Thailand information paper On estimating the risk of developing histamine poisoning from the consumption Thai fish sauces
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1 FFP/31 CRD 18 (original language only) Comments submitted by Thailand Agenda Item 3 Thailand information paper On estimating the risk of developing histamine poisoning from the consumption Thai fish sauces Executive Summary Fish sauce is a fundamental ingredient used in many Southeast Asian dishes, and is also used as a dipping condiment. The main species used in fish sauce production in Southeast Asia is Indian anchovy, which typically possesses high levels of free histidine and other amino acids. The amount of histamine produced in traditional fish sauce is related to the free histidine content in the raw materials. Due to the nature of raw materials and the production methods for traditional fish sauce, highs levels of histamine are found in many samples. This risk assessment on Histamine in Thai fish sauce was undertaken by the National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand and Dr. Tom Ross from the Tasmanian Institute of Agricultural Research (TIAR), School of Agricultural Science, University of Tasmania, Australia and the consumption data and the prevalence and concentration of histamine in Thai fish sauce data was provided by the National Bureau of Agricultural Commodity and Standards (ACFS), the Department of Fisheries, Ministry of Agriculture and Cooperative, respectively, to respond to the request of the Codex Committee on Fish and Fishery products for sound scientific advice as a basis for the development of guidelines for the control of histamine in fish sauce. Previous human trials and outbreak data were used to build a histamine dose-response model. Potential risks of developing histamine poisoning from consuming Thai fish sauce were investigated in this study using both deterministic and probabilistic approaches. Reference doses (RfD) were estimated from the No Observed Adverse Effect Level (NOAEL) and the Benchmark Dose (BMD) values. Based on this data, and frequencies and amounts of consumption, the risk of developing histamine poisoning from fish sauce among Thai consumers was estimated. From consumption of fish sauce alone yielded a very small histamine intake to consumers. Hence, there is no chance of developing histamine poisoning from fish sauce consumption alone when applying the standard of 400ppm histamine allowed in fish sauce. Different scenarios reflecting the effect of different histamine standards were also evaluated. Introduction
2 2 Fish sauce is a translucent brown liquid hydrolysate derived from a12-18 month fermentation of small fish and salt at a ratio of 3:1 in ambient temperature (Saisithi, 1994; Thongthai, 1999). During fermentation, protein hydrolysis is induced by endogenous proteinases in fish muscle and digestive tract as well as proteinases produced by halophilic bacteria (Gildberg and Thongthai. 2001). It has a salty flavor and unique fish aroma. Fish sauce is commonly used as seasoning for cooking Southeast Asia dishes and as a dipping condiment. The species most commonly used for fish sauce production in Thailand is Indian anchovy (Stolephorus spp.). Anchovies are normally caught and kept on board. Producers with good manufacturing practices would mix fish with salt after catch which retards histamine formation. However, some keep fish without salt for up to 8 hours before landing and transport to a factory in an open container without proper cooling system (Yongsawatdigul et al., 2004). Because anchovies have high levels of free histidine and this amino acid can be converted into histamine by enzyme called histidine decarboxylase released by bacteria. As a result, high histamine levels are usually found in fish sauce. To ensure the safety to the consumers and to give a sound scientific advice as a basis for the development of guidelines for the control of histamine in fish sauce, the risk assessment of histamine in Thai fish sauce was conducted. Scope and General Approach Risk assessment is an empirically based process that estimates the risk of adverse health effects from exposure of an individual or population to a chemical, physical or biological agent. The health risk assessment process usually involves four steps: hazard identification, hazard characterization and dose response assessment, exposure assessment and risk characterization (IPCS 1999). This risk assessment project had two main objectives: Estimate the risk of developing histamine poisoning from the consumption of histamine in Thai fish sauces Compare the risk of a current and an alternative histamine in fish sauce standards (200 and 400ppm) Data for this risk assessment were obtained from Thai national food consumption survey (ACFS. 2007), histamine in fish sauces report (Thai Department of Fishery), a histamine outbreak report (Bureau of Epidemiology, Ministry of Public Health, Thailand. 