FRAMEWORK FOR EXTRACTABLE AND LEACHABLE STUDIES IN DRUG PRODUCT DEVELOPMENT: A CASE STUDYAbstract
Extractables and leachables are compounds released from pharmaceutical packaging that can contaminate finished drug products under normal storage conditions. These compounds can potentially impact the efficacy, safety, and quality of the drug product. Despite regulatory requirements for conducting extractable and leachable studies for new drug product registrations, there are no internationally harmonized guidelines that address drug product categories and dosage forms, including design studies and approaches to safety assessment. This gap generates uncertainty, potentially delaying the regulatory approval of medicines and their access to patients. This case study discusses the impact of extractable and leachable studies on research and development routines and quality control in a Brazilian pharmaceutical industry. Herein, we proposed and applied a framework for developing an extractable and leachable study. After implementing the proposed framework, we observed a 43% reduction in execution time for the extractable and leachable studies. Additionally, safety limits for leachable compounds were determined based on toxicological evaluations to ensure patient safety. This article documents toxicological information for over 30 extractable and leachable substances identified in glass, elastomeric, and polymeric materials used in pharmaceutical applications. We also discuss the lack of specific guidance by regulatory authorities and future perspectives on extractable and leachable studies.
Keywords:
Extractables; Leachables; Packaging; Pharmaceutical development; Risk assessment; Safety evaluation.
INTRODUCTION
Packaging is a way of storing finished products, active pharmaceutical ingredients and excipients that are part of the medicine’s composition. To protect medicines, appropriate packaging must be used that does not interact with the stored product, thereby maintaining efficacy, quality, safety, and stability (WHO, 2002). Primary packaging represents the component that comes into direct contact with the product. Secondary packaging comes into direct contact with the primary packaging, providing an additional layer of protection. Moreover, tertiary packaging offers extra protection during the transport and/or storage of medicines (USP <1663>).
The selection of primary packaging materials must consider protection against external agents (e.g., humidity, light, temperature), biological contamination and physical damage. Additionally, the compatibility of packaging materials with the pharmaceutical form must consider the sorption and leaching processes that occur during storage for the sake of patient safety (EMA, 2017; WHO, 2002). The most common examples of primary drug packaging are ampules, tubes, bags, vials, bottles, flacons, cartridges, and syringes. On the other hand, secondary packaging includes cartons, sachets, cases, and overpouches.
Glass containers are widely used as primary packaging in various pharmaceutical dosage forms. They are composed principally of silicon dioxide, with varying amounts of other oxides (e.g., sodium, potassium, calcium, magnesium, aluminum, boron, and iron). All types of glass have the potential to leach elemental impurities into the product. In general, low levels of leachable substances have been reported at pH 4-8, while high levels of leachables can occur at pH > 9 or at elevated temperatures due to the dissolution process of borosilicate glass (Sacha et al., 2010; Srinivasan et al., 2019).
Plastics used in the production of drug packaging systems are composed of polymers with diverse chemical structures that include additives such as plasticizers, stabilizers, lubricants, antioxidants, dyes, and resins. The physicochemical properties of the additives that comprise the polymer composition in plastic packaging can be affected by the sterilization process, surface treatment, storage conditions, and the compatibility and permeability of excipients. The most commonly used polymers in the composition of plastic packaging for medicines are polyethylene (high and low density), polypropylene, polyethylene terephthalate, polyvinyl chloride, ethylene vinyl acetate and polyolefin (Cuadros-Rodríguez et al., 2020; Sacha et al., 2010; USP <661>).
Elastomers are polymers that can return to their initial state after being stretched or deformed. Closures contain natural or synthetic elastomers and inorganic and/or organic additives that control the vulcanization step and provide color and stabilization to the container. Synthetic rubbers include styrene-butadiene, neoprene [poly-(2-chloro-1,3-butadiene)], nitrile and butyl, which is the most used elastomer for the pharmaceutical area (Anvisa, 2019; Sacha et al., 2010).
Packaging materials must not be reactive, additive, or absorbent so as not to alter the identity, resistance, or quality of the medicinal product or pose a risk to the patient’s health. The evaluation of the packaging systems used to store the medicine is necessary for product release. In this sense, extractables and leachables (E&L) studies represent an experimental scenario for assessing the quality and safety of product packaging components.
Extractables studies involve extracting organic and inorganic chemical compounds from a packaging system using appropriate solvents (aqueous and/or organic) under forced experimental conditions that utilize time, temperature and extraction techniques, considering the mass ratio in relation to the solvent volume or surface area of the package (USP <1663>). In contrast, the leachable studies assess the migration of compounds from a packaging system into the medicine through direct contact under normal storage conditions. Leachables are normally derived from extractables, that is, a subset of the universe of compounds that are extracted from packaging. Furthermore, migrating compounds may also be present in medicines, but not through the direct action of the formulation in the container. The origin of these migrating compounds is derived from secondary and tertiary packaging or auxiliary components (USP <1664>).
