## 7 Risk Assessment
### Threshold of Toxicological Concern Approach  
The Cramer decision tree, developed by [PERSON] et al. [89] and later refined by EFSA [90], is a systematic approach for classifying chemical substances into one of three toxicity classes (Class I, II, or III) based on their structural features and potential toxicological concerns. This classification system evaluates molecular structure through a series of 33 questions that assess the presence of functional groups, structural alerts, and metabolic activation potential that may indicate toxicity. Class I (low toxicity) includes substances with simple chemical structures and efficient metabolic pathways (e.g., sugars and amino acids) that suggest low toxicity potential, assigned a TTC threshold (TDI values) of 30 ug/kg bw/day. Class II (intermediate toxicity) contains moderately complex structures (e.g., esters and alcohols) with some reactive groups but no significant toxicity alerts, assigned a threshold of 9 ug/kg bw/day. Class III (high toxicity) comprises substances with structural features suggesting potential toxicity (e.g., aromatic amines, conjugated double bonds, and heterocyclic compounds) or those that cannot be confidently assigned to Classes I or II, assigned the most conservative threshold of 1.5 ug/kg bw/day.  
This system has been widely adopted in NIAS risk assessments. For instance, [PERSON] et al. [20] reported that several NIAS, such as \(\epsilon\)-caprolactam oligomers, bis(2-hydroxyethyl) terephthalate (BHET), AA-DEG, and cyclic PES oligomers, were found in baby food at concentrations exceeding the 0.01 mg/kg migration limit set for NIAS by Regulation (EU) No 10/2011 [7]. In particular, \(\epsilon\)-caprolactam was detected in 67% of baby food samples (0.01-7.50 mg/kg), remaining below the SML of 15 mg/kg. However, its cyclic oligomers, BHET, and AA-DEG often exceeded the 0.01 mg/kg NIAS threshold. Other semi-quantified oligomers reached concentrations of up to 4 mg/kg in some samples, suggesting that consumer exposure was higher. To assess toxicological relevance, compounds were categorized according to the Cramer decision tree using the Toxtree software [91]. Cyclic oligomers were mainly classified as Cramer Class III, while linear oligomers were classified as Class II. For an average infant weighing 10 kg, this equates to a tolerable intake of 0.015 mg/day of Class III oligomers. However, the calculated intake for AA-DEG from a single baby food pouch exceeded this limit by over 30-fold, indicating a potential safety concern when applying the TTC concept to these substances. It should be noted that the TTC thresholds incorporate a 100-fold uncertainty factor to account for inter- and intra-species variability [90], yet such exceedances may still warrant further toxicological investigation, particularly in sensitive subpopulations.  
However, the TTC approach has limitations, particularly for genotoxic or bioaccumulative substances. In the case of potential genotoxic substances, Commission Regulation (EU) No 10/2011 sets a stricter migration limit of 0.00015 mg/kg. In the study by [42], 9 substances exceeded the general 0.01 mg/kg limit, while 45 exceeded 0.00015 mg/kg. Some compounds were IAS and complied with their SMLs, indicating no immediate risk from an rLDPE film. However, 291 unidentified substances were also detected, for which toxicity and genotoxic potential remain unknown. The authors recommended further studies to identify these compounds and assess their individual and mixture toxicity, ideally using bioassays to support comprehensive risk assessments. For such high-risk NIAS, the EFSA recommends a default threshold of 0.0025 \(\upmu\)g/kg bw/day [90]. In practical terms, these low thresholds pose significant analytical challenges, as the corresponding migration levels are often close to or below the limits of detection usually achievable with current NTA methods. As a result, screening workflows may fail to detect relevant compounds or require highly sensitive targeted follow-up analyses to confirm potential genotoxicants.