Migration Studies and Endocrine Disrupting Activities: Chemical Safety of Cosmetic Plastic Packaging.

The endocrine activity and endocrine disruptor (ED) chemical profiles of eleven plastic packaging materials covering five major polymer types (3PET, 1HDPE, 4LDPE, 2 PP, and 1SAN) were investigated using in vitro cell-based reporter-gene assays and a non-targeted chemical analysis using gas chromatography coupled to mass spectrometry (GC-MS). To mimic cosmetic contact, six simulants (acidic, alkaline, neutral water, ethanol 30%, glycerin, and paraffin) were used in migration assays performed by filling the packaging with simulant. After 1 month at 50 °C, simulants were concentrated by Solid Phase Extraction (SPE) or Liquid-Liquid Extraction (LLE). The migration profiles of seven major endocrine disrupting chemicals detected from GC-MS in the different materials and simulants were compared with Estrogen Receptor (ER) and Androgen Receptor (AR) activities. With low extraction of ED chemicals in aqueous simulants, no endocrine activities were recorded in the leachates. Paraffin was shown to be the most extracting simulant of antiandrogenic chemicals, while glycerin has estrogenic activities. Overall, ED chemical migration in paraffin was correlated with hormonal activity. The NIAS 2,4-di-tert-butyl phenol and 7,9-di-tert-butyl1-oxaspiro (4,5) deca-6,9-diene-2,8-dione were two major ED chemicals present in all polymers (principally in PP and PE) and in the highest quantity in paraffin simulant. The use of glycerin and liquid paraffin as cosmetic product simulants was demonstrated to be relevant and complementary for the safety assessment of released compounds with endocrine activities in this integrated strategy combining bioassays and analytical chemistry approaches.

Ethylhexadecyldimethylammonium bromide, a quaternary ammonium compound, controls inflammatory response through NRF2 pathway in a human immortalized keratinocyte cell line.

Many everyday products contain quaternary ammonium compounds (QAC) and some of them are known to be skin irritants such as benzalkonium chloride. Others, such as didecyldimethylammonium chloride, have been shown to cause allergic contact dermatitis. Ethylhexadecyldimethylammonium bromide (EHD) is a QAC for which sensitization potential is not clearly known. Therefore, we have studied its mechanism in human keratinocytes (KC), the main cells of the epidermis. We used the well-described human KC cell line KERTr exposed to EHD, cinnamaldehyde (CinA), a well-known skin sensitizer, and a mixture of both. Since chemical sensitizers are known to activate the transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF2), leading to cellular detoxification and suppressed proinflammatory cytokines, protein or mRNA expression of NRF2 pathway-related enzymes and pro-inflammatory cytokines were investigated by Western blot and RT-qPCR. The activity of the NRF2 pathway on inflammation was studied by RT-qPCR in NRF2-invalidated KERTr cells. We showed that EHD cannot induce the NRF2 pathway, unlike contact sensitizers like CinA. EHD triggers an inflammatory response by inducing the mRNA expression of pro-inflammatory cytokines such as IL-1ß or IL-6. Moreover, mixing EHD and CinA inhibits the effect of CinA on NRF2 expression and mitigates the inflammatory response induced by EHD alone. EHD treatment of KERTr cells in which NRF2 has been invalidated showed an exacerbation of the inflammatory response at the transcriptional level. Hence, EHD may elicit an inflammatory response in KC via the NF-κB pathway, which could lead to irritation when applied to the skin. This inflammation is negatively controlled by the basal activity of the NRF2 pathway.

Particle size distribution in the evaluation of the inhalation toxicity of cosmetic spray products.