The presence of leachable or migrating compounds may alter the quality, efficacy, and safety of medicines (Jenke, 2018). Leachable substances in medicines may present potential risks to patients due to their toxicity, concentration level and/or duration of treatment (Parris et al., 2020). Medicines may interact with packaging components through adsorption, absorption, permeation, leaching and extraction processes (Cuadros-Rodríguez et al., 2020).
Currently, there is no regulation on the evaluation and control of E&L studies. This regulatory gap creates uncertainty for drug manufacturers and health authorities due to the insufficient information required to meet regulatory expectations and conduct toxicological assessments of compounds. This article proposes and applies a comprehensive approach for assessing extractable and leachable studies. Thus, it provides a framework for conducting extractable and leachable studies, including toxicological safety assessment.
MATERIAL AND METHODS
The findings of this article are based on a case study conducted over more than two years. We evaluated three distinct types of packaging systems (e.g., glass, elastomer, and plastic) and four parenteral drug products. Thus, the database used in this article was selected based on its relevance in medicine [e.g., concentrations above the Analytical Evaluation Threshold (AET)]. The representative materials and medications were evaluated using multiple analytical techniques, including liquid chromatography coupled to mass spectrometry (LC-MS), headspace gas chromatography coupled to mass spectrometry (HS-GC-MS), gas chromatography coupled to mass spectrometry (GC-MS), and inductively coupled plasma mass spectrometry (ICP-MS), as described in the Product Quality Research Institute (PQRI) guidelines (PQRI, 2011b; PQRI 2011a).
RESULTS AND DISCUSSION
Framework for extractable and leachable studies
The framework of the E&L studies encompasses investigative studies across multiple stages, resulting in a suite of complex tests that are challenging to implement due to the use of multiple analytical instruments. Therefore, given the project’s complexity and the absence of specific guidelines, the design of the E&L studies proposed in Figure 1 is based on the recommendations and general guidance from the United States Pharmacopeia’s General Chapters (USP <1663>; USP <1664>) and the PQRI guidelines (PQRI 2011b; PQRI 2011a).
In the following sections, relevant points regarding the evaluation and development of E&L studies will be discussed. Data from published literature and expert reviews support the toxicological information.
Risk assessment
Before conducting analytical tests, it is necessary to perform a risk assessment, which involves a systematic process of organizing information to support informed decisions within a risk management process. It consists of identifying hazards, analyzing and evaluating the risks associated with exposure to these hazards (EMA 2023a). Within the context of extractables and leachables, risk assessment is essential. It aims to obtain information such as the main constituents of packaging materials, potential extractables previously reported by packaging material manufacturers, relevant risks previously reported due to the interaction of packaging material with the drug, and toxicological information about known extractables as potential leachables. Moreover, when toxicity data for a particular extractable or leachable compound and/or its mutagenic potential are not available, in silico approaches can be employed, as outlined in the ICH M7 guideline (EMA 2023b).
For a comprehensive risk assessment, this stage also establishes the Safety Concern Threshold (SCT) and the Analitical Evaluation Threshold (AET), which provide support in setting limits within which a leachable substance must be characterized and reported for toxicological evaluation. (USP <1663>; USP <1664>). The AET can be mathematically derived from the SCT or other limits, such as the Threshold of Toxicological Concern (TTC) or the Qualification Threshold (QT), based on factors including route of administration, maximum daily dose, and duration of drug treatment.
The SCT represents a value below which a leachable compound would have such a low dose that it would pose an insignificant toxicological concern for both carcinogenic and noncarcinogenic effects. The TTC is a level of exposure for all chemicals, regardless of whether specific toxicity data is available, below which there would be no appreciable risk to human health. Lastly, the QT is the threshold below which a given noncarcinogenic leachable is not considered for safety qualification (toxicological assessments) unless the leachable presents structure-activity-relationship (SAR) concerns (USP <1663>; USP <1664>).
Through the established relationship among the mentioned thresholds, it is possible to convert medication dosing into concentration units, thereby establishing a direct connection to the values set by the AET for assessing the leachables’ results. The commonly used SCT values for genotoxicity of organic compounds for orally inhaled, nasal, and parenteral drug products are 0.15 μg/day and 1.5 μg/day, respectively (PQRI 2006a). The TTC for mutagenic impurities is based on the treatment duration, ranging from 1.5 mg/ day (>10 years) up to 120 mg/day (≤1 month). The QT is fixed at 5 µg/day, representing a potential value for irritation or sensitization (EMA, 2023b; PQRI, 2021; Singh et al., 2021). The general formula for converting SCT into AET is presented below.
For liquid pharmaceutical forms:
For solid pharmaceutical forms:
According to PQRI (2006b), after calculating the AET, it is recommended to use a Correction Factor (CF) in the final AET calculation to enhance the accuracy of the estimated final concentration for the analytes (PQRI 2006b). Thus, the calculated AET value derived from the previous equations is divided by a CF equal to 2 or multiplied by 0.5. Since the standards used in estimating the quantification of leachable compounds are generic, employing the CF adds an extra level of safety for the patient.
