Fragrance Materials Research Articles

Assessment of genotoxic potential of fragrance materials in the chicken egg assays.

The genotoxic and clastogenic/aneugeneic potentials of four α,β-unsaturated aldehydes, 2-phenyl-2-butenal, nona-2-trans-6-cis-dienal, 2-methyl-2-pentenal, and p-methoxy cinnamaldehyde, which are used as fragrance materials, were assessed using the Chicken Egg Genotoxicity Assay (CEGA) and the Hen's egg micronucleus (HET-MN) assay, respectively. Selection of materials was based on their chemical structures and the results of their previous assessment in the regulatory in vitro and/or in vivo genotoxicity test battery. Three tested materials, 2-phenyl-2-butenal, nona-2-trans-6-cis-dienal, and 2-methyl-2-pentenal, were negative in both, CEGA and HET-MN assays. These findings were congruent with the results of regulatory in vivo genotoxicity assays. In contrast, p-methoxy cinnamaldehyde, which was also negative in the in vivo genotoxicity assays, produced evidence of DNA damage, including DNA strand breaks and DNA adducts in CEGA. However, no increase in the micronucleus formation in blood was reported in the HET-MN study. Such variation in responses between the CEGA and HET-MN assay can be attributed to differences in the dosing protocols. Pretreatment with a glutathione precursor, N-acetyl cysteine, negated positive outcomes produced by p-methoxy cinnamaldehyde in CEGA, indicating that difference in response observed in the chicken egg and rodent models can be attributed to rapid glutathione depletion. Overall, our findings support the conclusion that CEGA and/or HET-MN can be considered as a potential alternative to animal testing as follow-up strategies for assessment of genotoxic potential of fragrance materials with evidence of genotoxicity in vitro.

Fragrance Contact Allergy - A Review Focusing on Patch Testing.

Fragrance materials are widely used in various types of products in daily life and many of them can be contact sensitizers. Contact allergy to fragrances has been reported to be common worldwide. Unlike other groups of contact allergens such as metals and preservatives, fragrance materials in consumer products can be present as single fragrance chemicals or in the form of mixtures known as natural complex substances. Due to the complexity of the fragrance materials and the high number of fragrance substances known to cause contact sensitization, selecting suitable materials for patch testing is challenging. Emerging fragrance markers have been additionally introduced in different baseline series for screening to enhance the rate of fragrance contact allergy detection. Moreover, there have been continual updates on basic knowledge, clinical perspectives, sources of exposure, and regulations on the use of fragrance materials. Avoiding pitfalls while performing patch testing with fragrance test materials is also crucial and should not be overlooked. Therefore, this review aims to update knowledge to provide a high-quality holistic approach to fragrance contact allergy diagnosis and management.

Engineering Living Material for Controlled Fragrance Release Utilizing Kluyveromyces marxianus CBS6556 and Adaptive Hydrogel.

The demand for controllable fragrance materials is substantial owing to their potential to impart enduring scents in a variety of applications. However, the practical application of such materials has been limited by challenges in tunable morphogenesis, structural variability, and adaptability to diverse conditions. In our study, we introduce a hybrid living material that integrates a genetically engineered strain of Kluyveromyces marxianus CBS6556 with an adaptive hydrogel. The engineered K. marxianus achieved temperature stability in 2-phenylethanol (2-PE) and 2-phenylethyl acetate (2-PEAc) production by expressing relevant genes in the 2-PE metabolic pathway using the high-temperature preferential promoter SSE1. The enhanced water retention capacity supports the metabolic activities of the encapsulated yeast cells, ensuring their survival and functionality over an extended period. Fragrance-releasing living material (FLM) is designed to controllably emit fragrance 2-PE by adjusting the microbial concentration within the hydrogel matrix. The FLM exhibits versatile adhesion capabilities, effectively binding to a spectrum of surfaces such as wood, textiles, and glass as well as to natural substrates like leaves. This adaptability enhances the material's applicability across various settings. Furthermore, FLM can be crafted into various forms, including microbeads, fibers, and films. This research opens up new horizons for controlled fragrance release of living materials.

