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LLP “OO KAZAKH ACADEMY OF NUTRITION” RSE "Scientific and Practical Center of sanitary-epidemiological examination and monitoring” of the Ministry of Health of the Republic of Kazakhstan MATERIALS FOR HUMAN HEALTH RISK ASSESSMENT OF TETRACYCLINE INTAKE WITH FOOD Almaty, 2013 1 Content: INTRODUCTION………………………………………………………………….3 I. HAZARD IDENTIFICATION………………………………..…………………4 1.1 Body distribution……………………………………………………………6 1.2 Main effects………………………………………………...……………….6 II. HAZARD STATEMENT………………………………………..…………….10 2.1 Permissible exposure limit……………………………..………………….10 2.2 "Exposure-effect" dependency evaluation……………………………….12 III. EXPOSURE SSESSMENT…………………………………………………..14 IV. RISK SSESSMENT…………………………………………………………16 V. ASSESSMENT OF UNCERTAIN RESULTS…………..…………………..22 CONCLUSION…………………………….…………………………………….23 REGERENCE LIST……………………………………………………….…….24 2 INTRODUCTION Tetracyclines are broadly used antibacterial drugs that unite a group of substituted polyoxy-polycarbonyl hydroaromatic compounds which have the same structure and produced by bacteria of Streptomyces type. Due to chemical modification of these substances semisynthetic tetracyclines are obtained [1,2]. Basic tetracycline compounds were produced in 1948-1952. Main characteristics of representatives of this family are a general spectrum and mechanism of antimicrobial action, whole crossed resistance and close pharmacological characteristics. The differences relate to the degree of antibacterial effect, peculiarities of absorption, distribution, metabolism in the body, tolerability and some physicochemical properties [3]. 35 compounds of this group are described. According to the mechanism of biological action tetracyclines belong to the group of antibiotics that inhibit protein synthesis. Tetracycline within bacterial cells is an inhibitor of protein chain elongation and it prevents binding of another aa-t RNA. Tetracycline antibiotics are used in the treatment and prevention of bacterial infections of both human and animals. In animal husbandry, they are used as therapeutic and veterinary drugs, and growth promoters [4]. Tetracycline used as growth promoters in animal husbandry increase body weight by an average 4.5%, costs of feed per unit of gain is reduced by 5-8%, activates body's resistance and reduces animal feeding season. Intake of these products by poultry stimulates their increase, egg production, hatching egg quality, and contributes to a more efficient use of feed and better uptake of protein [5]. From the viewpoint of food hygiene the main danger of antibiotic residues for human is that it can get into human body through feed together with the agricultural products, which negatively affect intestinal microflora, lower resistance to infections, and increase resistance to antibiotics [4]. The legislation of the European Union, the USA, norms and standards of the Codex Alimentarius differ significantly in measurement of residual amount of 3 tetracycline (MRLs ≤0,1-1,2 mg/kg), which in its turn differ significantly from the requirements of the Customs Union regulations (≤0,01 mg/kg of product). Thus, according to the Codex Alimentarius and the EU legislation milk is normalized by no more than 100 µg/kg, in the United States it is not regulated, in the countries of the Eurasian Customs Union (CU) it is not accepted. This scientific work is done in accordance with the practical principles of risk analysis in terms of food safety, which can be used by governments of the Codex Alimentarius Commission (CAC/GL 62-2007) [45]. I. HAZARD IDENTIFICATION Hazard identification of tetracycline residues is carried out on the basis of results of various research studies available in the scientific literature, including those conducted in respect to humans. Tetracycline antibiotics amount to over 52% of all antibiotics used in animal husbandry and veterinary medicine in Europe [6]. Tetracyclines have a bacterial growth-inhibiting action in therapeutic dose. In animal husbandry the semisynthetic tetracyclines based on oxy- and chlortetracycline are used, including tetracycline carboxamide derivatives with a prolonged effect. They have several