2007) and many published scientific literature and reports produced by various organizations. Hazard Identification: Histamine Histamine is a biogenic amine Histamine formation in fish is controlled by three main factors: 1) the level of free histidine 2) contamination levels of histidine decarboxylating bacteria 3) temperature (NSW Food Authority, 2010). In high temperature, previous studies suggested that histidine decarboxylating bacteria such as the Enterobacteriaceae family (i.e. Klebsiella, Morganella and Hafnia) presented in the gills or gut cavity would release enzyme that convert histidine to histamine during its growth (Lehane and Olley, 1999). Histidine is naturally presented in high levels in anchovies and therefore high histamine is often found. Exposure assessment 2
3 The focus of the exposure assessment models was to account for distribution of the fish sauce consumption by the Thai population, the levels of histamine found in fish sauce and the amount of histamine intake from fish sauce consumption. 1. Fish sauce consumption The data of fish sauce consumption in Thailand was obtained from the national food consumption survey by the National Bureau of Agricultural Commodity and Food Standards (ACFS), Ministry of Agriculture Cooperative (ACFS, 2006). The national food consumption survey was carried out in The survey comprises 18,746 non-institutionalised persons (9,316 males and 9,430 females) aged range from years old from households across Thailand that were randomly selected based on a stratified three-stage sampling design. The survey includes 500 types of different food and drinks and the questionnaires were based on a quantitative amount and a frequency particular food is consumed. From the report, as shown in table, around 75% of the Thai population eat fish sauce and the average fish sauce consumption in Thailand is 9.07 g/capita/day. Table1: Fish sauce consumption by the Thai population Parameter Consumption amount (g/capita/day) Mean percentile percentile 21 Min 1.75 Max 63 Standard Dev Percentage of the eaters 74.9% 3 2. Histamine levels in Thai fish sauce 2.1. Histamine analysis A total of 230 samples of ready-to-eat, exported-grade Thai Fish sauce manufactured by 13 different companies were collected and examined by the Fish Inspection and Food Control Division, Department of Fishery, Ministry of Agriculture and Cooperatives, Thailand, from January 2007 to May Histamine levels were analyzed in duplicate following the Association of Official Analytical Chemists (A.O.A.C.) fluorometric method (A.O.A.C, 2006). In summary, samples were extracted in methanol (J.T. Baker). The methanol filtrate was loaded onto an 80x7mm Bio Rad AG 1x8, mesh ion exchange column. Histamine standards (0.5, 1.0, and 1.5μg) and sample eluant were derivatized with o-phthaldiadehyde (Merck) and fluorescence intensity was determined using a Turner Quantech Fluorometer (Barnstead/Turner International) at excitation wavelength of 360 nm and emission wavelength of 450 nm. (Turner model FL03 and FL 04, respectively) The prevalence and concentration of histamine in exported-grade Thai fish sauce The average of histamine concentration of 230 samples of exported-grade Thai fish sauce is mg/kg (or ppm) with a standard deviation of mg/kg as shown in table 2. 3