A Chronic Aquatic Hazard Assessment for the Perfume Raw Material Octahydro-tetramethyl-naphthalenyl-ethanone.

Octahydro-tetramethyl-naphthalenyl-ethanone (OTNE) is a high-production volume fragrance material used in various down-the-drain consumer products. To assess aquatic risk, the Research Institute for Fragrance Materials (RIFM) uses a tiered data-driven framework to determine a risk characterization ratio, where the ratio of the predicted-environmental concentration to the predicted-no-effect concentration (PNEC) of <1 indicates an acceptable level of risk. Owing to its high production volume and the conservative nature of the RIFM framework, RIFM identified the need to utilize a species sensitivity distribution (SSD) approach to reduce the PNEC uncertainty for OTNE. Adding to the existing Daphnia magna, Danio rerio, and Desmodesmus subspicatus chronic studies, eight new chronic toxicity studies were conducted on the following species: Navicula pelliculosa, Chironomus riparius, Lemna gibba, Ceriodaphnia dubia, Hyalella azteca, Pimephales promelas, Anabaena flos-aquae, and Daphnia pulex. All toxicity data were summarized as chronic 10% effect concentration estimates using the most sensitive biological response. Daphnia magna was the most sensitive (0.032 mg/L), and D. subspicatus was the least sensitive (>2.6 mg/L, the OTNE solubility limit). The 5th percentile hazardous concentration (HC5) derived from the cumulative probability distribution of the chronic toxicity values for the 11 species was determined to be 0.0498 mg/L (95% confidence interval 0.0097-0.1159 mg/L). A series of "leave-one-out" and "add-one-in" simulations indicated the SSD was stable and robust. Add-one-in simulations determined that the probability of finding a species sensitive enough to lower the HC5 two- or threefold was 1/504 and 1/15,300, respectively. Given the high statistical confidence in this robust SSD, an additional application factor protection is likely not necessary. Nevertheless, to further ensure the protection of the environment, an application factor of 2 to the HC5, resulting in a PNEC of 0.0249 mg/L, is recommended. When combined with environmental exposure information, the overall hazard assessment is suitable for a probabilistic environmental risk assessment. Environ Toxicol Chem 2024;43:1378-1389. © 2024 SETAC.

GARDskin dose-response assay and its application in conducting Quantitative Risk Assessment (QRA) for fragrance materials using a Next Generation Risk Assessment (NGRA) framework

Development of New Approach Methodologies (NAMs) capable of providing a No Expected Sensitization Induction Level (NESIL) value remains a high priority for the fragrance industry for conducting a Quantitative Risk Assesment (QRA) to evaluate dermal sensitization. The in vitro GARDskin assay was recently adopted by the OECD (TG 442E) for the hazard identification of skin sensitizers. Continuous potency predictions are derived using a modified protocol that incorporates dose-response measurements. Linear regression models have been developed to predict human NESIL values. The aim of the study was to evaluate the precision and reproducibility of the continuous potency predictions from the GARDskin Dose-Response (DR) assay and its application in conducting QRA for fragrance materials using a Next Generation Risk Assessment (NGRA) framework. Results indicated that the GARDskin Dose-Response model predicted human NESIL values with a good degree of concordance with published NESIL values, which were also reproducible in 3 separate experiments. Using Isocyclocitral as an example, a QRA was conducted to determine its safe use levels in different consumer product types using a NGRA framework. This study represents a major step towards the establishment of the assay to derive NESIL values for conducting QRA evaluations for fragrance materials using a NGRA framework.

Results of patch testing with five fragrance materials hitherto not tested: A dose-finding study in the clinical population.

