John A. Troutman

The incorporation of lysine into the peroxidase peptide reactivity assay for skin sensitization assessments.

To establish further a practical quantitative in chemico reactivity assay for screening contact allergens, lysine peptide was incorporated into a liquid chromatography and tandem mass spectrometry-based assay for reactivity assessments of hapten and pre-/pro-hapten chemical sensitizers. Loss of peptide was determined following 24 h coincubation with test chemical using a concentration-response study design. A total of 70 chemicals were tested in discrete reactions with cysteine or lysine peptide, in the presence and absence of horseradish peroxidase-hydrogen peroxide oxidation system. An empirically derived prediction model for discriminating sensitizers from nonsensitizers resulted in an accuracy of 83% for 26 haptens, 19 pre-/pro-haptens, and 25 nonsensitizers. Four sensitizers were shown to selectively react with lysine including two strong/extreme and two weak sensitizers. In addition, seven sensitizers were identified as having higher reactivity toward lysine compared with cysteine. The majority of sensitizing chemicals (27/45) were reactive toward both cysteine and lysine peptides. An estimate of the relative reactivity potency was determined based on the concentration of test chemical that depletes peptide at or above a threshold positive value. Here, we report the use of EC15 as one example to illustrate the use of the model for screening the skin sensitization potential of novel chemicals. Results from this initial assessment highlight the utility of lysine for assessing a chemicals potential to elicit sensitization reactions or induce hypersensitivity. This approach has the potential to qualitatively and quantitatively evaluate an important mechanism in contact allergy for hazard and quantitative risk assessments without animal testing.

Introduction of a methoxymethyl side chain into p-phenylenediamine attenuates its sensitizing potency and reduces the risk of allergy induction

The strong sensitizing potencies of the most important primary intermediates of oxidative hair dyes, p-phenylenediamine (PPD) and p-toluylenediamine (PTD, i.e. 2-methyl-PPD) are well established. They are considered as the key sensitizers in hair dye allergic contact dermatitis. While modification of their molecular structure is expected to alter their sensitizing properties, it may also impair their color performance. With introduction of a methoxymethyl side chain we found the primary intermediate 2-methoxymethyl-p-phenylenediamine (ME-PPD) with excellent hair coloring performance but significantly reduced sensitizing properties compared to PPD and PTD: In vitro, ME-PPD showed an attenuated innate immune response when analyzed for its protein reactivity and dendritic cell activation potential. In vivo, the effective concentration of ME-PPD necessary to induce an immune response 3-fold above vehicle control (EC3 value) in the local lymph node assay (LLNA) was 4.3%, indicating a moderate skin sensitizing potency compared to values of 0.1 and 0.17% for PPD and PTD, respectively. Finally, assessing the skin sensitizing potency of ME-PPD under consumer hair dye usage conditions through a quantitative risk assessment (QRA) indicated an allergy induction risk negligible compared to PPD or PTD.

Reactivity of chemical respiratory allergens in the Peroxidase Peptide Reactivity Assay

Sensitizing chemicals are commonly associated primarily with either skin or respiratory sensitization. In the Direct Peptide Reactivity Assay (DPRA), when compared with skin sensitizers, respiratory allergens have been demonstrated to selectively react with lysine rather than cysteine. The Peroxidase Peptide Reactivity Assay (PPRA) has been developed as a refinement to the DPRA. The PPRA incorporates dose-response analyses, mass spectroscopy for peptide detection and a horseradish peroxidase-hydrogen peroxide enzymatic system, increasing the potential to identify pro-haptens. In the investigations reported here, the PPRA was evaluated to determine whether it provides advantages for the identification of respiratory allergens. Twenty respiratory sensitizers, including five predicted to be pre-/pro-haptens were evaluated. The PPRA performed similarly to the DPRA with respect to identifying inherently reactive respiratory sensitizers. However, three respiratory sensitizers predicted to be pre-/pro-haptens (chlorhexidine, ethylenediamine and piperazine) were non-reactive and the general selectivity of the respiratory allergens for lysine was lost in the PPRA. Overall, the data indicate that the PPRA does not provide an advantage over the DPRA for discriminating allergens as either contact or respiratory sensitizers. Nevertheless, the PPRA provides a number of refinements to the DPRA that allow for an enhanced characterization of reactivity for both classes of chemical allergens.

