies related to the clinical effectiveness and/or safety of dietary supplements in infants and children are required, especially over the longer term. Because little data are

60 Shu Che, Colin Binns and Bruce Maycock available in this area, we suggest that parents exercise caution when giving their infants or young children dietary supplements. Before providing dietary supplements for them, parents should communicate with health professionals, such as pediatric doctors or dietitians. Wherever possible it is preferable to achieve nutrient intakes, including calcium from a varied diet rather than from supplements. References [1] Lee, W.T. and J. Jiang, Calcium requirements for Asian children and adolescents. Asia Pac. J. Clin. Nutr, 2008. 17 Suppl 1: p. 33-6. [2] National Health and Medical Research Council and Ministry of Health, Nutrient reference values for Australia and New Zealand: including recommended dietary intakes2006, Canberra. [3] Thacher, T.D. and S.A. Abrams, Relationship of calcium absorption with 25(OH)D and calcium intake in children with rickets. Nutr. Rev, 2010. 68(11): p. 682-8. [4] Voloc, A., et al., High prevalence of genu varum/valgum in European children with low vitamin D status and insufficient dairy products/calcium intakes. Eur. J. Endocrinol, 2010. 163(5): p. 811-7. [5] Baker, S.S., et al., American Academy of Pediatrics. Committee on Nutrition. Calcium requirements of infants, children, and adolescents. Pediatrics, 1999. 104(5 Pt 1): p. 1152-7. [6] Abrams, S.A., What are the risks and benefits to increasing dietary bone minerals and vitamin D intake in infants and small children? Annu. Rev. Nutr, 2011. 31: p. 285-97. [7] Frontela, C., et al., Effect of dephytinization on bioavailability of iron, calcium and zinc from infant cereals assessed in the Caco-2 cell model. World J. Gastroenterol, 2009. 15(16): p. 1977-84. [8] Gong, Q., et al., Relation of health and lactase deficiency situation in children aged 06 years. Chinese Journal of Child Health Care, 2008. 16(5): p. 567-569. [9] Yang, Y., et al., The prevalence of lactase deficiency and lactose intolerance in Chinese children of different ages. Chin. Med. J. (Engl), 2000. 113(12): p. 1129-32. [10] Sahi, T., Genetics and epidemiology of adult-type hypolactasia. Scand J. Gastroenterol Suppl, 1994. 202: p. 7-20. [11] Woteki, C.E., E. Weser, and E.A. Young, Lactose malabsorption in Mexican-American children. Am. J. Clin. Nutr, 1976. 29(1): p. 19-24. [12] Novotny, R., Motivators and barriers to consuming calcium-rich foods among Asian adolescents in Hawaii. Journal of nutrition education, 1999. 31(2): p. 99. [13] Vesa, T.H., Lactose intolerance. Journal of the American College of Nutrition, 2000. 19(suppl 2): p. 165S. [14] Abrams, S.A., I.J. Griffin, and P.M. Davila, Calcium and zinc absorption from lactose- containing and lactose-free infant formulas. Am. J. Clin. Nutr, 2002. 76(2): p. 442-6. [15] Di Stefano, M., et al., Lactose malabsorption and intolerance and peak bone mass. Gastroenterology, 2002. 122(7): p. 1793-9. [16] Stallings, V.A., et al., Bone mineral content and dietary calcium intake in children prescribed a low-lactose diet. J. Pediatr. Gastroenterol Nutr, 1994. 18(4): p. 440-5.

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In: Child Nutrition and Health ISBN: 978-1-62257-981-5 Editors: G. Cvercko and L. Predovnik © 2013 Nova Science Publishers, Inc. Chapter III Exposure of Slovenian Preschool Children to Preservatives and Polyphosphates Elizabeta Mičović, Mario Gorenjak, Gorazd Meško and Avrelija Cencič 1 Ministry of Agriculture and Environment of Republic of Slovenia in Food Safety Directorate, Food and Feed Safety Area and Consumer Protection 2Faculty of Medicine, University of Maribor and Junior Researcher at Faculty of Agriculture and Life Science, University of Maribor, Slovenia 3Professor of Criminology and Dean at the Faculty of Criminal Justice and Security, University of Maribor, Slovenia 4Head of the Department of Microbiology, Biochemistry, Molecular Biology and Biotechnology at the Faculty of Agriculture and Life Sciences, University of Maribor, Slovenia Abstract The purpose of this paper is to present exposure of preschool children to daily consumed food preservatives and polyphosphates: sorbic acid, benzoic acid, nitrate, nitrite, sulphur dioxide and polyphosphates. For exact exposure of chemicals in food, data of consumed food intake and concentration of observed chemicals in food are needed. Methodology: Among the randomly selected regions in Slovenia, we randomly selected kindergartens and children aged from 2-6 years. The study included 190 children, 98 boys and 92 girls. Anthropometric measurements of children were conducted, so data on the sex, age, measured weight and height of the children were available. The dietary intake was based on the 3-day-weighed record method. The data from databases obtained from the official control and monitoring of food additive content  Contact: [email protected].

