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研究生: 許沛君
Hsu, Pei-Chun
論文名稱: 以高通量體外評估再生食品接觸材之遷移物質毒性
High-throughput in vitro toxicity assessment of migrated substances from recycled food contact materials
指導教授: 陳秀玲
Chen, Hsiu-Ling
學位類別: 碩士
Master
系所名稱: 醫學院 - 食品安全衛生暨風險管理研究所
Department of Food Safety / Hygiene and Risk Management
論文出版年: 2024
畢業學年度: 112
語文別: 中文
論文頁數: 140
中文關鍵詞: 食品接觸材回收遷移試驗體外試驗高通量篩選
外文關鍵詞: Food contact materials, Recycling, Migration test, In vitro test, High-throughput screening
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  • 隨著全球人口數的增長以及疫情帶來的飲食模式改變,食品接觸材(Food contact materials, FCMs)特別是塑膠及紙類材料用量大幅上升,各國紛紛制定再生食品接觸材(recycled FCMs, rFCMs)指引,而於先前研究已發現,rFCMs中存在著不同的化學物質,包含有意添加物質(Intentionally added substances, IAS)如製造用劑、材質穩定劑,以及非有意添加物質(Non-intentionally added substances, NIAS)如金屬等污染物,可經由食品與rFCMs的接觸而發生物質遷移,IAS經研究顯示會影響生物之生理運作,主要影響肝、腎等器官,然而在NIAS尚未有全面性的了解,因此廣泛使用再生食品接觸材之前,其整體毒性有待評估。由於3R (Replacement, Reduction and Refinement)概念,因此本研究利用生物體外試驗結合高通量體外毒性評估技術以研究塑膠及紙類rFCMs材質、回收再生製程、不同食品及食品模擬物遷移物質安全性及rFCMs適用性。本研究包含塑膠原生Polyethylene terephthalate (virgin PET, vPET), rPolypropylene, rPolyethylene和 rPET共96個樣本,紙類包含原生紙及再生紙和紙板共51個樣本。利用中性紅試驗(Neutral red uptake assay, NRU assay)評估FCMs遷移物質暴露之細胞存活力,高內涵篩選(High-content screening, HCS)以定量人肝細胞(HepaRG)和人腎細胞(HK-2)之細胞損傷,最後與化學定性定量結果進行關聯性分析、化學物質與FCMs樣本之ToxPi優先排序及Gephi視覺化。研究結果顯示,塑膠FCMs於NRU試驗結果顯示清洗、高溫熔融及過濾等回收再生製程能降低終產品細胞毒性,於HCS也顯示肝腎細胞毒性不甚顯著,表示rFCMs安全性高;紙類FCMs於NRU試驗結果顯示原生紙安全性較再生紙高,於HCS分析結果顯示高油脂食品炸雞與酸性食品接觸遷移後,分別顯著造成肝與腎細胞毒性,紙類FCMs較適合使用於接觸乾性食品如地瓜,結合化學物質特性與毒理資料庫之ToxPi優先排序亦呈現相似之結論。本研究之結果可提供政府擬訂再生食品接觸材政策之科學性參考。

    Due to the circular economy, countries have begun to develop recycled FCMs (rFCMs). However, intentionally added substances (IAS) and non-intentionally added substances (NIAS) exist in rFCMs. IAS will affect the physiological system, but NIAS has not yet been fully evaluated. The toxicity of rFCMs needs to be investigated before widespread use. This study used a high-throughput in vitro toxicity screening technique to investigate the safety and suitability of plastic and paper rFCMs. Neutral red uptake assay (NRU assay) evaluated cell viability under exposure of FCMs migrated substances. High content screening (HCS) quantified the cell content of the human liver and human kidney cells. Correlation analysis between bioassay and chemical analysis, integration of both data for ToxPi prioritization and Gephi visualization of the FCMs samples were conducted. The NRU assay showed the recycling process can reduce the cytotoxicity of the final product, such as cleaning, high temperature melting. HCS assay also demonstrated that the toxicity was not very significant in plastics, meaning plastic rFCMs had a less threat in food safety. The vPaper was safer than the rPaper, rPaper contact with fatty food and acidic food significantly caused respectively liver and kidney cell toxicity, whereas rPaper were suitable to contact with dry food. The results were similar with ToxPi prioritization that combined chemical properties and toxicology databases. The current study could provide scientific information for the government to establish policies on rFCMs.