In vitro to in vivo extrapolation for high throughput prioritization and decision making

In vitro chemical safety testing methods offer the potential for efficient and economical tools to provide relevant assessments of human health risk. To realize this potential, methods are needed to relate in vitro effects to in vivo responses, i.e., in vitro to in vivo extrapolation (IVIVE). Currently available IVIVE approaches need to be refined before they can be utilized for regulatory decision-making. To explore the capabilities and limitations of IVIVE within this context, the U.S. Environmental Protection Agency Office of Research and Development and the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods co-organized a workshop and webinar series. Here, we integrate content from the webinars and workshop to discuss activities and resources that would promote inclusion of IVIVE in regulatory decision-making. We discuss properties of models that successfully generate predictions of in vivo doses from effective in vitro concentration, including the experimental systems that provide input parameters for these models, areas of success, and areas for improvement to reduce model uncertainty. Finally, we provide case studies on the uses of IVIVE in safety assessments, which highlight the respective differences, information requirements, and outcomes across various approaches when applied for decision-making.

Development of a physiologically-based pharmacokinetic model of 2-phenoxyethanol and its metabolite phenoxyacetic acid in rats and humans to address toxicokinetic uncertainty in risk assessment ☆

2-Phenoxyethanol (PhE) has been shown to induce hepatotoxicity, renal toxicity, and hemolysis at dosages ≥ 400 mg/kg/day in subchronic and chronic studies in multiple species. To reduce uncertainty associated with interspecies extrapolations and to evaluate the margin of exposure (MOE) for use of PhE in cosmetics and baby products, a physiologically-based pharmacokinetic (PBPK) model of PhE and its metabolite 2-phenoxyacetic acid (PhAA) was developed. The PBPK model incorporated key kinetic processes describing the absorption, distribution, metabolism and excretion of PhE and PhAA following oral and dermal exposures. Simulations of repeat dose rat studies facilitated the selection of systemic AUC as the appropriate dose metric for evaluating internal exposures to PhE and PhAA in rats and humans. Use of the PBPK model resulted in refinement of the total default UF for extrapolation of the animal data to humans from 100 to 25. Based on very conservative assumptions for product composition and aggregate product use, model-predicted exposures to PhE and PhAA resulting from adult and infant exposures to cosmetic products are significantly below the internal dose of PhE observed at the NOAEL dose in rats. Calculated MOEs for all exposure scenarios were above the PBPK-refined UF of 25.

Extrapolation of systemic bioavailability assessing skin absorption and epidermal and hepatic metabolism of aromatic amine hair dyes in vitro

Approaches to assess the role of absorption, metabolism and excretion of cosmetic ingredients that are based on the integration of different in vitro data are important for their safety assessment, specifically as it offers an opportunity to refine that safety assessment. In order to estimate systemic exposure (AUC) to aromatic amine hair dyes following typical product application conditions, skin penetration and epidermal and systemic metabolic conversion of the parent compound was assessed in human skin explants and human keratinocyte (HaCaT) and hepatocyte cultures. To estimate the amount of the aromatic amine that can reach the general circulation unchanged after passage through the skin the following toxicokinetically relevant parameters were applied: a) Michaelis-Menten kinetics to quantify the epidermal metabolism; b) the estimated keratinocyte cell abundance in the viable epidermis; c) the skin penetration rate; d) the calculated Mean Residence Time in the viable epidermis; e) the viable epidermis thickness and f) the skin permeability coefficient. In a next step, in vitro hepatocyte Km and Vmax values and whole liver mass and cell abundance were used to calculate the scaled intrinsic clearance, which was combined with liver blood flow and fraction of compound unbound in the blood to give hepatic clearance. The systemic exposure in the general circulation (AUC) was extrapolated using internal dose and hepatic clearance, and Cmax was extrapolated (conservative overestimation) using internal dose and volume of distribution, indicating that appropriate toxicokinetic information can be generated based solely on in vitro data. For the hair dye, p-phenylenediamine, these data were found to be in the same order of magnitude as those published for human volunteers.

A framework incorporating the impact of exposure scenarios and application conditions on risk assessment of chemicals applied to skin.