68 Elizabeta Mičović, Mario Gorenjak, Gorazd Meško et al. in consumed food were used to calculate estimated daily intake (EDI). Such estimated exposure of each preservative and polyphosphates EDI was compared with acceptable daily intake (ADI) and expressed as % of ADI. Results: average exposure to each preservative and polyphosphates EDI was not exceeding ADI. It is evident that average exposure to nitrites and sulphur dioxide is relatively high, while intake of benzoic acid, sorbic acid, nitrates and polyphosphates is not so high. The mean daily exposure of children to nitrites ranged from 12.8 % to 28.3 % ADI, to sulphur dioxide from 14.3 % to 21.4 % ADI, while to sorbic acid from 3.8 % to 4.5 % ADI and polyphosphates from 1.8 % to 3.9 % ADI. It is apparent that such exposure does not present any harm or threat to observed children although we should consider the fact that ADI for sum of preservatives and polyphosphates have not been set yet. Keywords: Preschool children, exposure, preservatives, polyphosphates, ADI 1. Introduction Environmental factors play a major role in determining the health and well-being of children. Accumulating evidence indicates that children, who comprise over one third of the world’s population, are among the most vulnerable of the world’s population and that environmental factors can affect children’s health quite differently from adults’ health (WHO 2006). One of the most important environmental factors, which have strong impact on children health, is food. Children have different susceptibilities towards food during their different life stages, giving their dynamic growth and developmental processes as well as physiological, metabolic, and behavioural differences. Children consume more food and beverages per kilogram of body weight than adults do. Also their dietary patterns are different and often less variable during different developmental stages. Children’s metabolic pathways may differ from those of adults. They have more years of future life and thus more time to develop chronic diseases that take decades to appear and that may be triggered by early environmental exposures. They are often unaware of environmental risks and generally have no voice in decision-making. The accumulating knowledge that children may be at increased risk at different developmental stages, with respect to both biological susceptibility and exposure, has raised awareness that new risk assessment approaches may be necessary in order to adequately protect children. Traditional risk assessment approaches and environmental health policies have focused mainly on adults and adult exposure patterns, utilizing data from adult humans or adult animals. There is a need to expand risk assessment paradigms to evaluate exposures relevant to children from preconception to adolescence, taking into account the specific susceptibilities at each developmental stage. The full spectrum of effects from childhood exposures cannot be predicted from adult data. Risk assessment approaches for exposures in children must be linked to life stages (WHO 2006). Almost in all food categories, preservatives are added to ensure longer shelf life of food products. It is very important that only proven preservatives can be used by food producers, in certain food product and in allowed quantities. Exposure to each preservative must not exceed acceptable daily intake (ADI), the amount of the substance considered to be safely consumed, daily, throughout a lifetime. This assessment is used to set the maximum amount of a particular additive permitted in a specific food, either as a specified number of grams or

Exposure of Slovenian Preschool Children to Preservatives ... 69 milligrams per kilogram or liter of the food or, if the ADI is very high or “non-specified”, at quantum satis - as much as is needed to achieve the required technological effect, according to good manufacturing practice. To ensure that consumers are not exceeding the ADI by consuming too much of, or too many products containing a particular additive, the EU legislation requires that intake studies be carried out to assess any changes in consumption patterns (Emerton and Choi 2008). The vast majority of toxicity studies and risk evaluations deal with single preservatives and single chemical. In reality, humans are exposed to large numbers of chemicals via multiple routes (Feron and Groten 2002; Feron et al 2002; Groten et al. 2004). So far, possible effect of mixture of all chemicals and interactions between them, so called ’cocktail effect’, have not been understood fully. Food additives are typically used in combination within processed foods and therefore collectively may have some adverse effects at the cellular level, even if their individual concentrations are below the ADI value (Lau et al. 2005). 2. Consumer as Possible Victim of Invisible Threats “Consumers by definition, include us all. They are the largest economic group, affecting and affected by almost every public and private economic decision. Furthermore, consumers are the only important group whose views are often not heard” (Kennedy 1962). Consumer is a person who buys goods or services for personal needs and not for resale or use in the production of other goods for resale (Consumer protection Act, 2004). Consumers expect a wide range of competitively priced, highly processed and convenient food products of consistently high quality. They expect it to be fresh, good looking, nutritious, wholesome, tasty and it must be primarily and absolutely safe. On the other hand, consumers have no direct means for the verification of their expectancies and have to rely completely on the food legislators and enforcement agencies (Anklam and Battaglia 2001). Consumers could be victims of food poisoning, food adulteration and food frauds, misleading regarding food content (labelling), misleading indications, misleading descriptions, misleading pictures, food packaging (Jin and Kato 2004; Gibson and Taylor 2005; Tombs 2008; Croall 2009). In today’s technological age, a reactive response to the consumer fraud is neither efficient nor effective (Holtfreter et al. 2005). Consumers are privileged to have human rights. However, they come with certain responsibilities too - to seek, to evaluate and to use available information on products and services, to make healthier and better decisions for themselves and for their children. In case of exposure to different chemicals in food, consumers know that food product consist additives, but they do not know the quantity of them. Total intake is unknown, so how could parents be sure that the consumed food is safe for their children? It is very important to consider different sensitivity of population, especially among infants and children.

