WORKSHOP. Understanding Formulation Design and Dermal Absorption: Technical, Scientific and Regulatory perspectives

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1 WORKSHOP Understanding Formulation Design and Dermal Absorption: Technical, Scientific and Regulatory perspectives 16th/17th November 2015, TNO Triskelion, Zeist, NL // University Utrecht, Utrecht, NL Program FINAL MINUTES Day 1 Day 2 Opening (Dr. Christian Strupp) Role of dermal absorption in risk Challenges in Radiochemistry (Dr. assessment (Dr. Manoj Aggarwal) Erzsebet Faigl Birkas / Laszlo Orha) Formulation Design (Dr. Gordon Bell) Introduction to in vitro and in vivo Target-directed Compound Delivery dermal absorption studies (Wilfred from Personal Care Products (Prof. Alex Maas) Lips) Practical demonstration of in vitro In vitro dermal absorption studies on dermal absorption studies (equipment complex formulations (Prof. Jon and technical aspects) (Wilfred Maas) Heylings) Tour to the dermal and bioanalytical Aggravation of local responses caused laboratories (with possibilities to by the chemical skin penetration exchange with experienced study enhancer Oleic acid (Dr. Berit Granum) directors) The EFSA Guidance on Dermal Absorption (Dr. Carsten Kneuer) Authority view on formulation composition (Dr. Ian Dewhurst) MEETING NOTES Day 1 E. Faigl & L. Orha (presentation 1): Radiosynthesis is challenging with respect to time, methods and resources. All radiosynthesis is based on Ba 14 CO 3, which is currently expensive due to monopole on production. Typically labelled intermediates are used to incorporate the radioactivity into the target molecules 1

2 synthesis routes have to be tailor made for each synthesis. Cases where alternative radiosynthesis approaches were applied were presented. W. Maas (Presentation 2): Practical aspects of dermal absorption studies were discussed. Use of radiolabeled material in dermal absorption studies is preferred in terms of sensitivity. Non-radiolabeled studies can be expensive (especially in vivo where the analytical method has to be validated for various matrices) Flow-through diffusion cells and split-thickness skin are most often used in-house, but other possibilities can be applied depending on the request of the customer and aim of the study Donor variability can be an issue. EFSA recommends use of 4 donors. A solution to reduce variability could be to add more replicates to better identify outliers. Considering statistical methodology, the error of the mean relates to uncertainty of the mean value estimate. On the contrary, the standard deviation describes variability of the individual data. Thus, it was suggested that SEM might be more appropriate as compared to use of SD as proposed by EFSA guidance in case CV exceeds >25%. When to use Franz cells? Franz cell (i.e. static rather than dynamic / flow-through set-up) is used at TNO Triskelion at the request of the sponsor. E.g. non-radiolabeled studies where low absorption is expected are generally performed in Franz cells. Recently TNO Triskelion has used Franz cells for nail penetration studies. How to avoid receptor fluid limiting absorption? Add bovine serum albumin (BSA) or polyethylene glycol (PEG) to increase solubility, check active ingredient solubility in receptor fluid to confirm that sink conditions are maintained, use of flow through system allows frequent replacement of liquid in diffusion cell. Could the receptor fluid constitution affect the number of outliers? Not expected with BSA, with PEG possibly after longer use it can influence dermal absorption, ethanol/water can be tricky in terms of variability and effect on dermal absorption. Addition of solvents into the receptor phase is not recommended as their back diffusion might influence the skin barrier function. Does the multichannel pump introduce variability? Variability is expected to be limited, every channel is checked for consistent volume flow and there are min/max criteria for calibration High rinses from the receptor compartment are an alert. If the active ingredient is stuck in the skin it might be released during rinsing of the receptor compartment, but then it was considered by the speaker as not relevant to add to the absorbed dose. Others expressed that high receptor rinse values indicate that there was a problem with the experimental set-up. Inappropriate skin thickness (extended dermis) might cause adhesion to the skin. If an active ingredient is piling up in vitro, then it might also occur in vivo. Piling up can happen for lipophilic active ingredients. The EFSA 75% rule: how to calculate RATA (relative absorption to total absorption, comparison of absorption halfway during the study to the final absorption) named t0.5 according to EFSA scientific report on assessment of new scientific studies on human in vitro dermal absorption (Adopted: 30 October 2015, published: 12 November 2015, doi: /j.efsa )? 2