Quantitative risk assessment (QRA) for skin sensitization is used to derive safe use levels of sensitising fragrance ingredients in products. Post-marketing surveillance of the prevalence of contact allergy to these ingredients provides relevant data to help evaluate the performance of these measures. To determine a suitable patch test concentration for five fragrance materials that had hitherto not been tested on a regular basis. These concentrations are then to be used in a surveillance study with patch testing consecutive patients over an extended monitoring period. Furaneol, CAS.3658-77-3; trans-2-hexenal, CAS.6728-26-3; 4,8-dimethyl-4,9-decadienal, CAS.71077-31-1; longifolene, CAS.475-20-7; benzaldehyde, CAS.10052-7, were patch tested with other fragrance allergens in four clinics. Patch testing was conducted in three rounds, starting with the lowest concentrations of the five ingredients. The doses were increased in the subsequent rounds if no late-appearing positive reactions and virtually no irritant reactions were reported. Overall, 373 patients were tested. No positive allergic reaction was reported to the five ingredients. Patch test results of other fragrance allergens are reported. The highest test concentrations are each considered safe for patch testing consecutive patients. Further surveillance based on these preparations will evaluate the hypothesis that QRA-driven consumer product levels of these fragrances can prevent sensitization.

Chemicals of Emerging Arctic Concern in north-western Spitsbergen snow: Distribution and sources

Personal care products contain chemicals that are considered of emerging concern in the Arctic. In this study, a selected group of personal care products was investigated in the snowpack on north-western Spitsbergen. We report a preliminary study on the spatial and seasonal distribution of 13 ingredients commonly found in personal care products, including fragrance materials, UV filters, BHT and BPA. Possible sources and deposition processes are discussed. Experimental analyses utilizing GC–MS/MS, were complemented with outputs from the HYSPLIT transport and dispersion model. The results reveal the presence of all selected compounds in the snow, both in proximity to and distant from the research village of Ny-Ålesund. For some of these chemicals this is the first time their presence is reported in snow in Svalbard. These chemicals show different partitioning behaviours between the particulate and dissolved phases, affecting their transport and deposition processes. Additionally, concentrations of certain compounds vary across different altitudes. It is observed the relevance of long-range atmospheric transport during winter at most sites, and, regardless of the proximity to human settlements, snow concentrations can be influenced by long-distance sources. This study highlights the need for detailed information on CEACs' physical-chemical properties, considering their potential impact on fresh and marine waters during the snowmelt under climate change.

Fragrance materials affect life history parameters and gene expression in Daphnia magna: An emerging issue for freshwater ecosystems

A chronic toxicity test (21 d exposure) with the model organism Daphnia magna was performed to study the single-compound and combined effects of four fragrance materials (FMs), including musk xylene (MX), Celestolide™ (ADBI), Galaxolide™ (HHCB), and ethylene brassylate (MT). Furthermore, the transcriptional responses of ten target genes related to detoxification, molting and reproduction (DHR96, P-gp, CYP360A8, GST, CYP314, EcRb, Vtg, CAT, GPX, and GCLC) were determined by performing a quantitative real-time polymerase chain reaction (qRT‒PCR) after juvenile D. magna was exposed for 48 h. The results showed that MX, ADBI and HHCB affected development and reproduction after chronic exposure at a concentration of 10 μg L−1. Conversely, MT did not affect reproduction, growth or molting during the 21 d exposure. In juvenile D. magna, gene expression was significantly altered by ADBI (DHR96, CYP260A8, and GCLC) and MX (DHR96, CYP360A8, EcRb, Vtg, CYP314, and GCLC) but not by HHCB. These results suggest that compared to biochemical measures, conventional biological endpoints provide more informative data regarding the effects of this FM. Compared to single substances in the chronic test, the mixture of the four FMs showed effects at lower concentrations and increased gene expression for EcRb and CYP314 during juvenile exposure, indicating a possible additive or synergistic effect of the four FMs compared to single compound exposure.

Utility of ToxTracker in animal alternative testing strategy for fragrance materials.