Purpose1. To develop a framework for exposure calculation via the dermal route to meet the needs of 21st century toxicity testing and refine current approaches; 2. To demonstrate the impact of exposure scenario and application conditions on the plasma concentration following dermal exposure.MethodA workflow connecting a dynamic skin penetration model with a generic whole-body physiologically-based pharmacokinetic (PBPK) model was developed. The impact of modifying exposure scenarios and application conditions on the simulated steady-state plasma concentration and exposure conversion factor was investigated for 9 chemicals tested previously in dermal animal studies which did not consider kinetics in their experimental designs.ResultsBy simulating the animal study scenarios and exposure conditions, we showed that 7 studies were conducted with finite dose exposures, 1 with both finite and infinite dose exposures (in these 8 studies, an increase in the animal dose resulted in an increase in the simulated steady-state plasma concentrations (Cp,ss)), while 1 study was conducted with infinite dose exposures only (an increase in the animal dose resulted in identical Cp,ss). Steady-state plasma concentrations were up to 30-fold higher following an infinite dose scenario vs. a finite dose scenario, and up to 40-fold higher with occlusion vs. without. Depending on the chemical, the presence of water as a vehicle increased or decreased the steady-state plasma concentration, the largest difference being a factor of 16.ConclusionsThe workflow linking Kasting’s model of skin penetration and whole-body PBPK enables estimation of plasma concentrations for various applied doses, exposure scenarios and application conditions. Consequently, it provides a quantitative, mechanistic tool to refine dermal exposure calculations methodology for further use in risk assessment.

Estimation of in vivo dose of dermally applied chemicals leading to estrogen/androgen receptor-mediated toxicity from in vitro data--Illustration with four reproductive toxicants.

We present a quantitative in vitro-in vivo extrapolation framework enabling the estimation of the external dermal exposure dose from in vitro experimental data relevant to a toxicity pathway of interest. The framework adapts elements of the biological pathway altering dose (BPAD) method [Judson et al. Chem Res Toxicol 2011;24:451] to the case of dermal exposure. Dermal doses of four toxicants equivalent to concentrations characterizing their effect on estrogen receptor α or androgen receptor activity in chemical-activated luciferase expression (CALUX) assays are estimated. The analysis shows that dermal BPADs, calculated from one in vitro concentration, can differ by up to a factor of 55, due to the impact applied dose and dermal exposure scenarios can have on skin permeation kinetics. These features should therefore be taken into account in risk assessment of dermally applied chemicals.

Development of growth equations from longitudinal studies of body weight and height in the full term and preterm neonate: From birth to four years postnatal age

Physiologically based pharmacokinetic (PBPK) models are developed from compound‐independent information to describe important anatomical and physiological characteristics of an individual or population of interest. Modeling pediatric populations is challenging because of the rapid changes that occur during growth, particularly in the first few weeks and months after birth. Neonates who are born premature pose several unique challenges in PBPK model development. To provide appropriate descriptions for body weight (BW) and height (Ht) for age and appropriate incremental gains in PBPK models of the developing preterm and full term neonate, anthropometric measurements collected longitudinally from 1,063 preterm and 158 full term neonates were combined with 2,872 cross‐sectional measurements obtained from the NHANES 2007–2010 survey. Age‐specific polynomial growth equations for BW and Ht were created for male and female neonates with corresponding gestational birth ages of 25, 28, 31, 34, and 40 weeks. Model‐predicted weights at birth were within 20% of published fetal/neonatal reference standards. In comparison to full term neonates, postnatal gains in BW and Ht were slower in preterm subgroups, particularly in those born at earlier gestational ages. Catch up growth for BW in neonates born at 25, 28, 31, and 34 weeks gestational age was complete by 13, 8, 6, and 2 months of life (males) and by 10, 6, 5, and 2 months of life (females), respectively. The polynomial growth equations reported in this paper represent extrauterine growth in full term and preterm neonates and differ from the intrauterine growth standards that were developed for the healthy unborn fetus.

Utilization of Peptide Reactivity Assays for the Prediction of Skin Sensitization

Due to the current ban on animal testing, the need for robust and reliable animal alternative test methods is critical. Reactivity of chemical allergens with proteins has long been established as a key step in induction of skin sensitization. Based on this, two in chemico approaches have been developed: the Direct Peptide Reactivity Assay (DPRA) and the Peroxidase Peptide Reactivity Assay (PPRA). Both assays utilize synthetic peptides which contain either a single cysteine or lysine amino acid as its nucleophilic center for assessing skin sensitization potential of chemicals. Chemical reactivity is determined by using analytical methods to measure the depletion of free peptide following a 24-h incubation of test chemical and peptide. By comparing reactivity data to local lymph node assay data, prediction models have been developed for both assays. These models allow for making hazard predictions and binning a chemical into reactivity categories. The DPRA has been thoroughly evaluated for its reproducibility, transferability, and accuracy under formal validation studies. The PPRA has not yet reached validated status but is undergoing interlaboratory evaluation. Although showing good correlation to established animal models, the data obtained from both of these assays should be considered in combination with other information in the context of integrated approaches such as weight of evidence or integrated testing strategies.