3 RATA can be calculated either individually (for each replicate first, averaged thereafter) or from the respective mean values. If no replicates show a clearly deviating absorption profile, calculation from the mean can work. Replicates showing clearly deviating absorption profiles might be excluded from calculations (not from reporting) if justified. There can be small differences in RATA as a way of calculation, especially when values are close to the 75% cut-off. ) Variability in absorption into the receptor fluid and consequently in RATA can be a result of donor variability. What drives donor variability? To avoid variability, skin is visually checked after receipt and if not visually OK discarded immediately (e.g. in case of stripes). Before freezing skin is dehydrated as much as possible. Based on membrane integrity test, only membranes with an acceptable Kp or other barrier integrity method used will be selected. Intrinsic structure of the skin, age, treatment, history and area are all factors that can contribute to variability. Skin from hand palms has low barrier permeability and is not useful for dermal absorption studies. Breast and abdomen can be regarded as appropriate, in comparison to the regions exposed mostly in reality e.g. face / forehead. Variability between donors is obvious and can be regarded as a good thing since everybody is different. With animal skin there is also variability, but thought to be less so compared to human skin. Can variability also occur between studies? Nowadays mostly only human skin studies are performed (if needed followed by rat in vitro or in parallel in combination with rat in vivo studies, for triple pack approach i.e. refining in vivo rat data by an interspecies correction factor derived from the in vitro comparison). Previously, human and rat skin were always performed in parallel. Separate experiments might increase variability. How is increased absorption between 8-10 h explained? This is called the washing-in effect: the active ingredient either laying on or being absorbed by the upper layers of the skin, as a result from washing can be mobilized. Generally the amount is thought to be negligible when taking into account the total absorption after 24 h. Are there formulation types that are more prone to washing-in effect? Difficult to say, organic based formulations might be but it is also dependent on the active ingredient. Usually the type of formulation is selected based on phys/chem of active ingredient. Since skin is analogous to bricks and mortar, there is room for formulation to migrate into the void spaces of the skin, hampering the skin wash. Sometimes washing-in effect is only observed for a single replicate in a study and not for the others. Practical demonstration: Should artificial sweat not be used when adding non-liquid formulations? Water, PBS and artificial sweat are being used to improve tissue contact/create a worst-case scenario for application of non-liquid formulations, which are according to the experience of the speaker selected based on request from sponsor or expertise. Note: the OECD guidance of 2004 recommends water to be used. An appropriate skin wash is important to reduce variability. Less efficient skin wash can result in high first tape strip values (both are not taken into account for calculation of absorption). You can never completely remove the formulation from the skin and it is not feasible to rinse until all formulation is removed. However, washing should always represent real life practice. There must be a balance between the number of washing steps and retaining integrity of the skin 3

4 membrane. Washing efficiency can be determined using Geiger Muller counter (not suitable for tritiated compounds) or calculated for a few replicates immediately after washing before continuing with the study. Day 2 M. Aggarwal (presentation 3): Results from two publications (Aggarwal et al. 2014, 2015), including data from 295 studies. Default values for absorption can be lower than current values in EFSA guideline and read across can be possible even if formulation ingredient content has changed > 25%. Re-entry studies where active ingredient is applied as dry residue are ongoing. Data interpretation as % of applied dose, should be amount expressed as µg/cm2 for risk assessment. An opinion was expressed by one of the participants as after workshop comment that µg/cm² may as well be misleading as compared to % of applied dose. For comparison to the real exposure the knowledge of skin load is detrimental which requires the knowledge on total area exposed. Change in dilution factor does not necessarily mean change in absorption by the same factor for spray dilutions. Solubility in skin /dermal dynamics should also be considered. First the increase in absorption (expressed as percentage of dose) with increasing dilution factor is linear, later the curve can be expected to flatten. This trend may be primarily due to a change in activity coefficient y being close to 1 in ideal highly diluted solution. Absolute concentrations of a.s. in concentrates may be quite different. It was suggested to group concentrates of the same range. The statistics must control for confounding effects. For example: certain formulation properties and types may have been chosen to account for the phys/chem properties of the a.s. Accordingly, apparent associations of formulation types with dermal absorption may be strongly influenced by the phys/chem properties of a.s. that are typically formulated in this type. An opinion was expressed by one of the participants as after workshop comment that discussion did not relate to the conclusions presented by M. Aggarwal. Therefore he wanted to state that the conclusion drawn remained valid after the discussion. G. Bell (presentation 4): Choice of formulation type is based on phys/chem, biological and toxicological properties of the active ingredient or combinations thereof. Several components in the product mainly aim to stabilize the product during production, for storage stability and during preparation of the in use product. Also, some co-formulants (e.g. surfactants, some oils/lipids) can serve to improve spreading and/or adhesion of the aerosol particles (spray droplets) on the plant surface, even during rainfall following application. Five commonly used formulations were presented as an 4