The acquisition of accurate results in E&L studies, particularly in the screening of extractables, poses a significant challenge due to the absence of reference standards and response factor variation, where compounds at the same concentration exhibit different signal intensities in the detector. The CF will effectively encompass the variability in the measurement of the unknown compounds due to the reduction of the threshold, consequently providing greater assurance and security in the results. However, this strategy may entail greater time and cost in the development of analytical methods, as well as the evaluation of more chromatographic signals that may not represent a real risk in the finished product (Jordi et al., 2020; Singh et al., 2021).
Extractable studies
Extractables are organic and inorganic chemical entities that are released from a pharmaceutical packaging/delivery system, packaging component, or packaging material and into a solvent under laboratory conditions (USP <1663>).
The extractable studies primarily aim to obtain a representative profile of potential extractables originating from packaging systems that constitute the finished product. To evaluate the extractables profile, it is necessary to subject packaging components to efficient sample preparation techniques that target the selective extraction of organic compounds (e.g., volatile, semi-volatile, and non-volatile) and inorganic compounds (e.g., metals and anions). This step is followed by analysis using efficient analytical techniques to detect, identify, and quantify these analytes.
The objective of the sample preparation technique is to subject packaging material components to intense laboratory conditions that are more aggressive than those encountered in real-use scenarios. For this purpose, extraction techniques utilize organic solvents of varying polarities (e.g., dichloromethane, hexane, and isopropanol) and aqueous solvents at different pH levels (e.g., pH 2.5 and pH 9.5) (PQRI 2011a; PQRI 2011b).
The goal is to assess the interaction of these extraction solvents with the chemical composition of packaging extracts. This interaction mainly depends on the solvent extraction process, the degree of interaction between the solvent and the material, material swelling, and the diffusion rate of material constituents (Li and Sobańtka, 2023). It is also recommended to assess the influence of time and temperature factors during the extraction step, as experimental conditions should be appropriate to avoid degrading the test materials or extractable compounds, while still generating significant quantities to identify the extractable compounds confidently (PQRI, 2011b; PQRI, 2011a). According to the literature, among the most commonly used sample preparation techniques for extracting potential extractables from packaging component materials are: (i) ultrasound-assisted extraction (UAE), (ii) reflux, (iii) Soxhlet extraction, (iv) microwave-assisted extraction (MAE), (v) pressurized liquid extraction (PLE), and (vi) supercritical fluid extraction (SFE) (Cuadros-Rodríguez et al., 2020; PQRI, 2011b; PQRI, 2011a).
After the extraction step, the extracts undergo multiple orthogonal analytical techniques to obtain a comprehensive profile of extractables. Therefore, for the analysis of volatile organic compounds, HS-GC/MS is employed. For the analysis of semi-volatile organic compounds, GC-MS is used. To analyze non-volatile organic compounds, LC-MS/MS is employed. Finally, for the evaluation of inorganic compounds, metals, and anions, techniques such as ICP-MS or inductively coupled plasma optical emission spectrometry (ICP-OES) and ionic chromatography (IC) are utilized, respectively (PQRI, 2011b; PQRI, 2011a; USP <1664>). It is important to mention that IC studies are not universally practiced in the screening of extractables and leachables since commonly evaluated potential extractables, such as common inorganic anions (e.g., chloride, bromide, nitrate, sulfate, phosphate, among others), and low molecular weight organic acids (e.g., acetic and formic acids) are generally considered safe. Thus, this information is rarely significant from a safety assessment perspective (Jenke, 2018).
At the end of the extractables study, a profile of compounds extracted from packaging components is obtained, which includes potential extractables derived from chemical additives of packaging with direct contact with the product and migrants from secondary and tertiary packaging (e.g., ink, adhesive, and varnish from labels) (PQRI, 2011b; PQRI, 2006b).
Once the profile of extractable compounds from packaging components is known, the next step is to evaluate the relevance of the identified risk to the finished product. In this context, the initial assessment involves determining whether the potential extractables are compounds known to be characteristic of the type of material used and whether there is information in the literature regarding their toxicological safety and defined exposure limits. Based on this information, evaluations are conducted to demonstrate the potential effects of extractables on a patient’s health if these extractables were leached into the analyzed finished product (Jenke, 2018). If the conclusion indicates an absence of real risks, conducting leachables studies on the finished product is not necessary. Thus, ensuring the safety and quality of the finished product can be achieved solely through screening performed in the extractables study. However, if it is not possible to ensure the safety of the finished product during the extractables study, leachables studies become necessary.
Leachable studies
Leachables are unintended organic and inorganic substances that diffuse from a packaging or delivery system, component, or material used in the manufacturing process into the pharmaceutical product under normal storage and usage conditions. Therefore, they should be evaluated throughout the stability study according to the analysis times planned in this study. Leachables have the potential to impact products in terms of safety, quality, and efficacy due to their interaction with the active pharmaceutical ingredient or excipients (USP <1664>). According to USP <1664>, the likelihood of interaction with packaging material depends on the pharmaceutical form associated with the route of administration (USP <1664>). In this regard, pharmaceuticals such as aerosols, inhalation sprays, parenterals, transdermal patches, and ophthalmic solutions and suspensions present a high to very high degree of risk associated with their route of administration. Furthermore, as per information from the USP <1663>, we should not assume that the low risk of administration and low probability of interaction between packaging and formulation will result in the absence of leachable compounds in the finished product (USP <1663>; USP <1664>). Other factors may influence the presence of undesirable compounds, such as the packaging structure (e.g., permeability, thickness, and composition) and/ or the physicochemical characteristics of migrating compounds that can penetrate through the packaging walls.
