Marine biotoxins in shellfish - okadaic acid and analogues1 Scientific Opinion of the Panel on Contaminants in the Food chain
Marine biotoxins in shellfish - okadaic acid and analogues1 Scientific Opinion of the Panel on Contaminants in the Food chain
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DOI：
10.2903/j.efsa.2008.589
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发表时间：
2008-01-01
期刊：
影响因子：
3.3
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中科院分区：
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冈田酸 (OA) 及其类似物、甲藻毒素（DTX1、DTX2 和 DTX3）共同构成 OA 毒素组。这些毒素具有亲脂性和热稳定性，由甲藻产生，存在于各种贝类中，主要存在于滤食性双壳类软体动物中，如牡蛎、贻贝、扇贝和蛤。 OA和DTX2仅在分子中一个甲基的位置上有所不同，而DTX1多了一个甲基，DTX3代表了OA、DTX1和DTX2的一系列饱和和不饱和脂肪酸酯化衍生物。OA族毒素会导致腹泻性贝类中毒(DSP)，其特征是腹泻、恶心、呕吐和腹痛等症状。人类在食用受污染的双壳类软体动物（例如贻贝、扇贝、牡蛎或蛤）后不久就会出现这些症状。假定丝氨酸/苏氨酸磷蛋白磷酸酶的抑制构成了OA组毒素的作用方式。OA组毒素的毒理学数据库有限并且主要包括对其急性毒性的研究。根据小鼠腹腔注射后的 LD50 实验，专家组确定了以下毒性当量因子 (TEF)：OA = 1、DTX1 = 1、DTX2 = 0.6。对于 DTX3，TEF 值等于相应的未酯化毒素（OA、DTX1 和 DTX2）的 TEF 值。果胶毒素经常与 OA 族毒素同时出现，目前已包含在 OA 族毒素的监管限值中，但它们与 OA 族毒素的作用机制不同。因此，它们的毒性不应表示为 OA 等效物，也不应包含在 OA 毒素组的监管限值中。尚未报道 OA 组毒素的长期毒性/致癌性实验，但 OA 被确定为啮齿类动物的肿瘤促进剂。 OA 在非标准体外试验中显示出一些遗传毒性的证据。这包括哺乳动物细胞系中非特异性 DNA 加合物形成的一些证据。然而，这些数据很难解释，专家小组指出，这些影响可能与这些测定中 OA 的细胞毒性有关。对于 DTX2 和 DTX3，没有可用的遗传毒性数据。专家组得出的结论是，OA 本身似乎不具有致突变性，但会诱导染色体水平的变化，并且在体外是非整倍体。专家组指出，这些影响可能与 OA 的细胞毒性有关。关于 OA 对动物或人类的慢性影响的数据不足以确定每日耐受摄入量 (TDI)。鉴于OA族毒素的急性毒性，专家组决定根据现有的人体数据建立急性参考剂量（ARfD）。考虑到各种人类病例报告中估计接触量的不确定性，专家组得出结论，人类疾病的最低观察到不良效应水平 (LOAEL) 约为 50 微克 OA 当量/人，这大约相当于成人 0.8 微克 OA 当量/公斤体重 (b.w.)。应用不确定性因子 3 将该 LOAEL 外推至未观察到的不良效应水平 (NOAEL)，从而得出 ARfD 为 0.3 μg OA 当量/kg b.w。专家组认为，没有必要对人类之间的差异应用额外的不确定性因素，因为这些数据是基于对来自不同国家的大量受影响的贝类消费者的观察，并被认为包括最敏感的个体。为了防止OA组毒素的急性影响，在贝类消费的健康风险评估中使用高份量而不是长期平均消费量非常重要。欧盟各地贝类物种的消费数据有限，因此 EFSA 要求成员国提供相关贝类物种的消费信息。根据五个成员国提供的数据，专家小组确定 400 克贝类肉为用于海洋生物毒素急性风险评估的高份量。值得注意的是，按照目前欧盟 160 微克 OA 当量/千克贝类肉的限量，含有 OA 族毒素的 400 克贝类肉将导致膳食暴露 64 微克毒素。对于 60 公斤的成年人来说，这相当于大约 1 微克/公斤体重。该数字超过 ARfD 约 3 倍，处于从人类案例研究得出的 LOAEL 范围内。因此，这种摄入量预计会对易受影响的消费者产生影响。根据消耗量和发生数据，大约有 20% 的机会超过 0.3 微克 OA 当量/千克体重的 ARfD。食用目前欧洲市场上出售的贝类时。因此，DSP 在现行立法和规定的控制参考方法下发生。专家组得出的结论是，为了使 60 公斤的成年人不超过 ARfD，400 克贝类中的毒素不应超过 18 微克，即 45 微克 OA 当量/公斤贝类肉。小鼠和大鼠生物测定法是欧盟官方规定的检测 OA 族毒素的参考方法。专家组的结论是，这两种方法都有缺陷，不适合评估当前的欧盟限制。目前欧盟监管限值为 160 微克 OA 当量/千克贝类肉，哺乳动物检测检测 OA 族毒素的能力有限，并且无法检测低于该水平的 OA 族毒素。此外，MBA 无法检测 DTX3。当前的欧盟立法允许更换生物测定法，前提是替代方法已根据国际公认的协议进行了验证。基于磷蛋白-磷酸酶测定和液相色谱-质谱 (LCMS) 的方法最有可能取代哺乳动物测定，并检测低于当前欧盟监管限值的 OA 组毒素水平。专家组指出，虽然根据公认的国际准则应用单一实验室验证来证明其适用性可能会推动出于监管目的实施海洋生物毒素替代仪器分析，但应尽可能规定方法性能标准，并应将实验室间试验验证作为长期目标。
Okadaic acid (OA) and its analogues, the dinophysis toxins (DTX1, DTX2, and DTX3), together form the group of OA-toxins. These toxins are lipophilic and heat stable, are produced by dinoflagellates and can be found in various species of shellfish, mainly in filter-feeding bivalve molluscs such as oysters, mussels, scallops, and clams. While OA and DTX2 only differ by the position of one methyl group in the molecule, DTX1 has one additional methyl group and DTX3 represents a wide range of derivatives of OA, DTX1 and DTX2 esterified with saturated and unsaturated fatty acids.OA-group toxins cause Diarrhoeic Shellfish Poisoning (DSP), which is characterised by symptoms such as diarrhoea, nausea, vomiting and abdominal pain. These symptoms may occur in humans shortly after consumption of contaminated bivalve molluscs such as mussels, scallops, oysters or clams. Inhibition of serine/threonine phosphoprotein phosphatases is assumed to constitute the mode of action of OA-group toxins.The toxicological database for OA-group toxins is limited and comprises mostly studies on their acute toxicity. Based on LD50 experiments following intraperitoneal injection in mice, the Panel established the following toxic equivalence factors (TEFs): OA = 1, DTX1 = 1, DTX2 = 0.6. For DTX3 the TEF values are equal to those of the corresponding unesterified toxins (OA, DTX1, and DTX2).Pectenotoxins frequently co-occur with OA-group toxins and are currently included in the regulatory limit for OA group toxins but they do not share the same mechanism of action as OA-group toxins. Therefore their toxicity should not be expressed as OA-equivalents and they should not be included in the regulatory limit for the group of OA toxins.No long-term toxicity/carcinogenicity experiments have been reported for OA-group toxins, but OA is identified as a tumour promoter in rodents. OA has shown some evidence for genotoxicity in non-standard in vitro assays. This includes some