While applying cosmetic sprays (pump sprays and propellant-based sprays) intended for use on the skin or hair, consumers may unintentionally inhale sprayed droplets/particles. Thus, it is essential to analyze the size distribution of sprayed droplets/particles because those less than 10 µm are considered to be respirable and may present a high systemic and local exposure risk. In this study, we investigated the droplet/particle size distribution of 78 cosmetic sprays by laser diffraction. Our results showed that the level of respirable droplets/particles released by pump sprays averaged 0.5% of all particles measured (0.00%-2.23%) and that released by propellant-based sprays averaged 15.25% (0.15%-32.27%). Dry shampoos (powder) released the highest percentage of respirable droplets/particles (16.66%-32.27%). A default value of 25% of respirable droplets/particles can also be suggested for dry shampoos. Droplet/particle size distribution was influenced by the spray dispensing system (pump or propellant-based), the product type (hairspray, sunscreen, etc.) and the galenic form (powder, oil, emulsion, etc.). However, it should be noted that more confidence is placed in the pump spray data due to the larger sample size. This study provides data on droplet/particle size, which may be used in a modelling approach to predict inhalation exposure. Therefore, it must be known and used, together with assessments of intrinsic and local toxicities to determine the margin of safety of the product by inhalation route, and to assess the risk of cosmetic sprays.

The Inflammatory Response in Human Keratinocytes Exposed to Cinnamaldehyde Is Regulated by Nrf2.

Keratinocytes (KC) play a crucial role in epidermal barrier function, notably through their metabolic activity and the detection of danger signals. Chemical sensitizers are known to activate the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2), leading to cellular detoxification and suppressed proinflammatory cytokines such as IL-1ß, a key cytokine in skin allergy. We investigated the role of Nrf2 in the control of the proinflammatory response in human KC following treatment with Cinnamaldehyde (CinA), a well-known skin sensitizer. We used the well-described human KC cell line KERTr exposed to CinA. Our results showed that 250 µM of CinA did not induce any Nrf2 accumulation but increased the expression of proinflammatory cytokines. In contrast, 100 µM of CinA induced a rapid accumulation of Nrf2, inhibited IL-1ß transcription, and downregulated the zymosan-induced proinflammatory response. Moreover, Nrf2 knockdown KERTr cells (KERTr ko) showed an increase in proinflammatory cytokines. Since the inhibition of Nrf2 has been shown to alter cellular metabolism, we performed metabolomic and seahorse analyses. The results showed a decrease in mitochondrial metabolism following KERTr ko exposure to CinA 100 µM. In conclusion, the fate of Nrf2 controls proinflammatory cytokine production in KCs that could be linked to its capacity to preserve mitochondrial metabolism upon chemical sensitizer exposure.

Surveillance of dermo-cosmetic products: a global cosmetovigilance system to optimise product development and consumer safety.

In the absence of formal marketing authorisation, the manufacturers of cosmetic products are responsible for their compliance with the cosmetic regulations. To present the key features of a structured, reactive, and rigorous global cosmetovigilance system through practical examples. During clinical development, adverse reactions are collected formally and analysed by cosmetovigilance experts. After commercialisation, information on reported adverse reactions is sought directly from the consumers. The results of allergological investigations are systematically requested. Pre- and post-marketing cases are analysed along with other sources of information (e.g. monitoring of the literature) to detect safety signals per product and per ingredient. A cosmetovigilance index (CVI) is calculated for each formula, based on the number of cases, causality level and number of commercialised units. Updated periodically, it is used to detect signals and select the best tolerated formulas to help formulating new products. Examples of safety issues raised during development or after commercialisation, and corresponding corrective actions, are presented. These actions include (but are not limited to) a safety watch to closely monitor adverse reactions, the modification of the formula or a change in the packaging. Cosmetovigilance data also impact future product development, as illustrated by the work done on sunscreens. Through the rigorous collection and analysis of adverse reactions during development and after commercialisation, the safety of dermo-cosmetic products can be improved by taking the appropriate corrective actions, monitoring their effectiveness and optimising future product development by focusing on the best tolerated formulas.

Safety assessment of cosmetics by read across applied to metabolomics data of in vitro skin and liver models.