70 Elizabeta Mičović, Mario Gorenjak, Gorazd Meško et al. 3. Food Safety The main challenge in the area of agriculture is to provide sufficient quantities of food, which have to be of good quality and safe. Food production and food consumption are the primary aspects of our lives and are therefore subject to our care. To achieve these goals agriculture and food industry have to use in their production different chemicals such as pesticides and food additives. Without the use of chemicals the yields of fields will reduce, food production and the earnings will be considerably smaller. While with using chemicals the benefits are visible, the risks are often invisible, even they effects on us and make us vulnerable (Beck 2001). Safe food is food that is free from not only toxins, pesticides, chemical and physical contaminants, but also from microbiological pathogens such as bacteria and viruses that can cause illness (Golob and Jamnik 2004). There are main concerns regarding food safety. Consuming food is daily routine activity throughout lifetime. It is very important that such food does not cause health risk to consumer. Possible hazards in food are microorganisms, viruses, contaminants (toxins, heavy metals), pesticides and other chemicals. Consumer could be concerned and afraid of such hazards, but in many cases one does not know exact quantity of exposure and exact effect on his health. Therefore, it is very important to be aware of possibility of invisible threats in food. Food industry has a strong motive to make profit and many opportunities to manage it. Food operators try to convince consumers that they need their products, so they use all sorts of food additives, ingredients and advertising tactics to achieve better sale (Cheftel 2005). Consumers have the legal right to be protected (Xu and Yuan 2009). The long-term health of consumers are also endangered by the use of foods and other consumer products of a vast range of chemicals and other substances that, while associated with long- term health risks, do not result in immediate harm. While there is a growing public concern about the number of foods and consumer issues, these facts have a lower political and governmental profile than the occupational health and safety or the safety of the environment (Croall 2009). The main authority concerns, regarding food safety, are to protect interests of public health, interests of food producers (economic view) and the consumer interests and their rights. It is not easy to make right decision and to achieve all that goals in practice at the same time. Recognizing possible invisible threats could assure better consumer protection. Furthermore, knowing all the risks of invisible threats in the food area help us to make corrective measures on time. These measures could make system for food safety more effective and give consumers better protection. The big challenge in the area of food safety for consumer health is recognition of possible invisible threats on time. This can help us to set up effective response regarding those threats and risk assessment. Identifying all potential hazards that have to be assessed, eliminating or reducing them to acceptable levels, are the most important activities for achieving consumer protection, specially their basic right to safety (Mičović 2010). 3.1. Risk Analysis Public health decisions on the plausible risks of chemical exposures can include several possible outcomes. The ultimate goal is to implement a risk management action that will

Exposure of Slovenian Preschool Children to Preservatives ... 71 produce the desired reduction of risk. A risk analysis paradigm is a formal representation of a process that distinguishes the scientific bases from the risk management objectives and generally contains a component where the probability of harm is estimated. The overall risk analysis process includes risk assessment, risk management and risk communication, and involves political, social economic and technical considerations. Moreover, there is consensus among scientists that risk assessment should be an independent scientific process, distinct from measures taken to control and manage the risk. Risk assessors are responsible for scientific evaluation whether a formal risk assessment is necessary or not (Benford 2001). As a probability calculation, a risk assessment will include both a statement of the objective under consideration (harm) and the basis for the assertion that the harm may occur (probability). Risk management is the decision-making process involving the consideration of political, social, economic and technical factors with relevant risk assessment information relating to a hazard so as to develop, analyze, select and implement appropriate risk mitigation options. Risk management comprises of three elements: risk evaluation, emission and exposure control and risk monitoring (WHO 2004). Risk management strategies may be regulatory, advisory or technological and take into account factors such as the size of the exposed population, resources required and available, costs of implementation and the scientific quality and certainty of the risk assessment. Risk managers are responsible for judgments concerning the acceptability of risk; they have to weigh risk against other factors including costs, benefit and social values, so called risk – benefit approach. Risk communication should include interactive exchange of information and opinions among risk assessors, risk managers, consumers and all other interested parties, often called stakeholders (Benford 2001). 3.1.1. Risk Assessment The procedures used to estimate exposure to chemicals contaminants in food are essentially the same as those used for food additives (DiNovi and Kuznesof 2006). Exposure assessment should cover the general population, as well as critical groups that are vulnerable or are expected to have exposures that are significantly different from those of the general population, for example infants, children, pregnant women, or the elderly (WHO 2005). Risk is defined as the chance or probability of an adverse health effect occurring and severity of that effect (Benford 2001). Risk assessment is a scientific process, conducted by scientific experts, who may begin with a statement of purpose intended to define the reasons that the risk assessment is required and support the aims of the subsequent stages of risk management. Chemical risk assessment often does not have a formal statement of purpose (Benford 2001). Generally, formal risk assessments are preceded by preliminary risk assessments. These are usually subjective and informal and may be initiated from inside or outside the risk assessment and scientific communities. A key consideration of these preliminary risk assessments is whether a formal risk assessment is necessary or not (WHO 2004). However, risk assessment may be defined implicitly in a generic form, as in the terms of reference of an expert committee, such as Joint FAO/WHO or JECFA. In this context, it concerns the definition of acceptable or tolerable levels of intake for a chemical in food that may require review and revision in the light of new information. It is very important fact, that risk assessment may need to be quantified differently for persons with different degrees of susceptibility. Risk assessment for chemical agents requires consideration of the factors mentioned before and these are generally encompassed within the stages of the overall risk