5 example. Each formulation (and active ingredient combination) has pros and cons. Examples of cons are crystallization and separation. Are there defined rules for listed formulation types? Sometimes there is overlap. Currently there is no guideline for terminology, for example a 50% SC can be a SL. Formulation preparation is difficult if it has to contain two or more a.i. s with different characteristics. The farmer does not care how the product is named, important is that it works. A. Lips (presentation 5: Surfactants can damage the stratum corneum and consequently affect dermal absorption. There have been major advances in modelling and prediction of dermal absorption, in particular in the development and implementation of conceptual model. Mass-transfer and heat transfer as basis for the modelling Transient models consider different compartments like e.g. product film stratum corneum viable epidermis, dermis with different modelling parameters/conditions individually. Modelling underpinned with molecular informatics and experiments on different exposure conditions (e.g. wet versus dried down) The models represent an infinite sink design, while pesticide exposure is finite.. Interactions like skin lightening or product sensory performance illustrated were mainly in the lower layers of the skin (below the barrier of the stratum corneum). This was thought to be less representative for pesticides, where the interest is on passing the barrier. Interactions may be due to lipid structure directed to aqueous environment. J. Heylings (presentation 6): Complex formulations can be problematic but this is surmountable by the use of modified diffusion cells and appropriately controlled non-radiolabelled techniques. Volatile actives in fumigants can be assessed using porous charcoal filter traps above the skin. Metals in fungicide products can be assessed using ICP-OES or ICP-MS but endogenous levels need to be measured in untreated controls. Important to mimic the actual worker exposure i.e. the concentrate for Mixer/Loaders and the field strength dilutions for Spray Operators. Guidelines for nanomaterial absorption is currently under consideration. There is published guidance for in vitro dermal absorption of nanomaterials in cosmetics (SCCS/1484/12). 5

6 Use of simulated sweat to improve tissue contact of powder or granule crop protection products may be regarded as worst-case dermal exposure. In general, small molecules with an intermediate polarity show high dermal absorption but the adjuvants, particularly surfactants can have significant impact on dermal absorption. Chemical structure gives a good first indication of dermal absorption potential of compounds, but the use of a finished product in OECD 428 studies makes it complicated to predict dermal absorption, due to the influence of the formulation on dermal uptake. B.Granum (presentation 7): Penetration enhancers in formulations may increase toxicity, e.g. sensitization by oleic acid. Why use tape strips prior to application in LLNA? This is not part of OECD guideline for local lymph node assay. But tape stripping prior to application promotes absorption. There is evidence that low levels of oleic acid have benefit effect and high levels are toxic. With high levels there is an increase in transcellular transport. Skin thickening was observed, is oleic acid a skin irritant? Self-classifications for oleic acid in ECHA registry indicate irritating potential of oleic acid at and above 10%. This could explain enhancement of sensitization effect, possibly mediated through penetration enhancement. Generally, the observations might support EFSA Guidance recommendations not to read-across absorption data to a formulation containing irritants. It is allowed to use up to 25% in cosmetics. Methyl salicylate (skin irritant) did not show a sensitization response. The boomerang shape of oleic acid might enhance absorption affecting lipid fluidity within the stratum corneum. How can mouse sensitization data be representative of human? The mouse test indicates only the sensitization potential of a compound. Its relevance for human needs to be addressed in the risk assessment taking into account the exposure conditions. What is in this regard the effect of tape stripping prior to application? Because then there are less lipids in the stratum corneum and thus an increase in absorption is expected. C. Kneuer (presentation 8): Requirements of EU-regulations for PPP with regard to dermal absorption assessment were clarified as well as the range of relevant current technical guidance on conduct and interpretation, including the EFSA Guidance on Dermal Absorption. Based on a formal mandate by the European Commission, EFSA currently reassesses the default values for dermal absorption recommended in the Guidance and other aspects of the document. Both data submissions by BfR (from 162 studies) and ECPA (publications M. Aggarwal) were evaluated with regard to their suitability as a scientific basis for such a revision. EFSA checked ECPA and BfR datasets and found some missing information, minor inconsistencies/mistakes. Overall, appropriate consistency with source data was shown. 6