distinct properties, which include high solubility in water at various values of pH (2,0 -8,5), fast absorption in gastrointestinal tract, higher concentration in the blood stream during the first hours after their administration. Antibacterial action of tetracyclines is based on suppression of protein synthesis due to dyspoiesis of a complex between soluble RNA and ribosome. Tetracyclines are active with respect to gram-positive organisms: Staphylococcus spp. (including Staphylococcus aureus, including producing betalactamase strains), Streptococcus spp. (including Streptococcus pneumoniae), Listeria spp., Bacillus anthracis, Clostridium spp., Actinomyces israelii; gramnegative micro-organisms: Haemophilus influenzae, Haemophilus ducreyi, 4 Bordetella pertussis, most enterobacteria: Escherichia coli, Enterobacter spp., including Enterobacter aerogenes, Klebsiella spp., Salmonella spp., Shigella spp., Yersinia pestis, Bartonella bacilliformis, Vibrio cholerae, Vibrio fetus, Rickettsi spp., Borrelia burgdorferi, Brucella spp., they are also active with respect to pathogens of venereal lymphogranuloma, Treponema spp. These micro-organisms are resistant to tetracycline: Pseudomonas aeruginosa, Proteus spp., Serratia spp., most strains Bacteroides spp. and mold fungi, viruses, beta-haemolytic streptococcus of А-group (including 44% of strains Streptococcus pyogenes and 74 % of strains Streptococcus faecalis) [7.] 1.1 Body distribution During parenteral administration tetracycline is well absorbed and penetrates into many organs and tissues. It poorly penetrates through the intact blood-brain barrier, but in case of cerebrosis and disease of brain membranes its concentration significantly increases within cerebrospinal fluid. Plasma binds with protein by 65%. It is selectively accumulated in bones, teeth, liver, spleen, and tumour tissues in significant numbers. It easy penetrates through the placental barrier and is excreted in breast milk. After intake 60-80% of the dose is absorbed from the gastrointestinal tract. It is cleared through the urine and fecal remaining unchanged (fecal - 20-50%, urine 20-69%). In case of disorder of excretory function of the kidneys, tetracycline concentration in blood increases, which can lead to accumulation [8]. Lipophilic doxycycline has 2-3 times longer half-life elimination compared to tetracycline. Despite good blotting capacity in digestive tract, residual tetracycline concentration in gut is always sufficient to affect the intestinal microflora [7,10]. 1.2 Main effects When a single dose of tetracycline equal to 75mg / kg was administered to white rats, plasma peak concentration (up to 3.6 mg/l) were identified in 2 hours after 5 administration, which decreased by 0.5 mg/l in 6 hours. Peak concentration in their liver and kidneys were identified in 2 hours after administration of that [9]. While intravenous injection of tetracycline hydrochloride (10 mg / kg) to dogs, the highest level was identified in its liver and kidneys. In case of peroral administration to dogs at a dose of 25 mg/kg, peak concentration amounted to 3 mg/ml in 2 hours, which reduced to a level of 0.27 in 24 hours after administration [11]. Antibiotics mainly excreted through the urine. Within 72 hours after intravenous administration of tetracycline to 2 rats and 1 dog in a dose of 15 mg or 4 mg/kg, respectively, 69.2% and 19.5% of antibiotics was identified in the urine [9]. High doses of tetracycline at the level of 2500 up to 7500 mg/kg of body weight may reduce the body weight and cause limited adiposis in rat’s liver [12]. Gonadotoxic, embryotoxic, teratogenic, mutagenic and carcinogenic effects of the doses studied have not been found. [13] It is noted that while raising the dose of the preparation injected, the content of tetracycline in bone tissue and its pigmentation also increases [14]. Within human body tetracycline is better absorbed in gastrointestinal tract (up to 66% of the dose taken). Maximum drug concentration appears in blood serum in 13 hours after ingestion. A significantly higher concentration may be achieved during intravenous injection. Абсорбция антибиотиков группы тетрациклина различается у человека