In this context, leachable studies are defined as laboratory investigations into the qualitative and quantitative nature of specific leachable profiles throughout the proposed shelf life of a given medication (USP <1664>). The leachable study is based on the extractables profile obtained during the extractables study. The goal of these studies is to identify and quantify the leachables according to defined analytical limit parameters systematically and rationally (USP <1664>).
One of the greatest difficulties in leachable studies is the potentially diverse profile of the compounds, which requires extensive knowledge of both analytical and toxicological methods. Another critical point is meeting extremely low analytical limits, which are defined based on the AET of the product, informing the minimum quantity in which leachables must be identified and quantified in the medicine. Thus, it is suggested that the quantification limit of the analytical method be equal to or less than half of the AET to ensure its ability to identify and quantify this limit adequately.
After the sample preparation step, the extracts undergo analysis using the same analytical techniques applied in the extractable study, as discussed above in the previous section. Therefore, the analytical methods should be capable of determining both the predefined target leachables in the extractable study and any new (or unspecified) leachables (e.g., screening by GC/MS or LC/MS) (USP <1664>). Additionally, due to the use of analytically challenging thresholds, dedicated target compound methods, such as selected ion monitoring mode, may be employed to achieve enhanced sensitivity. Lastly, to ensure the quality of the obtained results, system suitability tests and criteria should also be developed for each analytical method. The system suitability includes tests for method linearity, precision, sensitivity, and specificity as appropriate. These parameters should be evaluated with suitably constituted mixture(s) each time the quantitative leachables method is used. The tests should also use appropriate system suitability acceptance criteria based on the results of method validation. For instance, sensitivity can be confirmed by analyzing standards prepared at the analytical threshold (USP <1664>).
For leachables above the AET concentration without confirmed identity, their identification can be performed by coupling chromatographs to sequential mass spectrometry detectors, utilizing high-resolution mass analyzers such as quadrupole time-of-flight (QToF) or orbitrap for liquid chromatography (Vas et al., 2020). The confirmation of the target molecule’s identification can be verified using appropriate reference standards for analysis, an automated MS data library, or based on the molecule’s fragmentation profile.
Finally, once identified and quantified, the next step is to assess whether these leachables have concentrations below or above the determined AET for the product at the end of the stability study. If the leachables have concentrations below the AET, the leachable study is concluded, ensuring the quality and safety of the medication. For leachables that exhibit concentration exceeding the AET, conducting toxicological safety evaluation studies is necessary.
Toxicological safety assessment
The extractables and potential leachables that have been quantified through analytical studies and have concentrations exceeding the AET in the finished product should be identified and reported for toxicological evaluation. For compounds that exceed these limits, a health-based exposure limit (HBEL), such as a permitted daily exposure (PDE), should be established to demonstrate safety after exposure to the leachable (Masuda-Herrera et al., 2023). These assessments are necessary for regulatory submissions as part of evaluating product stability, quality, and patient safety. Information regarding the establishment of toxicological safety limits will be further discussed in the next section. Ultimately, if, after the toxicological safety assessment of the leachable(s), it is concluded that the quantity present in the medication represents a safe exposure limit for the patient, the E&L studies are concluded. Otherwise, it is necessary to replace the packaging material and initiate new studies.
Extractable and leachable report
After the extractable study, a report is drafted that includes information such as the rationale for conducting the extractable study, the choice of extraction conditions, the analytical methods used to generate the extractable profile, the extractable profile itself, the safety assessment of the extractable, and a list of target leachables. Upon completion of the leachable study, data related to leachables are attached to the extractables report. In addition to the information summarized in the extractables report, the leachable study design, a list of leachables and their concentrations, and a safety assessment of the leachables are provided.
Case study: Implementation framework for extractable and leachable studies.
The framework was implemented in 12 new drug product development projects, and its impact was monitored over a two-year period. The main evaluation included comparing execution times before and after implementation. The results showed a 74% reduction in execution time for extractable studies, a 24% reduction in execution time for leachable studies, and a 43% reduction in execution time for the E&L studies combined.
Case study: Safety limits for compounds observed in extractables and leachables studies.
We proposed safety limits for compounds identified in extractable studies from packaging materials that could potentially leach into drug products under storage conditions. Toxicity and pharmacokinetic data, including acute and chronic toxicity, genotoxicity, carcinogenicity, reproductive and developmental toxicity, as well as sensitization potential and ADME (absorption, distribution, metabolism, and excretion), were obtained from government databases and peer-reviewed scientific publications. Another approach, such as in silico predictions, was used in the absence of experimental data. There are tools available on the web that are used to identify potential structural analogues by read-across based on the principles of the European Chemicals Agency (ECHA, 2017). An expert review was conducted after all data collection was completed. Table I highlights the primary databases and web tools that can be used to assess the toxicological safety of extractable and leachable compounds.