72 Elizabeta Mičović, Mario Gorenjak, Gorazd Meško et al. assessment process, defined as hazard identification, hazard characterization, and exposure assessment risk-characterization (Benford 2001). Hazard identification is the process of identification of the type and nature of adverse health effects (human studies-epidemiology, animal – based toxicology studies, in- vitro toxicology studies, structure-activity studies (cell cultures, tissue slices). Hazard characterization involves the derivation of a level of exposure at or below which there would be no appreciable risk to health if the chemicals were to be consumed daily throughout life. Exposure assessment is evaluation of concentration or amount of a particular agent that reaches a target population: magnitude, frequency, duration, route, extent. Risk characterization is the stage of risk assessment that integrates information from exposure assessment and risk characterization into advice suitable for use in decision-making (Benford 2001). The basic concept underlying any chemical risk assessment is the dose-response relationship. As described by Paracelsus nearly 500 years ago, “All substances are poisons; there is none which is not a poison. The right dose differentiates a poison and a remedy” (Winter and Francis 1997). This means that any chemical substance is likely to produce some form(s) of harmful effect, if taken in sufficient quantity. Experts refer to a potential harmful effect as a hazard associated with that substance. The definition of hazard is “a biological, chemical or physical agent with the potential to cause an adverse health effect” (Unnevehr 2003; Raspor, 2004; Armstrong 2009). Whilst this may be appropriate with respect to pathogenic organisms, chemical substances may be associated with a number of different adverse health effects, not all of which would necessarily be expressed in a specific exposure scenario. Therefore, experts dealing with chemical substances prefer to define the potential health effects as individual hazards which need to be considered separately during the evaluation. The likelihood or risk of that hazard actually occurring in humans is dependent upon the quantity of chemical encountered or taken into the body, i.e. the exposure. The hazard is an inherent property of a chemical substance, but if there is no exposure, then there is no risk that anyone will suffer because of that hazard. Risk assessment is the process of determining whether a particular hazard will be expressed at a given exposure level, duration and timing within the life cycle, and if so the magnitude of any risk is estimated (Benford 2001). Among the first objectives of a risk assessment is the determination of the presence or absence of a cause-effect relationship. If there is sufficient plausibility for the presence of such a relationship, then dose - response modelling (DRM) information is needed. 3.1.2. Acceptable Daily Intake (ADI) and Estimated Daily Intake (EDI) of Food Additives The concept of the ADI, is internationally accepted today as the basis for estimation of safety of food additives and pesticides, for evaluation of contaminants and by this, for legislation in the area of food and drinking water. The ADI (Chart 1) is an estimate of the amount of a food additive, expressed on a body weight basis that can be ingested daily over a lifetime without appreciable health risk (WHO 1987). Although, ADI has derived from the safety assessment of each food additive, their combined adverse effects are unclear and have not been widely studied. Food additives are typically used in combination within processed foods and therefore, collectively may have some adverse effects at the cellular level, even if their individual concentrations are below the recommended ADI value (Lau et al. 2005).