4 4 Table2: Level of histamine (mg/kg) in Thai fish sauce Histamine Level (mg/kg) Manufacturer Average (N) SD Max Min A (27) B (11) C (4) D (11) E (4) F (19) G (36) H (56) I (10) J (31) K (30) L (21) M (48) Total (308) Source: Department of Fisheries, Ministry of Agriculture and Cooperatives 3. Histamine intake estimated from consumption of Thai fish sauce by the Thai population The histamine intake (mg) was calculated using fish sauce consumption data and the level of histamine found in fish sauce as followed: Histamine intake (mg) = Fish Sauce consumption (g/capita/day) x Histamine level (mg/kg or ppm)/1000 The estimates of how much a person would be exposed to histamine from fish sauce consumption were calculated based on two different approaches, deterministic (point estimated) and probabilistic approaches Deterministic approach Traditionally, the estimation of intake of chemicals from food is calculated based on a point estimate, usually the worst-case values from input variables such as 95 and percentile values (Spanjersberg et al., 2007). In this case, the 97.5 percentile value for amount of fish sauce consumption of 21 grams/capita/day was multiplied by the level of histamine concentration in fish sauce at 97.5 percentile, which is mg/kg to estimate the worst-case histamine intake value. The histamine intake calculated using a deterministic approach is 5.18 mg/capita/day Probabilistic approach Histamine intake from fish sauce was based on data for Thai consumption frequency and amount and reported levels in Thai fish sauce as shown in the previous sections. Variability and uncertainty in variables was taken into account via Monte Carlo simulation to estimate daily histamine intake from fish sauce. 4
5 5 x Histamine in Fish sauce (ppm) Daily fish sauce consumption (g/person/day) Daily histamine intake (mg/person/day) Ten simulations (10,000 iterations per simulation) were done program (version 5.5) software. Results were as followed: Table3: The values of daily histamine intake (mg/person) from fish sauce (based on 10,000 iterations) Min Max Average 97.5 percentile Hazard Characterization and Dose-response assessment There has never been a report in Thailand nor in other countries of a histamine poisoning outbreak caused by fish sauce consumption. It could be because the histamine poisoning symptoms can be mild and go away after sometimes and the cases were not reported or because the amount of fish sauce consumption is so low that the histamine intake from fish sauce does not cause any histamine poisoning to healthy consumers. In this step, the hazard characterization provides a description of how oral intake histamine can affect humans. Two selected human studies conducted by Motil and Scrimshaw (1979) and C. E. M. van Gelderen et al. (1992) were pooled together with the Thai epidemiological data of an incident of a histamine poisoning outbreak from consumption of homemade traditional fermented tuna in 2007 as shown in table. 5
6 6 Table4: Data from human studies and an epidemiological data used for histamine doseresponse assessment Dose (mg) Number of Subjects Number of Illnesses Rate of Observed Illness Reference Motil and Scrimshaw (1979) van Gelderen, C.E.M. (1992) Bureau of Epidemiology, Ministry of Public Health, Thailand (2007) Probability of Illness (Histamine Poisoning) Results volunteer studies Outbreak NOAEL 50 mg Histamine Intake (mg) 1. Determination of histamine s no-observe-adverse-effect level (NOAEL) For non-cancer toxicological chemicals, it is generally agreed that the effects only expressed when exposure exceeds a threshold level (Benford and Tennant. 1997). A NOAEL is traditionally used as a starting point in the determination of health based guidance values such as an acceptable daily intake (ADI) or a reference dose (RfD) (WHO. 1999). In this case, from figure 1, the NOAEL for histamine is 50 mg. 2. Determination of histamine s benchmark dose (BMD) 6
7 We used the BMD software (BMDS) version developed by the US.EPA. The BMD with its lower bound, BMDL, as initially defined as the dose causing a 1-10% increase in risk over background (Crump. 1984). In this case, dichotomous models were used to express an increase in incidence of 1, 5 and 10% above the control incident (BMR1, BMR5 and BMR10). An extra risk of 10% is the default for BMDS since the 10% response is at or near the limit of sensitivity in most cancer bioassays and in some non-cancer bioassays. Some studies chose lower BMR to increase sensitivity (US. EPA. 1995). The best-fit model was a gamma model and BMD 10 and BMDL 10 were determined Gamma Multi-Hit Model with 0.95 Confidence Level Gamma Multi-Hit BMD Lower Bound Fraction Affected :32 09/ BMDL BMD dose BMD 10 = mg BMDL 10 = mg Both NOAEL and BMD values for histamine were both around 50mg. For this case, we had decided that the