Relevant publicly available sources for obtaining information for toxicological safety assessment of extractable and leachable studies
The toxicological safety assessment was conducted based on studies published by Broschard et al. (2016) and Parris et al. (2020). Each limit was derived using the principles of PDE described in the ICH Q3D and Q3C guidance (EMA 2024; EMA 2022). PDE values for the parenteral route were expressed in milligrams per day (mg/day) using a conservative body weight of 50 kg. In all examples cited in this article, key studies were summarized in supplementary data. Table II summarizes PDE values for approximately 30 compounds commonly found in extractable and leachable studies.
The list in Table II results from E&L studies of four different injectable solutions, involving three types of packaging materials. A total of 142 potential extractables were detected, and 30 leachables were assessed for their toxicity. These compounds encompass a variety of chemical classes, including antioxidants and stabilizers, such as BHT and Irganox 1076, commonly used in polymers to prevent oxidative degradation (Khan et al., 2024); fatty acids, such as stearic acid, employed as plastic additives, processing aids, or contaminants, and their leaching from plastics is well-documented (Jenke, 2010); linear and branched alkenes, such as eicosane and octacosane, often associated with polyethylene oligomers (Jenke et al., 2006); Phthalate derivatives, such as DEHP, as plasticizers with known toxicological concerns (Bernard et al., 2014); and inorganic compounds, such as arsenic and silicon, which may originate from glass (Schaut et al., 2014). The identification and toxicological assessment of these substances are critical steps in ensuring the safety and suitability of packaging materials in parenteral drug products.
Thus, limits established for identified extractable and leachable substances in pharmaceutical products highlight the innovative nature of this study.
Technical report
At the end of the experimental stage, a detailed analysis must be performed between the determined concentration levels of the compounds in the finished product and the limits of toxicological assessments of the product under analysis for patient treatment. In cases where the concentrations of compounds exceed the safety limit, a strategy and discussion should be conducted to establish specific limits or modify packaging materials to ensure patient safety.
Regulatory aspects related to drug product packaging.
Currently, E&L studies are among the most discussed topics in pharmaceutical regulatory agencies worldwide, where the purpose of conducting these studies is to ensure that chemical compounds from packaging are not transferred to medicines at levels that pose a risk to the patient’s health.
Leachables are organic and inorganic substances that are potential impurities of medicines. These impurities have reference guides, such as ICH M7 (mutagenic impurities), ICH Q3C (residual solvents), and ICH Q3D (elemental impurities), which can be used for guidance in risk assessment (EMA 2024; EMA 2023b; EMA 2022). Generally, the required limits in leachable compounds are significantly more critical than the concentrations of these isolated impurities, which generate greater costs and time demands in the development of complex methods for E&L analyses.
The general chapters from USP do not establish specific extraction conditions, analytical determination, specifications, and acceptance criteria for packaging (USP <1663>; USP <1664>). The PQRI guidelines provide guidance on analysis methods, but do not address the regulatory aspects of product release (PQRI 2021; PQRI 2011a; PQRI 2011b). The absence of standards and more specific regulations for conducting studies on extractables and leachables leads to variability in the design of experiments, which consequently delays the release and regulatory approval of pharmaceutical products. The pharmaceutical industry and regulatory agencies highly regard the ICH Q3E guideline for assessing, controlling, and setting requirements, specific limits, and critical aspects of quality and safety in medicines (EMA, 2020).
Harmonization between guidelines, regulatory agencies, and pharmacopoeias will provide an opportunity for a global orientation in extractable and leachable studies, consequently leading to greater safety in the release of medicines by pharmaceutical industries.
Future trends in extractable and leachable studies
To mitigate the risks associated with extractable and leachable studies in medicines, pharmaceutical industries must adopt several strategies to reduce the transfer of potentially toxic compounds to the finished product. Some of these strategies may be related to the selection of packaging with quality assured by the manufacturer, the implementation of good manufacturing practices with greater criticality, and minimizing cross-contamination in production. Additionally, the development of a selective, precise, accurate, and robust analytical method is also essential.
CONCLUSION
Extractables and leachables are a concern in drug product development due to their potential impact on the quality of the drug product and patient safety. A risk assessment is necessary to evaluate the likelihood of compound extraction from packaging that could leach into drug products, thereby posing a risk to patients. Conducting E&L studies requires the development of specific regulations, monographs, and guides, along with a well-designed experimental strategy for extractions and analytical determinations. These studies are followed by a toxicological evaluation that considers patient safety and well-being. The results will be more reliable, and harmonized documents will contribute to faster regulatory approval.
ACKNOWLEDGMENTS
The authors are grateful for the financial support from Cristália Produtos Químicos Farmacêuticos LTDA.
Supplementary Information
Safety assessment of leachable 4,4’-thiobis(6-tert-butyl-m-cresol) and structural analogues4,4’-thiobis(6-tert-butyl-m-cresol) (CAS 96-69-5) (Supplementary Figure 1) is used in the rubber and plastics industry as an antioxidant. Additionally, it is also used as a stabilizer in polyethylene and polyolefin plastics used in food packaging materials (NTP, 1994).