и животных, показателей составляя только имеющих 4-9% у мышей и крыс [13]. Среди значительные различия между человеком и животными можно назвать: Absorption of tetracycline antibiotics of humans and animals is different, which accounts for 4-9% only for mice and rats [13]. Among the indicators that significantly differ between humans and animals there are: - number - time of during antibiotic which absorbed body collects in the antibiotic gastrointestinal peak tract; concentrations; - localization of antibiotic peak concentrations within the body; 6 - time during which antibiotic is cleared from the body. These differences must be considered in case of extrapolated results obtained from experiments, where laboratory animals were involved with respect to humans. If antibiotic gets in the human body the effects made on pathogenic flora cannot define its action, but it involves obligate and facultative intestinal microflora, causing a state of dysbiosis. Antibiotics also interact with the human body, which depending on the dose provides development of adverse changes at metabolic, morphological and functional level. [15]. Development of resistance to antibiotics and further transfer of the tetracycline-resistant strains of micro-organisms to human represent the major health concern. The pollution risk of food stock caused by antibiotic residues directly relates with development of microbial resistance. Both livestock and poultry, and crop production pollute antibiotics. Antibiotics cleared from animal organisms get into soil as organic fertilizers and then they accumulate in plant products. Nowadays representatives of both pathogenic and opportunistic microorganisms have polyresistance to antibiotics. Frequently, salmonella is the reason of hospital acquired disease and food poisoning. Antibiotic resistance was observed more frequently among salmonella found in humans, than among that found in farm animals and birds. In addition, 49.1% showed resistance to tetracycline [16]. It was shown that among clinical strains of microorganisms, enterobacteria had the highest percentage of antibiotic resistance. Strains are the most common among gram-negative micro-organisms, which are resistant to drugs 9-11 that is confirmed by data obtained in the Kazakh Academy of Nutrition from 1986 to 1999. Almost within all human waste there are antibiotic-resistant bacteria that can survive selectively during intake of the relevant drugs. (18). Normal microflora as human symbiont plays important role to support life of the organism and ensure its homeostasis. Proportion of species of intestinal microflora can be changed due to influence of tetracycline, even some or almost all the indigenous (autochthonous) strains can 7 disappear completely and opportunistic pathogens may rapidly multiply. Such changes are called dysbiosis [20, 21, 22, 23, 24, 25]. In medical practice, according to the International Classification of Diseases, Tenth revision [26], a number of diseases associated with an imbalance of intestinal microflora was identified: irritable bowel syndrome (IBS) with diarrhea (K58.0) without diarrhea (K58.9), constipation (K59.0), inflammatory bowel disease (IBD) (K50-K51). Microflora imbalance can often trigger development of pathology in various systems and organs, and cause secondary immunodeficiency states and compound the course of various diseases of infectious and non-infectious ethiology. [27,28,29,30,31,32,33]. It is considered that children that have the intestinal microflora imbalance, in addition to the above mentioned diseases can have: functional bowel disorder (K59.9), duodenitis (K29.8, K29.9), food allergy (T78.0, T78.1, T78.4), including atopic dermatitis (L20.8), iron deficiency anemia (D50), a general immunodeficiency (D83.9) [34,35,36]. Ingestion of antibiotic residues and tetracyclines, in particular, may represent effects of chemical factors of low intensity. Such stress on human body can cause long-term effects, and reduce body's resistance to adverse environmental and socially environmental factors. Generally, there are many scientific evidences collected that underestimate impact depth of small doses of biologically active