7 Recommendations to improve the value of the overall dataset were issued and are addressed as far as possible. A Scientific Report was published by EFSA on Nov 12th. Previous statistical analysis (Aggarwal et al., 2014, 2015) was criticised for the sequential approach taken, i.e. considering the impact of each factor such as physical state (e.g. solid vs. liquid), a.s. concentration, formulation type on dermal absorption separately. Lack of control for confounding effects could lead to inappropriate conclusions. Data analysis taking into account some of the potential confounding effects using a model-based approach (DA values quantile regression) indicated that DA among liquid concentrates depends on formulation type, thus not fully support ECPA conclusions. More detail is provided in the Scientific Report. According to presentation slides, exposure time ranged from 6-24 h for BfR dataset whereas EFSA Scientific report states 6-10 h. Which is correct? The presented figure (6-24h) is correct. The discrepancy will be followed up. What statistical model was used for the preliminary analysis of data? This should better be clarified directly by with statistician using the address provided on the final slide What are the differences between the various guideline and guidance documents of OECD and EFSA? Which is binding? EFSA Guidance (2012) and OECD Guidance Notes (2011) primarily but not exclusively provide recommendations for interpretation of data / assessment in absence of data, whereas OECD test guidelines and OECD Guidance for Conduct (all 2004) provide primarily instructions on how to perform an adequate study. Is 8 replicates per group recommended or required? If a study fulfils OECD criteria and is performed under GLP, it fulfils the criteria for mutual acceptance of data under the OECD MAD treaty. However, for specific fields of interest such as PPP, additional recommendations for test design can exist and rules for interpretation of the data may deviate, in particular if such recommendations are not respected. It was discussed that when using 2 replicates from each of 4 donors, 4 is the statistical unit. It was suggested to use 8 donors since technical duplicates are not relevant for statistical unit. The question is if we need to know experimental or donor variability? Standard deviation is currently based on 8 replicates from 4 donors acc. to EFSA Guidance. It was noted (proposed?) that, statistically, it should relate to the mean of the means of the two replicates per donor. The SCCS states that 8 replicates should be used (4 donors in duplicate). From statistical perspective none of the recommended replicate number of donors might be sufficient to cover the test system variability with an acceptable level of certainty. Leading to the question which level of certainty is acceptable? Can EFSA give an outlook on the new guidance document? - Currently there are many rules that are regarded as contributing to an overestimation of the absorbed dose. Which of these rules will be excluded? The results of the revision cannot be predicted. Crucial elements will be reviewed based on data by the Working Group. It will then be decided following public consultation and consultation with management bodies what is appropriate and what is not. The timeline for the revision is May 31 st 2017 with a draft for public consultation to be expected end

8 I. Dewhurst (presentation 9): When absorption is predicted, more than only changing formulation composition should be considered. More detailed information on the effect of different solvents and surfactants would be interesting. When evaluating available information for non-studied products consider more pragmatic approaches to overcome limitations in bridging opportunities. How can harmonization be achieved (as done for guidelines in the pharmaceutical industry)? Maybe the revised EFSA guidance. Will the required data be generated in time for revision? EFSA is in contact with OECD for harmonization. The OECD guideline covers all industries and allows a lot of flexibility, EFSA will focus on pesticides. Toxicity testing is expensive and the current default values/standards are considered too conservative by industry. What is the global perspective? The current default values cover all data points of the publications of M. Aggarwal and are better than the default value of 100%. There has to be confidence in decision for read across, currently supportive data is lacking. Quality data generation must improve. Post meeting note of participants: It is not just money : Animal testing must comply with the RRR principles (reduce replace refine) and need to be justified by a scientific need. Toxicity testing to fulfill a guideline tickbox is questionable if there is no material impact of the outcome (e.g. if the formulations could be assessed based on default assumptions as suggested by Aggarwal et al.). What is the effect of dilution? If the spray dilution contains a lot of water, the effect of the formulation characteristics is expected to be negligible, as supported by analysis for dilutions prepared from different formulation types presented in the publications of M. Aggarwal. The linear approach can be regarded as conservative. Changing in levels of surfactant might not have an effect on absorption for high dilution rates (level for high dilution rates not specified in the discussion). Case for a submission: use of default concentration for the concentrate and a read across for the field dilution. This may be accepted, but different scenarios should be presented. The ratio active ingredient to co-formulations should also be compared. The company should present good argumentation why read-across is possible/acceptable as outlined in the data requirements regulation. 8

