Periodic Reporting for period 1 - DermPlast (Assessment of human Dermal exposure to microPlastics additive chemicals and the risk arising from such exposure using innovative 3D-human skin equivalents)
Reporting period: 2021-11-03 to 2023-11-02
DermPlast aims to address a significant research gap represented by the lack of information on human exposure to toxic additive chemicals in Microplastics, especially as it relates to dermal exposure. Microplastics (MPs), defined as plastic particles of less than 5 mm in size have been detected in both marine and terrestrial ecosystems worldwide, with evidence suggesting uptake by animals and accumulation along the food chain. The ethical and technological challenges associated with both in vivo and in vitro studies using human tissues, limited analytical methodologies, strict restrictions on the use of laboratory animals in toxicological studies and large variabilities associated with the allometric scaling of dermatokinetic data from animals to humans due to inter-species variability are limiting current efforts for accurate risk assessment of microplastics. By delivering more effective and sustainable alternative approaches to study the bioavailability of toxic flame-retardant chemicals (FRs), DermPlast will benefit scientists, public health and policy-makers taking informed decisions to minimize human exposure to microplastics and their toxic additive chemicals within the context of legislative frameworks such as EU REACH. This will be achieved by applying in vitro 3-dimensional human skin equivalent (3D-HSE) model to study the dermal bioavailability of toxic brominated flame retardants (BFRs) in MPs.
In the first phase of DermPlast, we developed a quality assured standard protocol for determination of the dermal bioaccessibility of toxic additive BFRs (e.g. PBDEs and HBCDDs) in different types of MPs polymers including polyethylene, polypropylene and polystyrene. This protocol was successfully applied to provide first insights into the dermal bioaccessibility of additive BFR chemicals from MPs and assessed the subsequent human dermal exposure to these chemicals via skin contact with MPs and the risk arising from such exposure.
Following the successful implementation of the first phase, and based on our confirmation that BFRs are bioaccessible from MPs into human synthetic skin surface film liquid, we subsequently developed a protocol to study the dermal bioavailability of BFRs (i.e. absorption through the skin into the blood stream) using a commercially available 3-dimesional human skin equivalent (3D-HSE) model from EPISKIN™. Our dermal uptake experiments were carried out in a static diffusion cell configuration as recommended by the manufacturer. Before the commencement of the exposure to MPs, the skin tissues were equilibrated with the receptor fluid in 5% CO2 at 37 °C in an incubator. Our protocol complied with the current Organisation for Economic Co-operation and Development (OECD) guidelines for skin absorption testing. The protocol involved multi-step quality control/assurance control (QC/QA) measurements including physiological and histological testing, positive and negative experimental controls, in addition to regular procedural blanks.
DermPlast showed, for the first time, that BFRs e.g. PBDEs and HBCDDs leached from MPs into in vitro human skin surface film liquid (SSFL) and reported the fbioaccessible of PBDEs to range from 37 – 82 % and 17 – 54%, respectively for PE and PP MPs (Fig 1). The fbioaccessible of PBDEs were influenced by the particle size of MPs with MPs of particle size <4 mm and 0.45 mm fractions differing significantly (p < 0.05) in both the PE and PP MP particles, with PE presenting more bioaccessible fractions of PBDEs compared to PP MPs. Under the most physiologically relevant SSFL composition (1:1 sweat: sebum), the fbioaccessible of HBCDDs from PS MPs were 1.6 1.8 and 2.0 %, respectively for the α, β and γ-HBCDD isomers. We found the bioaccessibility of HBCDDs to be strongly correlated with their Log KOW, suggesting the influence of sebum on the release of HBCDDs from polystyrene MPs upon contact with the skin surface. We further employed the bioaccessible fractions of BFRs to estimate the worse-case human exposure to these flame retardants and found that pentaBDE congeners (BDE-47 and 99) and BDE-209 were the main contributors to the total exposure to PBDEs arising from dermal contact with PE and PP MPs. The daily exposure dose (DEDs) of PBDEs were lower than the US EPA reference doses (RfD) for penta-, octa- and deca-BDEs, respectively. While for exposure to PS MPs, the DED of HBCDD via dermal uptake from MPs, exceeded the lifetime average daily dose through direct skin contact with flame retarded curtains containing HBCDD.
DermPlast further provided first insight into the dermal bioavailability of PBDEs upon skin contact with different types of MPs containing flame retardant additives and showed that as much as 8 % of the exposure dose can be taken up by the skin for some PBDEs, however, the absorbed fraction i.e. the fraction of PBDEs available for circulation through the bloodstream, did not exceed 0.14 % of the dose of PBDE originally present in the MPs. These results confirmed for the first time that when MPs come in contact with the human skin, several toxic PBDE congeners are uptaken and absorbed through dermal barriers onto the bloodstream. We did not find any statistically significant difference between the absorbed fraction of PBDEs in both PE and PP MPs. We also established that while a wet skin condition results in the bioavailability of more PBDE congeners, there was no statistically significant difference between the absorbed mass of BDE 47 and BDE 99 in both wet and dry skin conditions (Fig. 2).