xenobiotics of anthropogenic origin. First, this is applied to tetracyclines, which reduce the adaptive capacity of people by affecting the environment and reducing the indicators of their sanitary and epidemiological welfare [37]. II. HAZARD STATEMENT 2.1 Permissible exposure limit 8 In 2006, the Expert Committee on Food Additives of FAO / WHO revised the acceptable daily intake of tetracyclines and changed it from 0-3 mg per kg of body weight per day to 0-30 mg per kg of body weight per day [26]. In 1990, when the acceptable daily intake was established (0-3 mkg per kg of body weight per day) an uncertainty factor at 10 was accepted due to variability of intestinal flora of humans. And it was assumed that the actual acceptable daily intake can be a little higher than the fixed one. Results of in vitro experiments about the dosage study, which is equivalent to 0,025; 0.25 and 2.5 mg/kg of body weight became the reason for revision. Based on these studies, it was concluded that the variability among individuals is small and now it is useless to apply uncertainty factor, therefore, the acceptable daily intake should take should be 0-30 micrograms per kg of body weight per day [37]. However, this decision is considered to be unproven due to reasons specified by the Russian FBSI "Federal Research Centre for Medical and Preventive Technologies of Public Health Risk Management" 1. Acceptable daily intake is established for food consumption terms on a daily basis throughout the whole life and it cannot be based on data, obtained during the short-term experiment in vitro without having a proper analysis of uncertainty that considers transfer of results obtained from in vitro experiment made on human, including data of short-term studies about influence through the whole life [28, 29]. 2. According to formula (1), recommended by FAO/WHO [29], we calculated acceptable daily intake of tetracycline and oxytetracycline for various types of micro-organisms. (1) 9 ADI = MIC50 x Mass of intestinal contents excreted (220 g) / Dose inhibiting the microorganisms growth x Margin factor x Individual’s weight (60 kg) while peroral administration Formula was developed based on modal value MIC50 (MIC50 – minimum antibiotic concentration, inhibiting increase of 50% cultures of certain microorganisms), margin factor to consider various types of variability, mass of intestinal contents, individual's weight and peroral dose of antibiotics, inhibiting micro-organisms growth. MIC50 values of tetracycline and oxytetracycline for 10 different micro-organisms were taken in accordance with WHO Food Additives Series 36, the margin factor value - 1, a dose that inhibits the microorganisms growth during peroral administration of tetracyclines - 0.6 mg, the mass of intestinal contents excreted 220g, and the average individual's weight - 60 kg. According to formula (1) the acceptable daily intake of tetracyclines obtained from minimum inhibitory concentration (MIC50) for some microorganisms ranged from 0.37 mg/kg of body weight in terms of Clostridium spp. to 195.6 mg/kg of body weight for Escherichia coli and Proteus spp. (Table 1). This high variability of results may indicate the need to include the acceptable daily intake of additional uncertainty factor into calculation, especially for the most sensitive groups of people, such as children. While carrying out health risk assessment considering the characteristics of these groups, they should be noted, including dismaturity of local gut-associated immunity of infants, chronic gastrointestinal diseases, changing immune status, gastrointestinal microbocenosis disorders, and dietary habits of people. Table 1 Micro-organism Escherichia coli Bifidobacterium spp. Bacteroides fragilis Dose that cause microbistatic mcg / kg 195.56 97.78 24.44 10 Eubacterium spp. Clostridium spp. Streptococcus spp. Fusobacterium spp. Lactobacillus spp. Proteus spp. Peptostreptococcus spp. 12.22 0.38 97.78 0.76 12.22 195.56 12.22 3. Results of the experiment in vitro became the reason why use of uncertainty factor was rejected while determining ADI (ADI) of tetracyclines [26]. It was shown that concentrations were studied, which were equal to doses of 0.025; 0.25 and 2.5 mg/kg of body weight and it was concluded that doses of 0.025 and 0.25 mg/kg of body weight are invalid. And we couldn't establish how equality of doses to levels of exposure to chemostat microflora was evaluated. Evaluation of the proposed ratio using the formula 1 showed that the results were inconsistent. This proves the high uncertainty of data, based on which there is not enough motivation in order to reject the proposed modifying factor of 10 and the acceptable daily dose of 3 mg/kg of body weight [37,38]. 