As a result of the cosmetics testing ban, safety evaluations of cosmetics ingredients must now be conducted using animal-free methods. A common approach is read across, which is mainly based on structural similarities but can also be conducted using biological endpoints. Here, metabolomics was used to assess biological effects to enable a read across between a candidate cosmetic ingredient, DIV665, only studied using in vitro assays, and a structurally similar reference compound, PA102, previously investigated using traditional in vivo toxicity methods. The (1) cutaneous distribution after topical application, (2) skin metabolism, (3) liver metabolism and (4) effect on the intracellular metabolomic profiles of in vitro skin and hepatic models, SkinEthic®RHE model and HepaRG® cells were investigated. The compounds exhibited similar skin penetration and skin and liver metabolism, with small differences attributed to their physicochemical properties. The effects of both compounds on the metabolome of RHE and HepaRG® cells were similarly small, both in terms of the metabolites modulated and the magnitude of changes. The patterns of metabolome changes did not fit with any known signature relating to a mode of action known to be linked to liver toxicity e.g. modification of the Krebs cycle, urea synthesis and lipid metabolism, were more reflective of transient adaptive responses. Overall, these studies indicate that PA102 is biologically similar to DIV665, allowing read across of safety endpoints, such as in vivo sub-chronic (but not reproduction toxicity) studies, for the former to be applied to DIV665. Based on this, in the absence of animal data (which is prohibited for new chemicals), it could be concluded that DIV665 applied according to the consumer topical use scenario, is similar to PA102, and is predicted to exhibit low local skin and systemic toxicity.

Identification of Potential Extractablesand Leachables in Cosmetic Plastic Packagingby Microchambers-Thermal Extraction and Pyrolysis-Gas Chromatography-Mass Spectrometry.

Most container-content interaction studies are carried out through migration tests on end products or simulants involving generally toxic solvents. This study was conducted with the aim of identifying potential leachables from materials used in cosmetic plastic packaging by using two approaches based on solvent-free extraction, i.e., solid-phase microextraction sampling and pyrolyzer/thermal desorption coupled with gas chromatography mass spectrometry. Volatile and semi-volatile intentionally and non-intentionally added substances were detected in seven packaging samples made of polypropylene, polyethylene, and styrene-acrylonitrile copolymer. Thirty-five compounds related to the polymers industry or packaging industry were identified, among them phthalates, alkanes, styrene, and cyanide derivates including degradation products, impurities, additives, plasticizers, and monomers. All except eight belong to the Cramer class I. These thermodesorption techniques are complementary to those used for migration tests.

Development of a HS-SPME-GC-MS method for the analysis of phthalates in glycerin and liquid paraffin: application to safety evaluation of cosmetic packagings.

Migration of molecules from packaging into products is a well-known phenomenon of which the studies in the food and medical industries are regulated in Europe by several legislations. However, for cosmetic packagings, there is no protocol nor specific migration limits available. The objective of this work was to use glycerin and liquid paraffin as cosmetic product simulants to perform a safety assessment on phthalates in 11 plastic packagings used in the cosmetic industry. To study these compounds in the matrices, 2 extraction techniques were compared: liquid-liquid extraction and solid-phase microextraction (SPME). The optimization of the 2 processes of extraction showed that SPME was more adapted to the study. Finally, samples of glycerin and liquid paraffin were analyzed by a SPME-GC-MS method to quantitate 10 regulated phthalates. In glycerin, only DEP was quantitated above the LOQ in 3 packagings, but the concentrations measured were under the set concentration threshold of 0.5 ppm. In liquid paraffin, DEP was quantitated above this concentration threshold. A safety evaluation was so performed by calculating the systemic exposure damage, and the results were finally considered to be safe for consumers.

In vitro measurement of skin sensitization hazard of mixtures and finished products: Results obtained with the SENS-IS assays.

Product safety evaluation in the EU is based on data mainly obtained on individual ingredients. However, mixture effects have been demonstrated in numerous skin sensitization studies due to the presence of irritating chemicals or to modification of dermal absorption. To evaluate the ability of the SENS-IS assay to detect such mixture effects, we performed three sets of experiments: First, the importance of the vehicle on absorption of individual ingredients was evaluated by testing the effect of commonly used cosmetic preparations on the sensitizing potential of 3 chemical allergens and 2 fragrance blends. The sensitizing potential of the 3 allergens was significantly reduced when tested in microemulsion while the "cleansing water" preparation significantly increased it. Water in oil, oil in water or oil preparations had significant but more moderate (enhancing or reducing) effects on the skin sensitization potency of the tested chemicals. We then analyzed the influence of irritants (SDS and Lactic acid) on the sensitizing potency of various allergens. The SENS-IS assay detected an enhancement of the potency of some allergens when mixed with non-irritating concentrations of irritant chemicals. We also tested the influence of mixing different sensitizers to analyze the effect of mixtures on the sensitization threshold. Some mixtures of chemicals, at doses that did not induce a positive signal in the SENS-IS assay alone, became positive, indicating a mixture effect. Finally we tested commercially available finished cosmetic products to find out that they were not all negative. These results indicate that the SENS-IS assay is a valuable source of information when analyzing mixture component effects and finished products.