To determine the utility of the ToxTracker assay in animal alternative testing strategies, the genotoxic potential of four fragrance materials (2-octen-4-one, lauric aldehyde, veratraldehyde, and p-methoxy cinnamaldehyde) were tested in the ToxTracker assay. These materials have been previously evaluated in an in vitro as well as in vivo micronucleus assay, conducted as per OECD guidelines. In addition to these studies, reconstructed human skin micronucleus studies were conducted on all four materials. All four materials were positive in an in vitro micronucleus assay but were negative in both in vivo and 3D skin micronucleus assays. The ToxTracker assay, in combination with in silico methods to predict metabolism was used to identify mechanisms for the misleading positive outcomes observed in the in vitro micronucleus assays. The results show that the ToxTracker assay, in conjunction with in silico predictions, can provide the information needed to aid in the identification of an appropriate animal alternative follow-up assay, for substances with positive results in the standard in vitro test battery. Thus, the ToxTracker assay is a valuable tool to identify the genotoxic potential of fragrance materials and can aid with replacing animal-based follow-up testing with appropriate animal alternative assay(s).

Top-Down Approach to Retention Time Prediction in Comprehensive Two-Dimensional Gas Chromatography-Mass Spectrometry.

In this contribution, we describe a novel modeling approach to predicting retention times (tr) in comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC × GC-ToF-MS) with a particular emphasis on the second-dimension (2D) retention time predictions (2tr). This approach is referred to as a "top-down" approach in that it breaks down the complete GC × GC separation into two independent one-dimensional gas chromatography separations (1D-GC). In this regard, both dimensions, that is, first dimension (1D) and second dimension (2D) are treated separately, and the cryogenic modulator is simply considered as a second consecutive injection device. Separate 1D-GC tr predictions are performed on both dimensions using the same flow rate as the one deployed in the conventional GC × GC system. The separate tr predictions are then combined to account for the two-dimensional separation. This model was applied to 24 analytes from 2 standard mixtures (Grob Test Mix and Fragrance Materials Test Mix) and assessed across 9 GC × GC chromatographic conditions. The experimental and predicted chromatographic retention space occupations were assessed by using the convex hull approach defined by the Delaunay triangulation. The predicted percentage of space occupation corresponded favorably with the experimental values. Furthermore, the top-down approach enabled an accurate prediction of the 2tr of all investigated analytes, providing an average 2tr modeling error of 0.26 ± 0.01 s.

Analysis of usage patterns and probabilistic risk assessment of personal care products in Korea.

In daily life, many people use large amounts of personal care products (PCPs) on a regular basis. For determining the risk to consumers, product usage amount, frequency of use, and co-use patterns are essential information. In this study, the PCP usage patterns of Korean consumers were analyzed, and the data were used to develop a probabilistic risk assessment. A web-based questionnaire was used to evaluate the PCP usage patterns of consumers. A model developed by Crème Global in conjunction with the Research Institute for Fragrance Materials was used to measure the systemic exposure to PCP ingredients. The use rates of 10 PCP family are compared with reported values obtained for populations in California and Switzerland which showed some distinct characteristics of Korean consumers. The co-use combinations of the 10 families of products frequently used by consumers were analyzed in an attempt to calculate aggregate exposure. Seven ingredients were selected and the aggregate risk assessment were performed based on bootstrapping analysis. The probability of the Hazard Quotient of all seven ingredients exceeding 1 was zero. The data of the usage patterns of Korean consumers reported here will be of value in developing more precise aggregate exposure risk analysis.

Evaluation of the anti-SARS-CoV-2 properties of essential oils and aromatic extracts

Essential oils and aromatic extracts (oleoresins, absolutes, concretes, resinoids) are often used as food flavorings and constituents of fragrance compositions. The flavor and fragrance industry observed significant growth in the sales of some natural materials during the COVID-19 outbreak. Some companies worldwide are making false claims regarding the effectiveness of their essential oils or blends (or indirectly point toward this conclusion) against coronaviruses, even though the available data on the activity of plant materials against highly pathogenic human coronaviruses are very scarce. Our exploratory study aimed to develop pioneering knowledge and provide the first experimental results on the inhibitory properties of hundreds of flavor and fragrance materials against SARS-CoV-2 main and papain-like proteases and the antiviral potential of the most active protease inhibitors. As essential oils are volatile products, they could provide an interesting therapeutic strategy for subsidiary inhalation in the long term.