uncertainty factor (UF) would be 1 because to use an uncertainty factor of 10 would overestimate the histamine poisoning cases, and will use the BMDL 10 instead of BMD 10 because BMDL 10 included uncertainty in itself. Therefore, the reference dose (RfD) in this case is 36.92mg (BMDL 10 ). (Note: considering the standard for fishery products is 200ppm, and if the serving size is around gram per person, depending on the types of products and countries, a person consumes just one serving of fish with histamine at 200ppm, he or she would get histamine from fish up to 40mg. This is already exceeding the calculated reference dose. Therefore, the BMDL 10 is quite restricted. Risk Characterization In this study, the risk of developing histamine poisoning from exposing to histamine in fish sauce is assessed by comparing the dietary exposure with an adequate safe exposure level, in this case, BMDL 10 or the RfD. The probabilistic approach estimates the probability that the exposure of an individual from a given population exceeds these limits. The risk can be expressed as Risk = #(Xi >RfD) N 7
8 Where #(Xi >RfD) denotes the number of exposure values that exceed the RfD and N denotes total number of iterations. The risk was calculated program version 5.5 (Palisade, 2009) using Monte Carlo simulation. One hundred simulations were done with 10,000 iterations per simulation. In summary table 2, the risk associated with exceeding NOAEL limit is shown based on different scenarios. Table 5: Estimation of the risk using probabilistic approach Scenario Mean risk per meal (SD) a 8 1. Fish sauce alone (a FS daily dose was consumed in 1 meal) 0.00 (0.00) 2. Fish alone b (a fish daily dose was consumed in 1 meal) 8.12 x 10-6 (0.4 x 10-5 ) 3. Fish + Fish sauce (a FS daily dose was consumed in 1 meal) 200ppm FS standard 400ppm FS standard 8.39 x 10-6 (0.46 x 10-5 ) 8.47 x 10-6 (0.52 x 10-5 ) a Risk per meal refers to the predicted risk of an individual becoming ill of histamine poisoning when he or she consumes a daily dose of fish sauce (FS) or a daily dose of a scombroid fish or a scombroid fish with fish sauce. The risk was estimated as a probability of the histamine intake to exceed the NOAEL limit of 50mg using Monte-Carlo simulations. b Assumption: a fresh scombroid fish had a lognormal distribution with an average of histamine concentration of 5ppm and standard deviation of 10ppm. Conclusion The contribution of fish sauce to daily histamine intake is small in comparison to the predicted intake from other sources such as scombroid-fish consumption. The risk of developing illness when applying a current standard of 200ppm histamine concentration allowed in fish sauce and an alternative standard of 400ppm did not greatly affect the risk to consumers and their risks were not significantly from eating fish alone (p>0.05). Hence, histamine intake from fish sauce consumption contributed very little to the total histamine intake. Under the various assumptions about acceptable histamine levels, and the consequences of changing acceptable levels, the contribution of fish sauce to total daily histamine intake, and to risk of illness, remains the same - i.e. it remains a minor contribution to the risk. These results will also be submitted to be published in an international journal. References Bureau of Epidemiology, Ministry of Public Health, Thailand "Surveillance of Histamine Poisoning outbreak from consumption of homemade traditional fermented tuna." Weekly Epidemiological Surveillance Report 38(36). IPCS International Program on Chemical Safety. WHO. Geneva. IPCS Principles for Modelling Dose-Response for the Risk Assessment of Chemicals. Environmental Health Criteria 239. Lehane, L., Olley, J Histamine fish poisoning revisited. International Journal of Food Microbiology. 58: Motil, K. J. and N. S. Scrimshaw "The role of exogenous histamine in scombroid poisoning." Toxicol. Lett. 3(2): NSW Food Authority Presence of histamine in anchovies report. The National Bureau of Agricultural Commodity and Standards (ACFS) Food Consumption Data of Thailand. Bangkok, Ministry of Agriculture and Cooperative. van Gelderen, C. E. M., Savelkoul, T.J.F., van Ginkel, L.A., van Dokkum, W "The effects of Histamine administered in fish samples to healthy volunteers." Clinical Toxicology 30(4):
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