Assessment of targeted non-intentionally added substances in cosmetics in contact with plastic packagings. Analytical and toxicological aspects.

Container-content interactions are common in the food and pharmaceutical industries. However, these studies are more complicated in the cosmetic industry, and it is necessary to ensure consumer safety. The objective of this work was to develop a strategy for the toxicological evaluation of leachables for cosmetic packagings. Eleven common plastic packagings were selected to evaluate interactions with 5 simulants (acidic, alkaline and neutral water, 30% and 96% ethanol) chosen to mimic cosmetics behavior. A GC-MS method was developed to screen for 12 non-intentionally added substances of particular concern: 10 phthalates, bisphenol A and distearyl thiodipropionate (European Pharmacopoeia plastic additive 17). Results were analyzed using a toxicological procedure established for this study. Some phthalates and bisphenol A were detected in several samples, but only one contaminant, diisobutyl phthalate (DiBP), was found to be above the set concentration threshold. Using toxicological data, this concentration was found to be safe for users. 96% ethanol appeared to be the strongest simulant in term of extraction, with a maximum concentration of 491 µg/L for DiBP in a 100% styrene-acrylonitrile copolymer packaging. In water simulants, less contaminants were extracted, with concentrations under 20 µg/L.

The THP-1 cell toolbox: a new concept integrating the key events of skin sensitization.

According to the current scientific consensus, one in vitro test is insufficient to cover the key events (KE) defined by the adverse outcome pathway (AOP) for skin sensitization. To address this issue we combined different end points in the same cell line to cover all KEs defined by the skin sensitization AOP. Since dendritic cells (DC) play a key role in the sensitization phase leading to the development of allergic contact dermatitis (ACD), we used THP-1 cells as a surrogate for DC. We measured ROS production and GSH depletion for KE1 (binding to proteins), Nrf2 activation pathway and gene expressions for KE2 (keratinocyte response), phenotype modifications using cell-surface markers and cytokine production for KE3 (DC activation), and T-cell proliferation for KE4 (T-cell activation). These measurements were performed using the THP-1 cell line and an original THP-1/T-cell co-culture system following exposure to a variety of chemicals, including irritant, non-sensitizers, and chemicals sensitizers (pro/prehaptens). Results showed that treatment with sensitizers such as cinnamaldehyde (100 µM) or methylisothiazolinone (150 µM) was able to trigger the three main key events (KE1, KE2, and KE3) of the sensitization phase of ACD in THP-1 cells. In addition, all sensitizers were able to induce T lymphocyte proliferation (KE4), while non-sensitizers and irritants did not. Our study shows for the first time that addressing the four main KE of skin sensitization AOP in a single cell line is an achievable task.

Consumption and exposure to finished cosmetic products: A systematic review.

According to the European Regulation (EC) No. 1223/2009, cosmetic products should be safe for human health when used under normal or foreseeable conditions of use. To perform a safety evaluation, consumption data of finished cosmetic product are necessary to assess the corresponding consumer's exposure. The aim of this review was to highlight consumption (percentage of users, frequency of use, amount used, number of products daily used, types of products co-used …) and exposure data to cosmetic products available in the literature. A systematic approach was used following the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) guidelines. Literature search was performed in February 2018 using Pubmed and Scopus databases. The following information was collected for the 82 publications included in this review: type of study, characteristics of the population (number, age, sex, region of origin), period of data collection, types of products studied, method(s) of data collection, consumption and/or exposure parameters obtained. Because of the high number of quantitative results obtained in the different studies, these data are not presented here. Readers interested in one or more studies are invited to consult the results available in the original publication(s). This work could be very useful for safety assessors or other persons working in the risk assessment field.

Consumption and exposure assessment to toothpaste in French families.