Exposure of Slovenian Preschool Children to Preservatives ... 73 Dose response modelling (DRM), used as quantitative risk assessment tool for public health recommendations about chemical exposures, can be described as a six-step process. The first four steps—data selection, model selection, statistical analyses, and parameter estimation—constitute dose–response analysis. The fifth step involves the integration of the results of the dose–response analysis with estimates of human exposure. The final step involves an assessment of the quality of the dose–response analysis and the sensitivity of model predictions to the assumptions used in the analysis. Chart 1. Acceptable daily intake of preservatives and polyphosphates Preservatives E number ADI (mg/kg BW/day) Sorbic acid E 200 0-25 (JECFA*, 1973) Benzoic acid E 210 0-5 (JECFA, 2002) Nitrate E 251 0-3.7 (SCF**; JECFA 2002) Nitrite E 250 0-0.07 (JECFA, 2002) Sulphur dioxide E 220 0-0.7(JECFA, 1973) Polyphosphates (E 450-452) 0-70(JECFA, 1981-2001) *, **Acceptable daily intakes have been derived from toxicological studies by the former EU Scientific Committee on Food (SCF) and by the WHO/FAO Joint Expert Committee on Food Additives and Contaminants Extrapolation is a fundamental problem in the quantitative health risk assessment of exposure to chemicals that are toxic to human in experimental systems. Adverse health effects of chemicals are, in the absence of human data, typically evaluated in laboratory animals at significantly higher doses than the levels to which humans may be exposed. Moreover, the data obtained in animals are very often misleading, as the animals used usually do not respond to the toxic compounds in the same way as humans (WHO 2004). The acceptable daily intake (ADI) is used widely to describe “safe” levels of intake; other terms that are used are the reference dose (RfD) and tolerable intakes that are expressed on either a daily (TDI or tolerable daily intake) or weekly basis. The weekly designation is used to stress the importance of limiting intake over a period of time for such substances (Herman and Younes 1999). In order to calculate an ADI using the data from toxicity studies, the lowest dose should ideally result in no effects under the conditions of the particular study. That dose may be termed as the No Observed Effect Level (NOEL). Observed effects are referred to assumptions and can not be made regarding effects not detectable by the methods used. Some effects observed in toxicity studies may represent adaptive responses with no implications for the health status of the animal and would generally not be used as the basis for establishing an ADI. Effects that are considered to result in harm to the animal are referred to as “adverse”, and therefore some expert committees use the expression No Observed Adverse Effect Level (NOAEL) (Benford 2001). When using this approach NOAELs are identified in the critical studies, to which appropriate safety or uncertainty factors are applied. Although the value of safety factors varies depending upon a number of factors, 100 is most often used, which is designed to account for interspecies and interspecies variations (Herman and Younes 1999).

74 Elizabeta Mičović, Mario Gorenjak, Gorazd Meško et al. ADI = NOAEL/ 100 Estimated daily intake (EDI) is value of chemical exposure, which can be determined by combining food consumption data with data on the concentration of additives in food. The resulting dietary exposure estimate is afterwards compared with the relevant toxicological or nutritional reference value for the food additive concern, for example acceptable daily intake (ADI) or tolerable daily intake (TDI) (WHO, 2005). Usually EDI is expressed as percentage of value of ADI for each food additive. If EDI is lower or the same as ADI, there is no concern regarding such exposure and we can assume that food additive intake among observed population represent no risk. If EDI is higher or the same as ADI, than exact risk assessment should be done on case-by-case base, and risk managers should decide and take effective measurements. 3.1.3. Preservatives and Polyphosphates For successful food production and selling food the most important are quality, safety, shelf life and price of the food product. Quality food product is source of suitable nutrients but it should has good taste and look, but above all it should be safe for consumption and must not be harmful for health of consumer (Lu 1991; Renwick 1996; Emerton and Choi 2008). Additives are substances which are intentionally added to food products and are therefore amenable to be limited or, if necessary, prohibited altogether (Huggett at al. 1998). They are not main ingredients in food products but they are added to food with aim to improve technological or sensory properties (look, taste, texture, longer shelf life). The authorization of food additives should be determined food, which these additives may be added, and the conditions under which they may be added. One principle is certain additives permitted for each sort of food and the other is ADI for this food additive. In certain food products, the addition of additives is prohibited (such as preservatives in dairy products) or limited (baby food). Therefore should be precise, what allowance should be additive to achieve the desired effect. The principle is to be tries with the lowest possible dose of additives to achieve the desired effect. It is very important to take into account the acceptable daily intake or other additive intake estimates and the probable daily intake from all sources. When the additive is used in food for groups of consumers with special dietary needs should be considered acceptable daily intake of a food additive for this group of consumers (Emerton and Choi 2008). Preservatives are certainly the most important group of additives, since they play an important role in ensuring food safety. They are used to protect against food poisoning, so as to prevent the development of microorganisms and thereby prolong the shelf life and achieve stability of food products (Emerton and Choi 2008). There are natural and unnatural preservatives (Simon and Ishiwata 2003) which prevent growth of bacteria, fungi and viruses and thus prevent food from spoilage. While food preservatives can increase stability of food, polyphosphates give certain product sensory properties (taste, mouth feel and texture). Thus, there are also polyphosphates, which are added to the meat industry in a variety of products in order to make the meat retained more water you add to food products according to a recipe. The production of meat products phosphate additives primarily use to an increase in binding of water, which naturally improves the taste. In cooked meat products and precipitated such added 0.1% to 0.5% polyphosphate (Emerton and Choi 2008; Uribari 2009).