The results obtained from the Ames test and chromosome aberration assay showed that 4,4’-thiobis(6-tert-butyl-m-cresol) is not genotoxic. An in vivo genotoxicity study using male and female Fischer 344 rats, followed by analysis of bone marrow cells for chromosomal events, revealed no significant increase in the incidence of chromosome aberrations. Therefore, 4,4’-thiobis (6-tert-butyl-m-cresol) is not clastogenic (ECHA, 2023a).
The disposition of 14[C]-labeled 4,4'-thiobis(6-tert-butyl-m-cresol) was studied in male Fischer 344 rats after a single oral dose of 5, 50 or 500 mg/kg and intravenous injection at a dose of 5 mg/kg. After oral ingestion, this compound was incompletely absorbed, and there was no dose-related decrease in the rate of absorption. However, the results of in situ administration via luminal perfusion showed that absorption in the small intestine was directly proportional, suggesting that retention of the compound in the stomach was related to the decline in absorption. After intravenous administration, a small percentage of the administered dose was detected in the liver, adipose tissue, skin, muscle, and blood. During chronic exposure, the compound may accumulate in these tissues. More than half of the administered dose of the compound was excreted on the first day, mainly via bile in the feces, and less than 2% was excreted in the urine. All radioactivity detected in bile was in the form of metabolites; thus, the main metabolite was monoglucuronic acid (NTP, 1994).
In acute oral toxicity studies of 4,4’-thiobis(6-tert-butyl-m-cresol), using the weight-of-evidence approach, it was possible to detect toxicity in rats. The LD50 values found were 2315, 2345, 2420 and 1450 mg/kg (ECHA, 2023a). In chronic toxicity studies of 4,4’-thiobis(6-tert-butyl-m-cresol), male and female F344/N rats showed a significant increase in weight and incidence of liver lesions in females at a dose of 45 mg/kg. The established oral NOAEL was 20 mg/kg/day (ECHA, 2023a).
A reproductive toxicity study in pregnant rats treated with 4,4'-thiobis(6-tert-butyl-m-cresol) at a dose of 485 mg/kg/day for 10 days during gestation showed an increase in maternal mortality and a decrease in pup survival percentage without affecting the number of viable litters, litter size, birth weight, or pup weight gain.
Since a single dose was assessed, the established LOAEL for maternal and developmental toxicity was 485 mg/kg/day.
A developmental toxicity study in white rabbits given 4,4'-thiobis(6-tert-butyl-m-cresol) orally at doses of 0, 0.2, 2, and 20 mg/kg from day 6 to day 8 of gestation showed that there was no significant increase in fetal anomalies. The only effect was a delay in maternal weight gain due to maternal toxicity, leading to an increased incidence of abortion in animals given high doses. The established NOAEL for maternal toxicity was 2 mg/kg/day, and the established NOAEL for developmental toxicity was 0.2 mg/kg/day (EPA, 2010). This study lacks information regarding guidelines for testing good laboratory practice conditions and the identity and quality of the substance evaluated, in addition to being poorly documented, i.e., classified with reliability grade 4 (ECHA, 2023a). For this reason, the NOAEL for maternal/developmental toxicity was not considered for calculating the PDE.
The PoD chosen for calculating the PDE was the oral NOAEL of 20 mg/kg/day obtained based on a chronic toxicity study in rats over 2 years. This study was classified as reliable (Klimisch score = 2). Thus, the established parenteral PDE was 2 mg/day, considering a safety factor of 500 and a body weight of 50 kg (Supplementary Table I).
Description of safety factor applied to the PDE for 4,4’-thiobis (6-tert-butyl-m-cresol)
Due to the limited toxicological data for extractables described above, data gap-filling by read-across to a surrogate structural analogue with toxicological data provides an alternative method for determining a PDE. A structural comparison was performed between three extractables (Supplementary Figure 2) and one analogue, [4,4’-thiobis(6-tert-butyl-m-cresol)], to determine the similarity coefficient using ChemMine Tools (Backman et al., 2011). The ChemMine Tools employ two algorithms to calculate similarity coefficients between two structures: the Atom Pair (AP) as a structural descriptor and the Maximum Common Structure (MCS) (Backman et al., 2011). AP is a type of structural descriptor defined by the shortest path between the non-hydrogen atoms of a molecule. The MCS algorithm is based on the maximum shared substructure between two compounds, providing a more accurate and sensitive similarity measure, especially for compounds with large size differences(Backman et al., 2011). Thus, the extractable molecules 1, 2, and 3 had MCS Tanimoto coefficients above 0.9, indicating a high similarity with the analogue molecule. Supplementary Table II showed Tanimoto coefficients for three extractables with limited toxicological data. Thus, based on the read-across approach, it is possible to determine the PDE for extractable molecules 1, 2, and 3.
Safety assessment of leachable methylcyclopentaneMethylcyclopentane (CAS 96-37-7) (Supplementary Figure 2) belongs to the class of organic compounds known as cycloalkanes and is a component of the naphthalene petroleum fraction (EPA, 2009). It is used as a solvent and intermediate in synthesis in the chemical industry, and is not considered highly toxic (Elliott et al., 1965). Studies have shown that methylcyclopentane can be extracted from plastic bags (Dorival-García et al., 2018) and pharmaceutical rubber (Yu et al., 2010).