The aim of the study was to assess the consumption and the exposure to toothpaste in French families leaving the consumers free to use their own product at home according to their habits. Consumption data were collected on 104 families. 206 adults (103 women and 103 men) and 195 children aged 2-17 participated in the study. Differences in toothpaste consumption depending on gender and on age were highlighted. As an example, frequency data were higher in adult women (2.0 day-1 on average) than in adult men (1.8 day-1 on average); amount per use data were higher in adult men (1.2 g on average) than in adult women (0.9 g on average). The frequency of use and the amount of toothpaste used per application increased with age. The exposure to toothpaste decreased with age. Children aged 2-6 were the most exposed to toothpaste with a P95 value equal to 8.2 mg/kg bw/day. Adult's P95 exposure value was equal to 2.8 mg/kg bw/day. Exposure values were in the same order of magnitude for both genders in children and in adults. These new data will be useful for safety assessors, especially children data which remain scarce.

Editor's Highlight: Fragrance Allergens Linalool and Limonene Allylic Hydroperoxides in Skin Allergy: Mechanisms of Action Focusing on Transcription Factor Nrf2.

Allergic contact dermatitis is regarded as the most frequent expression of immunotoxicity in humans. Many odorant terpenes commonly used in fragrance compositions are considered as weak skin sensitizers, whereas some of their autoxidation products, allylic hydroperoxides, are classified as strong sensitizers according to the local lymph node assay. However, the mechanism of their effects on the immune system remains unclear. Since dendritic cells play a key role in allergic contact dermatitis, we studied their activation by the frequently used linalool (LINA) and limonene (LIMO), and their respective sensitizing allylic hydroperoxides (LINA-OOH, LIMO-OOH). The THP-1 cell-line was used as a surrogate for dendritic cells, the model currently employed in the validated h-CLAT in vitro test. Our data showed that allylic hydroperoxides behave differently. Both LINA-OOH and LIMO-OOH oxidized cell surface thiols 30 min after stimulation. However, the oxidative stress induced by LINA-OOH was stronger, with a higher decreased GSH/GSSG ratio and a stronger reactive species production. Moreover, LINA-OOH induced a stronger Nrf2 accumulation in correlation with nqo1 and ho-1 gene expression, 2 Nrf2 target genes. Regarding signaling pathways involved in these effects, P38 mitogen-activated protein kinase and P-ERK were activated in response to LINA-OOH but not with LIMO-OOH. CD54 and CD86 were induced 24-h postexposure. In contrast, LINA and LIMO did not modify THP-1 phenotype. This work underlies that autoxidation forming allylic hydroperoxide (ROOH) does not lead to equal chemical reactivity since LINA-OOH appears to be a stronger activator than LIMO-OOH, in regard to oxidative stress and Nrf2 pathway activation.

Chemical analysis and potential endocrine activities of aluminium coatings intended to be in contact with cosmetic water.

The objective of the work was to check the presence of Non-Intended Added Substances (NIAS) with hormonal activities in aluminium coatings extracts coded: AA, BBF, MC and RR, furnished by four different suppliers. Water samples were prepared at room temperature or at 40°C for three months to verify the storage effect on the coatings. Solid phase extraction was used to concentrate and to extract coating substances. Hormonal activities were checked in vitro using reporter gene bioassays. Except BBF, all extracts induced a weak but significant estrogenic agonist activity in the human cell line. Using an estrogenic antagonist (ICI-182, 780), the answer was demonstrated specific in the bioassay. RR was the only extract to induce a concentration dependent anti-androgenic response in the MDA-KB2 cell line. Analysis performed using GC-MS and HPLC-MS detected 12 substances in most of the extracts. 8 NIAS were present. Among them, 4 were identified with certainty: HMBT, BGA, DCU and BPA. Estrogenic potency was BPA>DCU>BGA>HMBT. HMBT was also anti-androgenic at high concentration. Combining chemical analysis and bioassays data, we demonstrated that in the RR and the RR40 extracts, the observed estrogenic response was mainly due to BPA, the anti-androgenic activity of RR could be due to a synergism between HMBT and BPA. For MC and AA, estrogenic responses appear to be due to the presence of DCU. Except BBF, storage conditions tended to increase estrogenic activities in all extracts. However, in term of risk assessment, activities observed were negligible. This work demonstrated that sensitive bioassays are pertinent tools in complement to chemical analysis to monitor and check the presence of NIAS with hormonal activity in coating extracts.