Exposure of Slovenian Preschool Children to Preservatives ... 75 In addition to the meat industry, where polyphosphates represent one of the key technological elements of the food additives used in the manufacture of cheeses, bakery products and a variety of drinks. Some studies linking intake of preservatives to the occurrence of allergic reaction, food intolerance and urticaria (Yang and Purchase 1985; Hannuskela and Haahtela 1987; Schultz-Ehrenburg and Gilde 1987; Steinman, Le Roux and Potter 1993; Hawkins and Katelaris 2000; Merget and Korn 2005, Andersson, Knutsson, Hagberg, Nilsson, Karlsson, Alfredsson and Torén 2006; Michaëlsson and Juhlin 2006), so it is very important to find out exact exposure to food additives. 4. Study among Preschool Children in Slovenia The purpose of research was to assess if preservatives and polyphosphates intake could be possible threat to the health of preschool children. It is assumed that intake of each food additive separately could not be so high or greater than ADI. However, it is unknown the total intake of all food additives per day. It is assumed that higher exposure to food additives is in relation to appearance with possible harm reactions among observed children. In the first place, it is necessary to find out the exact quantity of consumed food and then exposure to food additives, in our case preservatives and polyphosphates, added to food. 4.1. Methodology 4.1.1. Observed Population Among by randomly selected regions in Slovenia, we selected kindergartens and all children aged 2-6 years. Initially, the study included 250 children, representing all children of one section of selected kindergartens. Due to incomplete data, 60 children were excluded from observation. Therefore, the total number of children used in this study were 190, 98 boys and 92 girls. Anthropometric measurements of experimental children groups were conducted as for example data regarding their age and gender, weight and height. In the selected group, Boys and girls were divided into two groups regarding their age: 2-3.9 years and 3.9-6 years. 4.1.2. Food Consumption Data The dietary intake is generally based on the 3-day-weighed record method in the kindergartens and homes. In the kindergartens subject to the research, we have weighed quantities of individual foods and dishes, randomly chosen and offered to the observed children. Quantity of food eaten by children at home, were collected by questionnaire, which were answered by parents. Data were calculated by average day and body weight of each child included into research. Food intake (g) = food offered (g) - waste food on the plate (g)

76 Elizabeta Mičović, Mario Gorenjak, Gorazd Meško et al. From these data, we have obtained the exact amount of food eaten by each child in three days. Specific types of food it has divided regarding to their characteristics in the following categories as shown Chart 2. It was considered those food categories in a particular food or otherwise adds any additives, which are the subject of our research. These are mainly processed industrially prepared food. Chart 2. Food categories included to study Food category Food product coca-cola, ice-tea Non-alcoholic beverages margarine, butter Fat spreads bread, cookies, pastry Bakery products dried fruits, jams, candied fruits Fruit products chocolate like products, cream cakes, milk cakes Confectionary products salami, pate, hot-dogs Meat products salty snacks Snacks 4.1.3. Estimated Daily Intake of Preservatives and Polyphosphates (EDI) Additives included in the research are preservatives: sorbic acid (E 200), benzoic acid (E 210), nitrate (E 251), nitrite (E 250), and emulsifiers: polyphosphates (E 450-452); To estimate the daily intake of preservatives and polyphosphates we had to combine food consumption data with data on the concentration of observed additives in food. EDI = food additive concentration x food consumption per body weight Calculation of the average daily consumption of specific groups of food per body weight for each child was made. The data from databases obtained from the official control and monitoring on food additives content in different food categories were used. To calculate how much of each additive, the children daily ingested per kg body weight, food additives were added together, equal and shared with the child's body weight expressed in kg and thus received the additive value in mg/kg body weight. 4.1.4. Comparison between ADI and EDI To assess safety of food additives exposure, ADI of each food additive have been carried out. The estimated daily intake (EDI) of each additive is acceptable and safe if its value is lower or the same as ADI. To determine safety assessment among observed children regarding exposure to preservatives and polyphosphates, EDI was compared with ADI, and expressed in percentage of ADI. 5. Results 5.1. Characteristic of Observed Sample The study included 250 preschool children, but, as shown in Chart 3, we have 60 excluded from consideration. The reason for this was that in the absence of some children in

