Lead impurities in traditional herbal medicinal products with Plantago lanceolata L., folium (Ribwort Plantain leaves) available in Polish pharmacies – toxicological risk assessment for adults

Abstract Traditional herbal medicinal products (THMP) with Plantago lanceolata L., folium (Ribwort Plantain Leaves) are well-known and very popular among the European population. The aim of our study was the toxicological risk assessment for adults of Pb impurities in THMP with Plantago lanceolata L. obtained from pharmacies in Poland. The estimations of Pb impurities in the single dose (22.1 − 50.7 ng/single dose) and in the daily dose (ng/day) were based on the worst-case scenario (WCS), i.e. based on the posology recommended by manufacturers. The estimated daily exposure (ng/day) meets the standards of the ICH Q3D (R1) guideline on elemental impurities (5.0 µg/day) according to the Pb levels (all results were below 250 ng/day). It can be concluded that all investigated THMPs with Plantago lanceolata L., folium available in pharmacies from Poland should not represent any health risk to the adults. Graphical Abstract


Introduction
The studies about elemental impurities in final natural pharmaceutical products containing plant materials are very rare but extremely important subject. Especially, this is important for many readers working in different areas related to safety of traditional herbal medicinal products (THMP), pharmaceutical botany, toxicology, phytopharmacy, safety assessment, quality control and other regulatory issues. Based on review of all European Union herbal monographs (n ¼ 167; June 2022) (EMA 2022), a very important but not studied plant applied in the European pharmaceutical industry is Plantago lanceolata L., folium (Ribwort Plantain). Mentioned plant is a species of the genus Plantago in the Plantaginaceae botanical family. It is a common perennial weed of arable fields and grassland (Bond et al. 2007), ubiquitous across Europe and North and Central Asia (Sagar and Harper 1964).
In Europe, various herbal products with Plantago lanceolata with a traditional indication are available; however, most of them do not fulfil the requirement of a medicinal use for at least 30 years. Hence, in European pharmacies there are only a few registered THMP with Plantago lanceolata L., folium. These products are well known and popular among European population as a demulcent for the symptomatic treatment of irritations of oral and pharyngeal mucosa with associated dry cough (EMA 2011). However, what about the potential elemental impurities in such products? The scientific literature review from last few years shows there is limited studies about element levels in Plantago lanceolata L., folium (Ribwort Plantain leaves), e.g. (Nadg orska-Socha et al. 2013;Skrynetska et al. 2019;Jurowski et al. 2022). Hence, the aim of our short communication was toxicological studies (analysis and risk assessment for adults) of lead impurities in THMP with Plantago lanceolata L., folium (Ribwort Plantain leaves) as registered phytopharmaceuticals in EU available in Polish pharmacies. It should be underlined that only a few independent manufacturers produce these kinds of pharmaceutical products in Poland (only products manufactured in Poland have been investigated; we did not consider products from other EU countries), hence there is a relatively low number of available samples for studies (n ¼ 5). The description of the analysed samples (PL1-PL5) is briefly summarised in supplementary materials 1 (Table S1). The levels of Pb impurities in investigated THMP samples were determined by atomic absorption spectrometry (AAS technique); the details about experimental conditions and methodology are described in supplementary materials 1 (Tables  S2 and S3). The justification of our studies is the fact that monitoring of heavy metals impurities (like Pb) in final phytopharmaceutical products should be mandatory due to ICH Q3D R1 elemental impurity guideline (ICH Q3D R1 2019). To the best of our knowledge, this short communication is the first study about Pb impurities levels in final THMP with P. lanceolata L., folium (Ribwort Plantain leaves) available in pharmacies in Europe.