2.2 "Exposure-effect" dependency evaluation Development of intestinal microflora imbalance due to tetracycline Относительная численность кишечной микрофлоры, % concentration according to research studies in vitro shown in Fig. 1. 100% Бифидобактерии 90% Лактобактерии 80% 70% Бактероиды 60% 2% 50% 1% 40% 0% Фузобактерии Пептострептококки 0 30% 5 10 15 20 25 30 35 20% E.Coli типичные Факультативная флора 10% Клостридии 0% 0 5 10 15 20 25 30 35 концентрация, мкг/мл 11 Figure 1 - The dependence of the relative abundance of the intestinal microflora (%) on the concentration of tetracycline Changes in the ratio of facultative and obligate intestinal flora are presented in Figure 2. 100% 90% Доля факультативной флоры Share of facultative flora 80% 70% 60% 50% 40% 30% 20% 10% 0% 0% 20% 40% 60% 80% 100% Shareоблигатной of obligate flora Доля флоры Figure 2 - Tthe ratio of facultative and obligate intestinal microflora Data of research studies on diseases associated with an imbalance of intestinal microflora of adults and children have shown that changes in composition of intestinal microflora for adults result in: dysbiosis 1st degree – from 18,6 to 34,8%, dysbiosis 2nd degree – from 24,2 to 45,7%, dysbiosis 3rd degree – from 27,0 to 56,3%. For children: dysbiosis 1st degree – from 2,0 to 74,0%, dysbiosis 2nd degree – from 26,0 to 84,0% and dysbiosis 3rd degree – from 0,0 to 39,1% [39, 40, 45, 46]. III. EXPOSURE ASSESSMENT 12 The exposure assessment was provided on the base of maximum residue level (MRL) of tetracycline for different animal origin food stuff groups taking into the account the daily consumption average level of corresponding foods. Thus according to the Sanitary Regulations and Standards of the Republic of Kazakhstan from 2001 and according to the data from the ‘Agency of Statistics of the Republic of Kazakhstan’ the following the daily animal origin food content is received into the food basket for different age groups of citizens of the Republic of Kazakhstan (table A1). Table А1 The daily animal origin food content in the food basket for different age groups of citizens of the Republic of Kazakhstan (gram/day). Products Meat and meat products Until 3 years old 65 From 3 to 7 years old 100 School aged Teenagers Adults 95 100 150 635 570 570 592 579 17 17 25 30 55 15 15 17 764 27 50 25 789 27 60 35 812 55 100 50 927 38 185 70 1084 Milk, fermented milk products Poultry and poultry products Egg Fish and seafood Animal oils and fats Total Values of maximum residue levels of tetracycline for adults, for different types of animal tissues and exposition types, recommended by WHO (1990), FAO/WHO (1998) and accepted in the USA. (Table А2). Table А2 MRL values for various types of animal tissues and exposition types Food Milk, mkg/l Soft tissue, mkg/kg Fat tissue, mkg/kg Eggs, mkg/kg Liver, mkg/kg Kidney, mkg/kg ТС WHO 1990 10 10 10 10 10 10 100 100 10 200 300 600 FAO/WHO 1998, Кодекс алиментариус 100 200 400 600 1200 USA 300 2000 10 200 300 600 13 According to submitted data the maximum daily incoming of tetracycline with the food staff for different age group citizens of RoK was calculated (Table A3). Table А3 The maximum daily incoming of tetracycline with the food staff for different age group citizens of RoK in mcg/kgs for the value of ТС, WHO (1990), FAO/WHO (1998)/ CODEX Alimentarius and accepted in the USA. Until 3 years old ТС WHO (1990) FAO/WHO (1998) USA 8.29 86.87 92.17 184.87 From 3 to 7 years old 7.89 79.45 84.75 393.55 School aged Teenagers Adults 8.12 80.85 86.15 388.85 9.27 103.70 115.70 421.10 10.84 106.60 115.60 542.40 Taking into account the average mass of eliminating content