Exposure of Slovenian Preschool Children to Preservatives ... 77 kindergarten at the time of carrying out research or incomplete information on foods eaten at home, provided by the parents. That was the deal involved only 190 children, 98 boys and 92 girls aged 2 to 6 years. The population of children was divided into two age groups: 2 to 3.9 years and 4-6 years, were divided by gender: girls (F) and boys (M). In the first age group was 31 boys and 20 girls in the second age group was 67 boys and 72 girls. The average body weight of each considered children in the first group of boys was 16.86 kg ± 1.74 kg and among girls 14.95 kg ± 1.44 kg. In other age group, average weight was 20.61 kg ± 4.03 kg among boys, and 19.60 kg ± 2.96 kg among girls. Chart 3. Observed population Group Gender No. of Avg. body Standard deviation 2-3,9 y children weight (Kg) 4-6 y M 31 16.86 1.74 F 20 14.95 1.44 M 67 20.61 4.03 F 72 19.60 2.96 5.2. Food Consumption Data Weighing three-day method was used to determine the food intake data among observed children in kindergarten. Quantity of food eaten by children at home, were collected by questionnaire, which were answered by parents. Consumed food were classified into food categories shown in Chart 2, and calculated the average intake of each food category. We performed the anthropometric measurements of observed children, and take into account body weight to calculate the intake of food per kilogram of body weight of each child. Chart 4 therefore shows the average daily intake of all consumed food in three days, expressed in grams per kg body weight (g/kg BW). In the first age group boys consumed significantly more food than girls. In another age group 4-6 years, the difference in quantity of consumed food among girls and boys is not so relevant. Regarding the above results, it is interesting fact that the intake per kilogram of body weight is higher among smaller and younger children. This is very important regarding of risk assessment and set the acceptable daily intake of additive. In case of the same amount of consumed food is exposure to food additives for lighter child higher than exposure to equal quantity of additives for child with higher body weight. Figure 1 shows the average intake of food categories among observed children. It is clear that children consume most bakery products and dairy products, which is understandable. Bakery products and milk products are basic foodstuffs included in a daily nutrition of preschool children and include bread, breakfast cereals, cereal grains, cereals, milk and yogurt. Compared to other food it is obviously that quite large intake of consumed food belongs to confectionery products, such as sweets, chocolate, chocolate-like products, lollipops, chewing gums and meat products such as salami, hot dogs and pate.

78 Elizabeta Mičović, Mario Gorenjak, Gorazd Meško et al. Chart 4. Average daily intake of all consumed food Group Gender Average daily Standard deviation Average intake 2-3.9 y intake (g) of average daily (g/kg BW) 4-6 y M intake F 350.63 83.19 20.79 M 318.51 65.05 21.30 F 336.82 61.79 16.34 330.61 65.51 16.86 Figure 1. Average intake of food category among observed children. These products are very popular among children and they like them. Among older children we noticed increasing of consumed fat spreads, bakery products, fruit products, confectionery, meat products and snacks and decreasing intake of dairy products. This is logical, because parents offer dairy products in the meals more often to younger children. Relatively high quantity soft drinks (1.79 ± 5.44 g/kg BW) were consumed by girls aged 2 to 3.9 years, which is clearly shown in Figure 1. Category of fat spreads and dairy products were excluded from further analysis because the observed preservatives and polyphosphates were not having been identified in official control in these categories of food. 5.3. Estimated Daily Intake of Food Additives Levels of preservatives and polyphosphates in different food products are known from official inspections and analysis reports on content of food additives in different food

Exposure of Slovenian Preschool Children to Preservatives ... 79 products. Food products were set in food categories shown in Chart 2 and Figure 1. The results show that preservatives are added into bakery products (sorbic acid and benzoic acid), non-alcoholic beverages (sorbic acid and benzoic acid), fruit products (sulphur dioxide) and meat products (nitrites, nitrates, and polyphosphates). From results of analysis reports, we can calculate exact quantity of preservatives and polyphosphates added in consumed food. As Chart 5 shows, the higher intake of preservatives and polyphosphates expressed in mg/kg body weight, are among older children, 4-6 years old girls. It is very interesting, that the highest daily intake of preservatives and polyphosphates belongs to polyphosphates, commonly added to meat products. Chart 5. Average intake of preservatives and polyphosphates (mg/kg BW) Benzoic Sorbic SO2 Polypho Nitrites Nitrates sphates mg/Kg mg/Kg Group acid acid BW mg/Kg/BW mg/Kg BW TOTAL SD mg/kg mg/kg BW 2-3.9 years BW BW 0.126 0.027 2.577 0.60 old M 0.024 1.022 1.368 0.01 2.627 0.58 2-3.9 years 0.1 0.024 3.362 0.85 old F 0.122 1.13 1.242 0.009 3.935 1.08 4-6 years old 0.127 0.041 M 0.065 1.008 2.106 0.015 4-6 years old 0.15 0.053 F 0.032 0.945 0.503 2.735 0.02 0.145 TOTAL 0.243 4.105 0.02 0.01 SD 0.04 0.08 7.451 0.054 0.70 0.01 Figure 2. Average intake of benzoic acid.

80 Elizabeta Mičović, Mario Gorenjak, Gorazd Meško et al. Figure 3. Average intake of sorbic acid. The average daily intake varied between 1.242 to 2.735 mg/kg BW. This fact is not so unusual, regarding to high value of the ADI for polyphosphates (0-70mg/kg BW/day). It is obviously that this result goes in line with quantity of consumed meat products by girls 4-6 years old. In comparison with the intakes of other preservatives is also fairly high daily intake of sorbic acid, which is on average from 0,945 to 1,130 mg/kg BW/day. Figures 2-7 show the average amount consumed each observed preservatives and polyphosphates among children included in the study and it is obviously that average intakes of each preservative do not exceed the ADI. Figure 4. Average intake of sulphur dioxide.