Results and discussion
2.1. The lead impurity profile in THMP with Plantago lanceolata L., folium (Ribwort Plantain leaves) from Polish pharmacies The lead impurity profiles of all investigated THMP samples with Plantago lanceolata L., folium (n ¼ 5; PL1 -PL5) are shown in in supplementary materials S1 ( Figure S1).
Additionally, the descriptive statistics of Pb impurity level in analysed samples are briefly summarised in supplementary materials S1 (Table S4). The highest level of Pb was observed in sample PL1 (5.10 ± 0.11 lg/L) and the lowest levels was in sample PL5 (2.20 ± 0.09 lg/L). Overall, the impurity profile of Pb shows that lead impurities were present in all investigated samples (in the range: 2.20 À 5.10 lg/L), but at a relatively low level (< 5.5 lg/L). The raw results for samples PL3 (2.6 ± 0.08 lg/L), PL4 (2.4 ± 0.09 lg/L) and PL5 (2.2 ± 0.07 lg/L) are at a very similar level. The ratio between the highest value and the lowest value is 2.32; values of kurtosis and skewness confirms the distribution of results and their consistency.
The research on these products has already been the subject of our research, but only for Cd impurities (Jurowski et al. 2022). Based on an extensive review of the scientific literature, there is excellent review about trace elements in Plantago lanceolata L., used for herbal and food preparations (Drava et al. 2019). However, mentioned article describes only raw materials (roots, plants), hence it is not suitable for comparison with our results about final pharmaceutical products with Plantago lanceolata L. (our research concerns the product available in pharmacieshigh dilution, no information on the origin of the raw materials, no information on quality control, etc.).
However, obtained results are characterised by scientific value (pioneering research being a benchmark for other researchers) and value for entities controlling and monitoring the quality of THMP in each country) and for safety assessors, phytotoxicologists and pharmaceutical botanists.
The fact that research is pioneering and is extremely valuable from a regulatory point of view (in particularregulatory toxicology). In this context, the results obtained should be compared with the recommended/applicable impurities limit relevant for regulatory toxicology. In this situation, it is possible to compare our results with two important sources: the permissible limit set by FAO/WHO for herbal medicines and the level limits recommended by the ICH Q3D R1 guideline on elemental impurities in final pharmaceutical products. Our results show that all samples contained Pb levels below the permissible limit set by FAO/WHO for herbal medicines (10 mg/kg or 10 mg/L (World Health Organization 2006)). Additionally, considering the level limits for Pb in pharmaceuticals via the oral route recommended by the ICH Q3D R1 guideline (0.5 lg/g) (ICH Q3D R1 2019), all the investigated samples also meet the requirements.

The estimation of Pb impurities exposure for human health in THMP with
Plantago lanceolata L., folium (Ribwort Plantain leaves) from Polish pharmacies The estimation of Pb impurities exposure for human health (adults) in THMP with Plantago lanceolata L., folium from Polish pharmacies was calculated based on a 'worst-case scenario' described clearly in supplementary materials 1 (SM 1). The obtained results for estimated exposure of Pb to which the patient is exposed for single dose and maximal daily dose of the THMP with P. lanceolata L., folium (Ribwort Plantain leaves) available in Polish pharmacies are described in Table 1.
The obtained results show that the estimated maximum daily exposure of this element is similar among analysed samples (88.4 À 232.5 ng/day). Based on the integrated exposure uptake biokinetic (IEUBK) model (Hogan et al. 1998) with the assumption of 100% absorption, the oral intake of this element is 5.0 lg/day, hence the permitted daily intake (PDE) value for this heavy metal is 5.0 lg/day. Based on the value of PDE for Pb for oral pharmaceutical products suggested by the ICH Q3D guideline (ICH Q3D R1 2019), all investigated products meet the requirements. What is more, our results indicated that all analysed THMP with P. lanceolata L., folium are characterised by results much lower than PDE value for Pb.

Conclusions
There is no doubt that Pb is one of the most harmful heavy metals for human and is ubiquitous in environment (ICH Q3D R1 2019; Jurowski et al. 2019), especially in many plants which can be used as raw materials in phytopharmaceutical industry. Our results show that Pb impurities were present in all investigated samples (in the range: 2.20 À 5.10 lg/L). However, the level of Pb impurities in all samples is relatively low (<5.5 lg/L) and below the permissible limit set by FAO/WHO for herbal medicines (10 mg/kg or 10 mg/L) (World Health Organization 2006)). Additionally, all investigated samples meet also the requirements recommended by the ICH Q3D R1 guideline on elemental impurities in final pharmaceutical products (0.5 lg/g) (ICH Q3D R1 2019). What is more, our calculation about estimated exposure of Pb to which the patient is exposed for single dose and maximal daily dose of the THMP with P. lanceolata L., folium (Ribwort Plantain leaves) available in Polish pharmacies confirm safety of these products (all values are much lower than PDE value for Pb (5.0 lg/day).
To the best of our knowledge, this article is the first study about Pb impurities levels in final THMP with P. lanceolata L., folium (Ribwort Plantain leave 6u s) available in pharmacies in Europe. Control and monitoring of elemental impurities in THMP are extremely rare topic, hence we hope this article will be interesting for a wide range of readers.