from the bowel of adults which is 220 gr [46,47], and the average mass of eliminating content from the bowel of children (50 gr) [47], the concentration of tetracycline in gastro-intestinal tract is calculated. Table А4 The concentration of tetracycline in gastro-intestinal tract for the different age group of citizens of the Republic of Kazakhstan for the values of ТС, WHO (1990), FAO/WHO (1998) and accepted in the USA. Until 3 years old ТС WHO (1990) FAO/WHO (1998) USA 0.17 1.74 1.84 7.70 From 3 to 7 years old 0.16 1.59 1.70 7.87 School aged Teenagers Adults 0.16 1.62 1.72 7.78 0.04 0.47 0.53 1.91 0.05 0.48 0.53 2.47 Thus, the concentration of tetracycline in gastro-intestinal tract of adult people varied from 0,05 mcg/g to 2,47mcg/g. The concentration of tetracycline in the gastro-intestinal tract of children varied from 0,16 mcg/g to 7,87mcg/g. 14 The obtained data can be used for calculating the risk characterization of forming resistant strains of microorganisms in gastro-intestinal tract of people and distortion of balance of bowel flora with increasing part of pathogenic and potentially pathogenic strains. IV. RISK CHARACTERISATION Using of antibiotics enables the grow of resistant flora both pathogenic and potentially pathogenic, in this case the high bacterial and bacterio-static activity of drugs is saved towards the obligate anaerobic flora (bifid bacterium, bacteroid, clostridium, fusobacterium, peptostreptococcus, lactic bacteria). Level of potentially-pathogenic (commensal) microflora (Escherichia coli, Salmonella spp., Campylobacter spp. и Еnterococci) is estimated figure of resistant induction of bacteria on the back of antibiotics treatment and indirect indicator of expected resistance of pathogenic flora. Commensal stability monitoring is the prevalence indicator of antibiotic resistance in population [19]. Forming changes of bowel microbial landscape spectrum on the back of tetracycline influence in the form of ratio distortion between anaerobic and aerobiotic flora with growth inhibition of obligatory microorganisms (lactic bacteria, fusobacterium, peptostreptococcus, clostridium) brings to increasing of Escherichia coli, Salmonella spp., Campylobacter spp. and Enterococci, staphylococcus proportion, which shows the bowel microflora disbalance [19, 23, 24, 25, 26]. The assessment of health risk under the tetracycline influence, incoming with food, was provided separately for each age group. The calculations were conducted by maximum residue concentrations of tetracycline for four values, recommended by ТС, WHO (1990), FAO/WHO (1998) and accepted in the USA (Table А4). In the result of modeling the bowel microflora balance distortion, there was established that the values, recommended by TC are the most harmless for health for all the age groups of people at which the content of potentially pathogenic 15 microorganisms is not more than 5% from the total amount of bacteria. The values, recommended by WHO (1990) and FAO/WHO (1998)/ Codex Alimentarius are harmless for only two age groups (teenagers and adults). The values of residue tetracycline in the animal origin food, recommended by WHO (1990) and FAO/WHO (1998) are dangerous to health of children up to 3 years and from 3 to 7 years. The realization of values, recommended by WHO (1990) and FAO/WHO (1998) and accepted in the USA can be related to inhibiting obligatory microflora and brings to bowel microflora disbalance of different heaviness. (Tables А7, А8 and А9). According to the WHO data, the minimum inhibiting concentration of tetracycline Mic90 (the concentration at which the 90% of bacteria is inhibited) was determined. In the Table A5 the composition of normal microflora and the minimum inhibiting tetracycline concentration data is shown Mic90 [13]. Тable А5 The MIC90 tetracycline values towards the human bowel microflora. The proportion in normal Mic90, Bacteria type bowel microflora, % мcg/ml Bifidus bacteria spp. 85 32 Bateroides spp. 4.99 32 Clostridia spp. 0.01 32 Lactic bacteria 4 32 Peptostreptococcus 2 32 E. Coli (coliform 1 64 bacterium) The results of calculations of parameters for every bowel microflora bacteria, for every age group, by the norms, recommended ТС, WHO (1990), FAO/WHO (1998) and accepted in the USA are shown in the Tables е А6, А7, А8 and А9. 