9 Draft outcome of the Breakout Groups Question 1: What is the impact of formulants in concentrated products on dermal penetration? Which chemistry is important and which can essentially be disregarded? Impact of local effects (irritation and sensitization) on dermal penetration? Local effects: sensitizing and irritating properties will likely increase dermal absorption. Majority agreed. Chemistry that can be disregarded: currently there is no chemistry that can be solidly disregarded. Any waiver needs to care about all aspects. Changes in formulation should be discussed for example in regard to solubility of the active ingredient. Function of formulants added need to be discussed: (leaf penetration enhancer or formulation stabilizer) Historical experience on certain formulation changes should be discussed Effects of drying of the sample on the skin should be looked at Side discussion: effect of skin temperature on absorption: majority convinced that there is an effect. Question 2: What is the impact of dilution on the formulation environment and dermal absorption? Abstract from Simon Wilkinson: can help to understand the influence of the dilution on formulation environment Phys/chem properties: key questions to be addressed are: solubility in the diluted test sample completely dissolved or suspended? In the aqueous phase or an oily environment, still in droplets of oil in the aqueous matrix? Is there a matrix change during drying of the sample on the skin? What is the microscopical appearance of the active ingredient in the sample? Are there data available from formulation development (efficiency, surface behavior) that can help understand? Absorbed dose (amounts) per area is more meaningful to use in risk assessment than % absorption of a finite dose o Different definitions of absorption are around it would be helpful to have definitions on - reference was made to OECD GD 28 and will be include in minutes Dilution of a product mostly expected to reduce flux, but effect for toxicological risk assessment might be small due to the achieved systemic dose dilution may not have a big effect for the risk assessment Surfactants: what is the effect of the surfactant mostly dependent on the concentration. If the intention of the addition is to increase leaf penetration, there is a higher likelihood that dermal penetration is affected, while if it is formulation (concentrate) stability maybe not. One participant expressed as an after workshop comment the opinion that a surfactant is likely to effect at rather low concentrations as assumed to cumulate at the interphase between skin and the solution. This presumed relationship is also reflected in the CLPregulation where lower concentration limits are in place for endpoints like skin irritation/corrosion. 9

10 Thermodynamics, kinetics, surface tension and wettability are to be addressed. Irritant properties for diluted products are unlikely to be relevant Question 3: How strong is the influence of the concentration of the active substance compared to the formulation environment on dermal absorption? Formulation type of the concentrate could have minimal impact on dermal absorption from the diluted (spray) if the concentration of the ai is constant no agreement reached in the WG Spray dilution: o In which phase of the spray is the active dissolved or suspended o Solubility of active substance in water vs. solubility (fraction) of a.i. in spray dilution vs. solubility of the a.i. in the concentrate. Side question: Solubility in certain types of water may be interesting to look at (tap, distilled and deionized), as solubility data may only be available in one of these. o Hydrodynamics, thermodynamics and concentration may affect rate of absorption (flux) it is important to understand if a rate limited condition (infinite or finite sink under the skin) is in place in the study. Guideline requests that no limiting factors are in place limiting the solubility in the receptor fluid however the product phase may be rate limiting. Can be addressed by investigating the absorption profile (flattening of the absorption curve) o Level of dilution formulation environment is not critical after a certain dilution factor. o Weight of evidence should be used on these key parameters in the decision making process. o Concentration is likely a more influencing factor than composition of the spray, but cannot be generalized. o concentrate o Depending on the a.i. s and properties o Formulation environment is critical o Solubility in formulation Presentations of the workshop may be downloaded via one of the following links: Available here for about 2-weeks, and will be automatically deleted thereafter. 10