    摘要 i Extended Abstract ii 誌謝 v 目錄 vi 表目錄 x 圖目錄 xi 中英文縮寫對照表 iii 一、緒論 1 1-1 研究背景 1 1-2 研究問題 3 1-3 研究目的 3 1-4 研究意義 4 二、文獻回顧 5 2-1 rFCMs使用及管理現況 5 2-1.1 國際 5 2-1.2 臺灣 7 2-2 FCMs存在之化學物質 8 2-2.1 塑膠 9 2-2.2 紙類 11 2-3 FCMs生物毒理研究 13 2-3.1 鄰苯二甲酸酯類(PAEs) 14 2-3.2 全氟烷基化合物(PFAS) 15 2-3.3 有機磷酸酯(OPEs) 16 2-3.4 金屬與類金屬 16 2-4 生物試驗 17 2-4.1 體外試驗 18 2-4.2 高通量篩選(HTS) 19 2-5 化學與毒理優先排序 21 2-6 數據視覺化 21 三、研究材料與方法 22 3-1 研究架構 22 3-2 FCMs遷移試驗樣本 24 3-2.1 食品模擬物及真實食品 24 3-2.2 再生食品接觸材製程 24 3-2.3 樣本前處理 24 3-2.4 FCMs樣本及試驗條件 25 3-3 細胞培養 26 3-4 中性紅攝取試驗 27 3-5 高通量毒性篩選─高內涵篩選 28 3-5.1 高內涵篩選方法建立與優化 28 3-5.2 肝腎毒性評判標準 30 3-6 統計分析 31 3-7 FCMs化學及毒理優先排序 31 3-8 FCMs樣本數據視覺化 33 四、結果與討論 34 4-1 中性紅攝取試驗標準 34 4-2 塑膠FCMs 35 4-2.1 中性紅試驗 35 4-2.2 高內涵篩選 42 4-2.2.1 人類肝細胞HepaRG 42 4-2.2.2 人類腎細胞HK-2 47 4-2.2.3 塑膠FCMs樣本之肝腎細胞毒性 52 4-3 紙類FCMs 54 4-3.1 中性紅試驗 54 4-3.2 高內涵篩選 56 4-3.2.1 人類肝細胞HepaRG 56 (一) 食品模擬物 56 (二) 真實食品 62 4-3.2.2 人類腎細胞HK-2 66 (一) 食品模擬物 66 (二) 真實食品 72 4-3.2.3 紙類FCMs樣本之肝腎細胞毒性 76 4-4 生物試驗與化學定性定量之相關性分析 80 4-4.1 中性紅試驗 80 4-4.2 高內涵篩選 80 4-4.2.1 人類肝細胞HepaRG 80 4-4.2.2 人類腎細胞HK-2 84 4-5 FCMs化學及毒理優先排序 88 4-5.1 塑膠FCMs 88 4-6 FCMs樣本數據視覺化 94 4-6.1 塑膠FCMs 94 4-6.2 紙類FCMs 95 五、結論與建議 97 5-1 結論 97 5-2 建議 98 5-3 研究限制 98 5-3.1 樣本種類的代表性 98 5-3.2 回收再生製程的評估 99 5-3.3 樣本處理與NIAS的潛在影響 99 5-3.4 體外試驗的限制 99 參考文獻 100 六、附錄 110 附錄一 各國食品模擬物代表性列表 110 附錄二 再生食品接觸材製程 111 –塑膠rFCMs 111 –紙類rFCMs 112 附錄三 遷移樣本列表 113 表A 塑膠FCMs遷移樣本列表 113 表B 紙類FCMs遷移樣本列表 114 附錄四 食品接觸材ToxPi優先排序數據 115 表C 食品接觸材終產品化學物質清單與數據 115 表D 食品接觸材終產品化學物質Peak area、濃度與ToxPi scores 120

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