16 Table А6 Changes in intestinal microflora affected by tetracycline residues according to the standards of the CU 84.58 0.16 84.58 4.96 0.17 4.97 0.16 Clostridia spp. 0.01 0.17 0.01 Lactic bacteria 3.66 0.17 Peptostreptococcus 1.99 E. Coli (coliform bacterium) Opportunistic pathogenic microflora 1.00 84.55 Bateroides spp. 3.83 0.16 84.89 4.97 0.16 0.16 0.01 3.68 0.16 0.17 1.99 0.17 1.00 3.78 Share in a normal flora of an intestinal tract, % number of tetracycline in a stomach 0.17 Bifidus bacteria spp. Bacteria type Share in a normal flora of an intestinal tract, % adults number of tetracycline in a stomach Share in a normal flora of an intestinal tract, % Share in a normal flora of an intestinal tract, % teenagers number of tetracycline in a stomach Share in a normal flora of an intestinal tract, % School aged children number of tetracycline in a stomach children from 3 to 7 years old number of tetracycline in a stomach children until 3 years old 0.04 84.87 0.05 4.98 0.04 4.98 0.05 0.16 0.01 0.04 0.01 0.05 3.68 0.16 3.92 0.04 3.90 0.05 0.16 1.99 0.16 2.00 0.04 2.00 0.05 0.16 1.00 0.16 1.00 0.04 1.00 0.05 3.78 3.20 3.24 17 Table А7 Changes in intestinal microflora affected by tetracycline residues according to the standards of WHO (1990) Share in a normal flora of an intestinal tract, % 80.78 1.59 80.70 1.62 83.75 4.72 1.74 4.74 1.59 4.74 1.62 Clostridia spp. 0.01 1.74 0.01 1.59 0.01 Lactic bacteria 3.78 1.74 3.80 1.59 Peptostreptococcus 1.89 1.74 1.90 E. Coli (coliform bacterium) Opportunistic pathogenic microflora 0.97 1.74 0.98 Bifidus bacteria spp. 80.38 Bateroides spp. Bacteria type 8.25 7.79 Share in a normal flora of an intestinal tract, % number of tetracycline in a stomach Share in a normal flora of an intestinal tract, % Share in a normal flora of an intestinal tract, % adults 0.47 83.73 0.48 4.92 0.47 4.92 0.48 1.62 0.01 0.47 0.01 0.48 3.80 1.62 3.94 0.47 3.94 0.48 1.59 1.90 1.62 1.97 0.47 1.97 0.48 1.59 0.97 1.62 0.99 0.47 0.99 0.48 7.89 4.42 number of tetracycline in a stomach 1.74 teenagers number of tetracycline in a stomach Share in a normal flora of an intestinal tract, % School aged children number of tetracycline in a stomach children from 3 to 7 years old number of tetracycline in a stomach children until 3 years old 4.45 18 Table А8 Changes in intestinal microflora affected by tetracycline residues according to the standards of FAO/WHO (1998) and Codex Alimentarius Share in a normal flora of an intestinal tract, % adults Share in a normal flora of an intestinal tract, % number of tetracycline in a stomach Share in a normal flora of an intestinal tract, % teenagers number of tetracycline in a stomach Share in a normal flora of an intestinal tract, % School aged children number of tetracycline in a stomach Bacteria type children from 3 to 7 years old number of tetracycline in a stomach Share in a normal flora of an intestinal tract, % number of tetracycline in a stomach children until 3 years old Bifidus bacteria spp. 80.11 1.84 80.48 1.70 80.43 1.72 83.59 0.53 83.59 0.53 Bateroides spp. 4.70 1.84 4.72 1.70 4.72 1.72 4.91 0.53 4.91 0.53 Clostridia spp. 0.01 1.84 0.01 1.70 0.01 1.72 0.01 0.53 0.01 0.53 Lactic bacteria 3.77 1.84 3.79 1.70 3.79 1.72 3.93 0.53 3.93 0.53 Peptostreptococcus 1.89 1.84 1.89 1.70 1.89 1.72 1.97 0.53 1.97 0.53 E. Coli (coliform bacterium) Opportunistic pathogenic microflora 0.97 1.84 0.97 1.70 0.97 1.72 0.99 0.53 0.99 0.53 8.55 8.13 8.19 4.60 4.60 19 Table А9 Changes in intestinal microflora affected by tetracycline residues according to the standards adopted in the United States Bifidus bacteria spp. 64.55 7.7 64.10 7.87 64.33 7.78 79.93 1.91 78.44 2.47 Bateroides spp. 3.79 7.7 3.76 7.87 3.78 7.78 4.69 1.91 4.60 2.47 Clostridia spp. 0.01 7.7 0.01 7.87 0.01 7.78 0.01 1.91 0.01 2.47 Lactic bacteria 3.04 7.7 3.02 7.87 3.03 7.78 3.76 1.91 3.69 2.47 Peptostreptococcus 1.52 7.7 1.51 7.87 1.51 7.78 1.88 1.91 1.85 2.47 E. Coli (coliform bacterium) Opportunistic pathogenic microflora 0.88 7.7 0.88 7.87 0.88 7.78 0.97 1.91 0.96 2.47 Bacteria type 26.22 26.73 Share in a normal