Comparison and validation of an in vitro skin sensitization strategy using a data set of 33 chemical references.

Allergic contact dermatitis (ACD) is an adverse health effect that develops following repeated exposure to skin sensitizing chemicals. An animal testing ban has been applied in EU, leading to development of reliably predictive non-animal methods. Several in vitro methods have been developed as alternatives but one single non-animal test method is not been sufficient to fully address since the LLNA test ban. Here, we have selected an ITS (Integrated Testing Strategy) for skin sensitization which focuses on three in vitro methods that covered the first three steps of the AOP (DPRA, SENS-IS or h-CLAT). The aim of this study was to compare these three methods due to the WoE approach based on a 2-out-of-3-assessment. The results of 33 references were compared to in vivo data (especially human). We have shown that tested firstly DPRA and SENS-IS have permitted to conclude on 29 of 33 chemicals, whereas DPRA and h-CLAT on 25, and SENS-IS and h-CLAT on 23. With this sequence, DPRA and SENS-IS and then h-CLAT in case of equivocal results, we conclude more quickly by performing fewer tests. Thereby, we have shown that it is better to follow a preferential sequence than testing chemicals simultaneously with these three methods.

Consumption and exposure assessment to cosmetic products for children under 2 years old.

Today, the use of personal care products is an integral part of daily life. Little information about children's consumption and exposure to cosmetic products is available. The aim of the study was to assess the consumption and the exposure of French babies aged 0-23 months old to seven common baby care products: shampoo, shower gel, cleansing water, cleansing milk, moisturizing cream, bottom cream and wipes. Consumption and exposure were assessed using small age intervals in order to identify any differences. Exposure was calculated using a probabilistic method. These original data will be useful for safety assessors and safety agencies in order to protect consumers.

Baby care product development: artificial urine in vitro assay is useful for cosmetic product assessment.

As a result of infants' inability to control urination, the skin of the diaper area has special needs for protection from irritating effects of urine and prevention of diaper dermatitis such as products for cleansing and protection of the skin. Several in vitro models are currently available to assess tolerance. In vitro testing using artificial urine allows the protective effects of diaper-region cosmetics to be ascertained. Thus, a new model defined as "artificial urine in vitro assay" has been added to our traditional pre-clinical in vitro testing program. IL1-α is a highly active and pleiotropic pro-inflammatory cytokine. It plays a key role in inflammation and is the biological mirror of irritation induced by diaper dermatitis. This study determines, on human skin explants, if a cosmetic formula is (1) tolerated equally as well in the presence of artificial urine as in its absence and (2) is able to decrease IL1-α production induced by artificial urine or Sodium Dodecyl Sulfate. 31 tests including 17 in-house formulas, 10 bench-markers and 4 combinations of products were performed and data obtained are represented on a simple four-point scale (from practically non protective to very protective). It allows determination of formula-type groups that will have predictable protective properties in subsequent clinical trials and comparison with competitors' products. It is a useful aid in the formulation stage and provides readily-useable data for the cosmetic risk assessment.

Retrospective exposure data for baby and children care products: an analysis of 48 clinical studies.

To conduct a reliable safety assessment, accurate exposure information for cosmetic products and ingredients is needed. The aim of the present retrospective study was to determine the amount per application and the daily exposure for some of the most commonly used baby cosmetic products. Consumption data from 48 clinical studies performed on 1481 babies and children (from 0 to 10 years old) were reviewed and used to conduct a probabilistic evaluation of dermal exposure. Six categories of products were reviewed: rinseoff products for hair and body; rinse-off products for the whole body; leave-on products for face and body; cleansers for face and body; diaper dermatitis treatment products; shampoos. Subjects were provided with products and recorded detailed daily usage information over a 1-4-week period, depending on the study. Products were weighed at the start and upon completion of each study in order to determine the total amount of product used. The mean, median, standard deviation and 95th percentile were calculated for daily consumption and exposure, for several age groups from 0 to 10 years old. This study provides current baby cosmetic exposure information for commonly used products which will be useful for risk assessment purposes.