Ames test results with methylcyclopentane were uniformly negative in strains of TA100, TA98, TA135, TA1535, and TA97, with or without metabolic activation (EPA, 2009). There are no ADME studies available for methylcyclopentane. A metabolism study of commercial hexane (structural analogue) was used to evaluate these pharmacokinetic parameters. According to this study, in rats, n-hexane was metabolized and excreted within 168 hours after intravenous bolus administration, inhalation exposure or dermal application. Exhaled breath and urine were the major routes of excretion. While n-hexane was widely distributed in body tissues, neither n-hexane nor its metabolites were significantly concentrated in any of these tissues. It was found that n-hexane was extensively metabolized, and several radiolabeled metabolites were excreted in the urine. Moreover, n-hexane and its radiolabeled metabolites disappeared from the blood of rats with a half-life of approximately 9-10 hours. No significant differences were observed between males and females in the rates and routes of metabolism and excretion of the test compounds. Repeated inhalation exposure had no apparent effect on the rates or pathways of excretion of n-hexane or its metabolites. Bioaccumulation of n-hexane or its metabolites is not expected due to extensive metabolism and excretion via urine, and the absence of significant tissue concentrations observed (ECHA, 2023b).
In the nephrotoxicity study groups of ten males Fischer 344 rats were administred 500 or 2000 mg/kg methylcyclopentane (98% purity) by gavage once daily, 5 days/week, for 4 weeks. A negative control group received isotonic saline at a dose of 2000 mg/kg/day. Animals were observed twice daily for mortality and clinical signs of toxicity. Body weights were measured before dosing on day 1 and at the time of the scheduled sacrifice. Gross necropsies were performed on all animals after moribund condition, death, or terminal sacrifice. Only the kidney was evaluated histopathologically. In the high-dose group, 4/10 rats died. One of the 10 low-dose rats died; no clinical signs were observed in the surviving low-dose rats. No deaths or clinical signs were reported among control rats. Body weight was significantly reduced by 16% in the high-dose group and 8% in the low-dose group. Kidney weights were similar to those of controls in both groups. Necropsy findings observed in the high-dose group were prominent hepatic lobular patterns in 2/10 rats, punctate patterns in the renal cortex in 2/10 rats, and a pale elevated area in a non-glandular section of the stomach in 1/10 rats, and in both the lowand high-dose groups, mottled lung in 1/10 of each group and discolored pancreas in 1/10 rats of each group. Histological examinations of the kidney did not indicate any methylcyclopentane-related nephropathy. Stomach irritation (observed during gross necropsy) or other portal-of-entry effects were not noted since these tissues were not examined histologically. For this review, a NOAEL of 500 mg/kg dose and a LOAEL of 2000 mg/ kg dose for the body weight parameter are considered (EPA, 2009).
Comparative neurotoxicity study of five components of commercial hexane (each >99% pure) in which a group of 5-7 male Wistar rats were treated with methylcyclopentane by daily gavage in olive oil for 8 weeks at doses of 0.4 mL for the first 4 weeks, 0.6 mL for the following 2 weeks, and 1.2 mL for the final 2 weeks of the study. Considering the mean increase in body weight over the 8 weeks of the study, the doses of methylcyclopentane can be estimated at 800, 1,050, and 2,020 mg/kg/day for weeks 1-4, 5-6, and 7-8, respectively. A group of 5-7 control animals received olive oil alone. Peripheral nerve activity was measured in the tail of unanesthetized animals. Motor nerve conduction velocity, motor distal latency, and mixed nerve conduction velocity were measured. Body weight and conduction velocities were measured at the beginning of the experiment and then every 2 weeks until the experiment was terminated. No mortality or clinical signs of toxicity were observed. Body weight gain in treated animals was similar to that of the control group. Motor nerve conduction velocity and mixed nerve conduction velocity (proximal, but not distal, portion of the tail nerve) were significantly reduced in treated rats at 8 weeks. Distal latency was not affected at either time point. The investigators characterized these proximal effects as a slight, although statistically significant (p < 0.05), difference from the control in the impairment of peripheral nerve function by methylcyclopentane.
The point of departure (PoD) considered the oral NOAEL value of 500 mg/kg/day obtained from a nephrotoxicity study in rats, in which an 8% reduction in body weight gain was observed at a low dose (EPA, 2009). Although confidence in the study is low due to suspicious dosimetry, given the potential volatilization of the substance under study, this study was chosen because it was one of the few studies examining chronic toxicity, two-generation reproductive toxicity, and developmental toxicity. Based on this study, an exposure limit of 0.4 mg/kg/day eas derived. The NOEAL was initially adjusted from the 5-day/week dosing schedule (500 x 5/7 = 375 mg/kg/day) (EPA, 2009), and subsequently divided by a sefety factor of 1000. Considering individuals weighing 50 kg, the resulting proposed exposure limit is 20 mg/day (Supplementary Table III).