flora of an intestinal tract, % 26.46 8.76 number of tetracycline in a stomach adults number of tetracycline in a stomach teenagers number of tetracycline in a stomach School aged children Share in a normal flora of an intestinal tract, % Share in a normal flora of an intestinal tract, % number of tetracycline in a stomach children from 3 to 7 years old Share in a normal flora of an intestinal tract, % Share in a normal flora of an intestinal tract, % number of tetracycline in a stomach children until 3 years old 10.45 20 These data indicate that the least changes in intestinal microflora are set due to tetracycline residues identified in food at the level of 10 mkg/kg (0.01 mg/kg). The greatest changes in species composition of normal microflora that is reduction of percentage of symbiontic microorganisms and increase of a number of potentially pathogenic flora, are fixed at acceptable levels of antibiotic residues in food accepted in the US. V. ASSESSMENT OF UNCERTAIN RESULTS The analyses of shown materials mean the following possible uncertainties: - the absence or lack of the information about possible effects of tetracycline chronic effects on human organism, especially to the most sensible group of people (children, pregnant women, people with diseases of gastro-intestinal tract, old people) uncertainties, related to undergoing the experiments with animals and in vitro on human; - the parameters, used for assessment the exposition and risk estimation (parameters uncertainties, related to food consumption. For instance, due to food preferences of some group of people, as the main food in the Republic of Kazakhstan are considered the meat and milk products); - the gaps in the scientific theory, necessary for prediction on the base of causal connections (limited amount of researches, by the results of which the model parameters were estimated, modified factors using or refuse to use them, using of the generalized, average information for big population and partial imagination of microflora balance distortion forming regulations and health problems of citizens of RoK, connected to it. Therewith, the health risks underevaluating of RoK citizens is possible, which is related to the absence of knowledge about the by-product usage, especially, liver and nephros of farm animals). Generally, the uncertainty of the results of researches is characterized as high. 21 CONCLUSION The Republic of Kazakhstan considers that the refusal of uncertainty coefficient 10 at the rating of tetracycline ADI is not sufficiently proved. Taking into account the aspects of meat and dairy products using in the Republic, the tetracycline acceptable residual quantities establishment on the level of 100 mcg/l in milk, 100 mcg/kgs in muscular tissues, 10 mcg/kgs in fat tissues, 200 mcg/kgs in eggs, 300 mcg/kgs in liver, 600 mcg/ kgs in nephros, can bring to the additional disease risks of digestive organs, anemia, dermatitis, allergy and immune deficiency of most sensible group of people (children, pregnant women, people with diseases of gastro-intestinal tract, old people). Especially, increasing of dysbiosis proportion and amount of bowel microflora resistant to antibiotic are made uneasy. On the base of abovementioned, the Republic of Kazakhstan considers, that the tetracycline acceptable residual quantities establishment in food on the degree of 10 mcg/kgs (0,01 mg/kgs) is proved and will not bring to increasing the health risks of citizens. 22 Reference list 1. Antibiotic chemistry, 3 publication., т. 1, М., 1961, pages 180-268; 2. Nazashin S.M., Fomina I.P., Rational antibiotic treatment, 4 publication., М., 1982, pages.183-208. 3.Chernuh А. М., Kivman G.Ya., Tetracycline group antibiotics, М., 1962. Control of antibiotic resistance from the position of food safety in Europe, WHO, European Regional Bureau, page. 1, 2011 4. Chepurnoi I.P. Nutrition and health. 3-rd publication. М., 2008, с. 208 5. 9-th Moscow International Congress on veterinary science. A.N.Panin’s report, FGBU “Vserossiyskiy Gosudarstvenny Centr Kachestva I Standartizacii lekarstvennyh sredstv dlya zhivotnyh i kormov ” 18 april, 2011. 6. 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