As a result of the innovation and novelty of this work and the differences in MP polymer types as well as the wide variability in the physicochemical properties of the target BFRs, we applied and optimised a mathematical model to fit the dermal bioavailability data. This model enabled the identification of the linear absorption range and the determination of compound-specific dermatokinetic parameters including the dermal flux (Jss, ng cm-2.h-1) the permeability constant (KP, cm h-1) and lag time (Tlag, h). These parameters are crucial in risk assessment as they are essential to generate valuable information on the dermal absorption and risk of toxic BFR chemical additives under diverse, real-life exposure scenarios.
Following the successful implementation of the first phase, and based on our confirmation that BFRs are bioaccessible from MPs into human synthetic skin surface film liquid, we subsequently developed a protocol to study the dermal bioavailability of BFRs (i.e. absorption through the skin into the blood stream) using a commercially available 3-dimesional human skin equivalent (3D-HSE) model from EPISKIN™. Our dermal uptake experiments were carried out in a static diffusion cell configuration as recommended by the manufacturer. Before the commencement of the exposure to MPs, the skin tissues were equilibrated with the receptor fluid in 5% CO2 at 37 °C in an incubator. Our protocol complied with the current Organisation for Economic Co-operation and Development (OECD) guidelines for skin absorption testing. The protocol involved multi-step quality control/assurance control (QC/QA) measurements including physiological and histological testing, positive and negative experimental controls, in addition to regular procedural blanks.
DermPlast showed, for the first time, that BFRs e.g. PBDEs and HBCDDs leached from MPs into in vitro human skin surface film liquid (SSFL) and reported the fbioaccessible of PBDEs to range from 37 – 82 % and 17 – 54%, respectively for PE and PP MPs (Fig 1). The fbioaccessible of PBDEs were influenced by the particle size of MPs with MPs of particle size <4 mm and 0.45 mm fractions differing significantly (p < 0.05) in both the PE and PP MP particles, with PE presenting more bioaccessible fractions of PBDEs compared to PP MPs. Under the most physiologically relevant SSFL composition (1:1 sweat: sebum), the fbioaccessible of HBCDDs from PS MPs were 1.6 1.8 and 2.0 %, respectively for the α, β and γ-HBCDD isomers. We found the bioaccessibility of HBCDDs to be strongly correlated with their Log KOW, suggesting the influence of sebum on the release of HBCDDs from polystyrene MPs upon contact with the skin surface. We further employed the bioaccessible fractions of BFRs to estimate the worse-case human exposure to these flame retardants and found that pentaBDE congeners (BDE-47 and 99) and BDE-209 were the main contributors to the total exposure to PBDEs arising from dermal contact with PE and PP MPs. The daily exposure dose (DEDs) of PBDEs were lower than the US EPA reference doses (RfD) for penta-, octa- and deca-BDEs, respectively. While for exposure to PS MPs, the DED of HBCDD via dermal uptake from MPs, exceeded the lifetime average daily dose through direct skin contact with flame retarded curtains containing HBCDD.
DermPlast further provided first insight into the dermal bioavailability of PBDEs upon skin contact with different types of MPs containing flame retardant additives and showed that as much as 8 % of the exposure dose can be taken up by the skin for some PBDEs, however, the absorbed fraction i.e. the fraction of PBDEs available for circulation through the bloodstream, did not exceed 0.14 % of the dose of PBDE originally present in the MPs. These results confirmed for the first time that when MPs come in contact with the human skin, several toxic PBDE congeners are uptaken and absorbed through dermal barriers onto the bloodstream. We did not find any statistically significant difference between the absorbed fraction of PBDEs in both PE and PP MPs. We also established that while a wet skin condition results in the bioavailability of more PBDE congeners, there was no statistically significant difference between the absorbed mass of BDE 47 and BDE 99 in both wet and dry skin conditions (Fig. 2).
As a result of the innovation and novelty of this work and the differences in MP polymer types as well as the wide variability in the physicochemical properties of the target BFRs, we applied and optimised a mathematical model to fit the dermal bioavailability data. This model enabled the identification of the linear absorption range and the determination of compound-specific dermatokinetic parameters including the dermal flux (Jss, ng cm-2.h-1) the permeability constant (KP, cm h-1) and lag time (Tlag, h). These parameters are crucial in risk assessment as they are essential to generate valuable information on the dermal absorption and risk of toxic BFR chemical additives under diverse, real-life exposure scenarios.
Overall, we successfully delivered an efficient, effective, reproducible and sustainable in vitro approach, alternative to animal testing and human tissues, for studying human dermal absorption of toxic chemicals in microplastics for the first time. In addition, DermPlast, delivered the first experimentally determined risk assessment, arising from human exposure to toxic BFRs in microplastics.