Safety assessment of leachable SiliconSilicon (Si) (CAS 7440-21-3) is the second most abundant element in the Earth's crust and occurs naturally in foods as silica dioxide (SiO2, silica) and silicates. High levels of silica are found in foods derived from plants, particularly cereals, while lower levels are found in foods derived from animal sources (EFSA 2009). This element serves as the initial substrate for producing silicones and glasses used in the pharmaceutical industry. Additionally, biologically, in nutritional quantities, silicon provides benefits for bone health and cognitive function (Prescha et al., 2019).
There is no information available on the mutagenicity of silicon. Therefore, a comparative analysis was conducted with synthetic amorphous silica. In vivo and in vitro studies with amorphous silica suggest that silicon is non-mutagenic (ECHA, 2023c).
After ingestion, amorphous silicon dioxide has negligible effects on tissues and urine. Silicon in different forms is ubiquitous in the environment (food, water, beverages). Normal dietary intake of silicon is 20-50 mg/day. It appears to be in a highly available form, as demonstrated by a high proportion of dietary silicon excreted in urine. Differences in dietary intake likely explain the variability in urinary silicon levels among individuals. Although in neutral solutions elemental silicon and silicon dioxide dissolve slowly, in acidic solutions, it is significantly less. Thus, for example, in the stomach, the release of silicon from silicon particles is likely to be low, which may affect absorption in the gastrointestinal tract (ECHA, 2023c).
Jungdaohsignh et al. (2002) studied dietary silicon intake in a cohort of 5,209 men and women, as well as the gastrointestinal absorption of silicon in 3-8 healthy subjects. Silicon was readily available from food and, in many cases, showed similar absorption to silicon from fluids. For example, urinary excretion of silicon (as an indicator of absorption) was 41-86% from cornflakes, white rice, and brown rice and 50-86% from mineral waters (Jugdaohsingh et al., 2002). Animal data on the acute toxicity of synthetic amorphous silica can be used to extrapolate the assessment for elemental silicon. These data do not reveal acute toxicity by oral, inhalation, or dermal routes(ECHA, 2023c). According to the available data, the acute oral toxicity of synthetic amorphous silica in rats is very low. No signs of toxicity were observed at doses up to 5000 mg SiO2/kg.
Repeated-dose toxicity studies on synthetic amorphous silicon dioxide and calcium silicate have demonstrated that silicon ions do not induce systemic toxicity in target organs following oral exposure. Therefore, no systemic toxicity is expected from oral exposure to silicon (ECHA, 2023c). In this study, micronized silica gel (SYLIOD®) was administered in the diet of B6C3F1 mice and Fisher rats at doses of 0, 1.25, 2.5, and 5%) (equivalent to 0, 2500, 5000, and 10000 mg/kg/day for mice and 0, 325, 1200, and 2500 mg/kg/day) for 93 and 103 weeks, respectively. The results showed that there was no biological damage or any other significant change in body weight, food consumption or physical characteristics. Dietary administration of SYLIOD® to animals did not cause macroscopic or microscopic changes in the tissues examined. The occasional presence of some neoplasias did not reveal a consistent dose-related trend in the animals. It can be concluded that dietary administration of micronized silica is safe, with no long-term toxic effects (ECHA, 2023c). The oral NOEAL established by this study was 2500 mg/kg/day for rats (ECHA, 2023c)
The available animal data on the developmental toxicity of synthetic amorphous silica, calcium silicate and sodium aluminum silicate, which can be used for read-across comparison, do not suggest developmental toxicity or teratogenicity. The inherent physicochemical properties and ubiquitous nature of the silicon ion suggest that there is no structural alert indicating any potential for developmental toxicity (ECHA, 2023c).
Silica and synthetic amorphous silica have not been evaluated for their skin sensitizing properties. Long-term use of synthetic amorphous silica, without any reported cases of sensitization, supports the lack of sensitizing properties of silicon. Therefore, silicon is not a skin or respiratory sensitizer(ECHA, 2023c)
The PoD is the NOAEL value of 2500 mg SiO2/kg obtained in a repeated-dose oral toxicity study in rats (ECHA, 2023c), which was conducted in accordance with OECD guideline 452 (Chronic Toxicity Studies). This study was considered reliable with restrictions (Klimisch of 2), as it meets the read-across criteria for synthetic amorphous silica. To calculate the PDE, the NOAEL value was converted to its equivalent amount of silicon, which is 1168.57 mg/kg (2500 mg/ kg × 28.085 g/mol / 60.085 g/mol = 1168.57 mg/kg). Thus, the proposed parenteral PDE for silicon is 584.28 mg/day, considering a safety factor of 100 and a body weight of 50 kg (Supplementary Table IV).
The EVM (Expert Group Vitamin and Mineral) has established the limit for the use of silicon dioxide in dietary supplements at 1500 mg SiO2/day, equivalent to 700 mg Si/day for adults over a lifetime (12 mg Si/kg/day for adults weighing 60 kg) (EFSA, 2009). Therefore, it is possible to conclude that the established parenteral PDE for silicon of 584.28 mg/day is safe.
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Supplementary Figure 1
Supplementary Figure 2
Supplementary Figure 3
AUTHORS' CONTRIBUTIONS
DATA AVAILABILITY STATEMENT
Use of data not disclosed.
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