evidence for unspecific DNA-adduct formation in mammalian cell lines. However, the data are difficult to interpret, and the Panel noted that these effects may be related to the cytotoxicity of OA in these assays. For DTX2 and DTX3 no genotoxicity data are available. The Panel concluded that OA appears to be not mutagenic per se, but induces changes at the chromosome level and is aneugenic in vitro. The Panel noted that these effects may be related to cytotoxicity of OA.The data on the chronic effects of OA in animals or humans were insufficient for a tolerable daily intake (TDI) to be established. In view of the acute toxicity of OA-group toxins, the Panel decided to establish an acute reference dose (ARfD) based on the available human data. Taking into account the uncertainties in the estimated exposure in the various human case reports, the Panel concluded that a lowest-observed-adverse-effect-level (LOAEL) for human illness is in the region of 50 mu g OA equivalents/person, this approximates to 0.8 mu g OA equivalents/kg bodyweight (b.w.) for adults. An uncertainty factor of three was applied to extrapolate this LOAEL to a no-observed-adverse-effect-level (NOAEL) which resulted in an ARfD of 0.3 mu g OA equivalents/kg b.w. The Panel considered it not necessary to apply an additional uncertainty factor for the variation among humans as the data are based on observations in a rather large number of affected shellfish consumers, originating from various countries, and considered to comprise the most sensitive individuals.In order to protect against the acute effects of OA-group toxins, it is important to use a high portion size rather than a long-term average consumption in the health risk assessment of shellfish consumption. Consumption data for shellfish species across the EU, were limited, therefore EFSA requested the Member States to provide information on consumption of relevant shellfish species. Based on data provided by five Member States, the Panel identified 400 g of shellfish meat as the high portion size to be used in the acute risk assessment of marine biotoxins.was noted that a 400 g portion of shellfish meat containing OA-group toxins at the current EU limit of 160 mu g OA equivalents/kg shellfish meat would result in a dietary exposure of 64 mu g toxin. For a 60 kg adult this is equivalent to approximately 1 mu g/kg b.w. This figure exceeds the ARfD by approximately 3-fold and is in the region of the LOAEL as derived from the human case studies. Therefore, this intake would be expected to exert effects in susceptible consumers. Based on the consumption and occurrence data, there is an approximately 20% chance of exceeding the ARfD of 0.3 mu g OA equivalents/kg b.w. when consuming shellfish currently available on the European market. Thus DSP occurs under the current legislation and the prescribed reference methods for control. The Panel concluded that in order for a 60 kg adult to not exceed the ARfD, a 400 g portion of shellfish should not contain more than 18 mu g toxin, i.e. 45 mu g OA equivalents/kg shellfish meat.The mouse and the rat bioassay are the officially prescribed reference methods in the EU for the detection of OA-group toxins. The Panel concluded that both methods have shortcomings that make them inappropriate for assessing the current EU limit. The mammalian assays have limited capability to detect OA-group toxins at the current EU regulatory limit of 160 mu g OA equivalents/kg shellfish meat, and are not capable of detecting OA-group toxins below this level. In addition, the MBA are not able to detect DTX3.The current EU legislation permits the replacement of the bioassays, provided that the alternative methods have been validated according to an internationally recognised protocol. The phosphoprotein-phosphatase assays and liquid chromatography-mass spectrometry (LCMS) based methods have the greatest potential to replace the mammalian assays, and to detect levels of OA-group toxins below the current EU regulatory limit. The Panel noted that, while application of single laboratory validation according to recognised international guidelines to demonstrate their fitness-for-purpose can be an impetus for implementation of alternative instrumental analyses of marine biotoxins for regulatory purposes, method performance criteria should be stipulated where possible and validation by interlaboratory trials should be the long-term objective.