Exposure of Slovenian Preschool Children to Preservatives ... 81 Although average intakes above additives do not exceed the ADI, we have found eight children who have been exposed to higher levels of intake than ADI, namely exposure to sulphur dioxide, which is 1.048 to 1.930 mg/kg BW. ADI of sulphur dioxide is 0-0.7 mg/kg BW. Estimated daily intakes of other preservatives and polyphosphates among the individual child do not exceed the ADI. Figure 5. Average intake of polyphosphates. Figure 6. Average intake of nitrites.

82 Elizabeta Mičović, Mario Gorenjak, Gorazd Meško et al. Figure 7. Average intake of nitrates. Figure 8. EDI–ADI relationship of preservatives and polyphosphates.

Exposure of Slovenian Preschool Children to Preservatives ... 83 Figure 9. Average total intakes of preservatives and polyphosphates. 5.4. Results of Comparison between EDI and ADI of Preservatives and Polyphosphates Results of safety assessment regarding relation between EDI and ADI of preservatives and polyphosphates show that average intake of each additive are acceptable and safe. Figure 8 presents EDI - ADI relationship of preservatives and polyphosphates. It is evident that exposure to nitrite and sulphur dioxide is relatively high, the highest among older children. Intake of benzoic acid, sorbic acid, nitrates and polyphosphates is not so high. It is interesting that EDI to sulphur dioxide and nitrite is relatively high in compare to other preservatives and polyphosphates, and it is in range 14.3 to 21.4 % ADI for sulphur dioxide and 21.4 to 28.3 % ADI for nitrite. It could be concluded that basic food products that are consumed very often by children (few times a day) contain sulphur dioxide and nitrites. Such food products are fruit products and meat products. 5.5. Results of Total Intake of Preservatives and Polyphosphates Although average intake of each preservative and polyphosphates do not exceed ADI and we can conclude that such exposure is acceptable and safe, we wonder about exposure of total intake - preservatives and polyphosphates together. We calculated sums of intakes of all observed preservatives and polyphosphates and we expressed them for each group of children in Figure 9. Results show that sum of total intake of preservatives and polyphosphates for each group of children is in range from 2.63 to 3.36 mg/kg BW. Total intake is higher among older children, especially among girls from 4-6 years old. Unfortunately we cannot compare these intakes with any ADI, because ADI for mixtures of additives have not been set yet. Anyway, values in consideration with average children’s body weight tell us enough for themselves.

84 Elizabeta Mičović, Mario Gorenjak, Gorazd Meško et al. Conclusion The human health risk assessment of food constituents is an internationally agreed and well-established process, being an integral part of the risk analysis process, which also includes risk management and risk communication. These three elements are separate tasks, performed by different players, however each of them is very important. With exchanging knowledge and cooperation, they are parts of an interactive process. The risk assessment of chemicals in food is a purely scientific process that requires expertise in toxicology, nutrition and exposure assessment. The risk management includes an identification of the food safety problem, consideration of its magnitude, seriousness, and consequently the way of handling it. In this process, the risk manager may include cost-benefit considerations before deciding how to manage the case (ban the compound, introduce limitations, provide specific dietary advice or accept the status quo). Finally, the risk analysis must include a clear and interactive risk communication with consumers, food industry and other stakeholders. In this study an example of risk assessment is presented, special exposure assessment of preservatives and polyphosphates among preschool children in Slovenia. Ultimately 190 children attending kindergarten (of which 92 girls and 98 boys aged 2-6 years) took part in the study. The results showed that nutrition regarding the quantity of food consumed among Slovenian preschool children does not differ between regions in Slovenia. It is obvious that people responsible for preparing meals in kindergartens follow the guidelines for healthy nutrition recommended by Ministry of Health of Republic of Slovenia. Food habit questionnaires answered by parents of children participants at home showed a big difference between children nutrition in kindergartens and that at home. The observation of individual child included in the study showed the differences in pattern of food consumption at home. Parents have very different levels of knowledge regarding ingredients included in food products, healthy nutrition and possible effect of food additives on children health. Some of them are aware of the importance of healthy balanced diet and nutrition. They offer their children food products without food additives, or they rarely offer them processed foods containing additives in relatively small quantities.. They also combine meals at home with food that has not been eaten in the kindergarten. However, there are some parents, who do not pay attention to food included into meals at home and composition of meals in kindergarten. The study’s results show, that their children were exposed to higher level of preservatives and polyphosphates. It is apparent that such exposure does not present any harm or threat to observed children, regarding comparison between estimated daily intake (EDI) and acceptable daily intake (ADI) of each food additive. The other very important problem is the total intake of all food additives daily consumed by children: preservatives, polyphosphates, sweeteners and colours together. The fact is that ADI for sum of food additives has not been set yet. According to the study’s results, exposure to preservatives and polyphosphates is relatively high among observed children. We must not forget that children consume also other food additives, such as colours and sweeteners. We strongly believe that future study should lead to exposure assessments of mixture of chemicals and interactive influence among them. We must not forget that children are

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