The BlueScreen HC assay to predict the genotoxic potential of fragrance materials

BlueScreen HC is a mammalian cell-based assay for measuring the genotoxicity and cytotoxicity of chemical compounds and mixtures. The BlueScreen HC assay has been utilized at the Research Institute for Fragrance Materials in a safety assessment program as a screening tool to prioritize fragrance materials for higher-tier testing, as supporting evidence when using a read-across approach, and as evidence to adjust the threshold of toxicological concern. Predictive values for the BlueScreen HC assay were evaluated based on the ability of the assay to predict the outcome of in vitro and in vivo mutagenicity and chromosomal damage genotoxicity assays. A set of 371 fragrance materials was assessed in the BlueScreen HC assay along with existing or newly generated in vitro and in vivo genotoxicity data. Based on a weight-of-evidence approach, the majority of materials in the data set were deemed negative and concluded not to have the potential to be genotoxic, while only a small proportion of materials were determined to show genotoxic effects in these assays. Analysis of the data set showed a combination of high positive agreement but low negative agreement between BlueScreen HC results, in vitro regulatory genotoxicity assays, and higher-tier test results. The BlueScreen HC assay did not generate any false negatives, thereby providing robustness when utilizing it as a high-throughput screening tool to evaluate the large inventory of fragrance materials. From the perspective of protecting public health, it is desirable to have no or minimal false negatives, as a false-negative result may incorrectly indicate the lack of a genotoxicity hazard. However, the assay did have a high percentage of false-positive results, resulting in poor positive predictivity of the in vitro genotoxicity test battery outcome. Overall, the assay generated 100% negative predictivity and 3.9% positive predictivity. In addition to the data set of 371 fragrance materials, 30 natural complex substances were evaluated for BlueScreen HC, Ames, and in vitro micronucleus assay, and a good correlation in all three assays was observed. Overall, while a positive result may have to be further investigated, these findings suggest that the BlueScreen HC assay can be a valuable screening tool to detect the genotoxic potential of fragrance materials and mixtures.

Perfumes and associated allergens: A brief review

A perfume (Latin, perfumare for smoke through) is essentially a cosmetic product to be used on the human body for its pleasing scent to mask body odor and have a feeling of freshness. A perfume is composed of fragrances which are aromatic compounds and essential oils, and solvents and fixatives. These fragrances are chemicals or are derived mostly from botanical sources. A perfume that usually consists of a few to hundreds of fragrance materials remains a major source of contact allergy from them. Everyone comes in contact with fragrance materials in daily life either directly from the application of a product to the skin, mostly cosmetics, or occasionally after contact with fragrance-containing household or other personal care products. Cosmetics and personal care products such as personal perfume(s), deodorant, aftershave lotion/gel, eau de cologne, and eau de toilette with the highest ever concentrations of fragrances remain the major source of contact sensitivity from fragrances. The adverse effects associated with the use of perfumes are both allergic and irritant contact dermatitis, pigmented contact dermatitis, phototoxicity and photoallergy, and contact urticaria. Systemic toxicity may also occur following inhalation and ingestion of fragrance(s) causing respiratory illness, headache, and attacks of migraine. However, allergic contact dermatitis and pigmented contact dermatitis remain by far the commonest cutaneous adverse effects. Colophony, Myroxylon pereirae, lanolin, formaldehyde, vanillin, musk mix, rose oil, cetostearyl alcohol, and more than 160 fragrances, both single and complex mixtures of chemicals (methyl ionones), or essential oils (Evernia furfuracea (treemoss) extract, E. prunastri (oakmoss) extract, Ferula galbaniflua gum, Narcissus poeticus flower extract, and Viola odorata leaf extract) have caused cosmetic contact sensitivity in most instances. Ylang-ylang oil, jasmine absolute, Cananga oil, benzyl salicylate, hydroxyl citronellal, sandalwood, and geraniol are common fragrances to cause pigmentary changes. This review highlights the composition of perfumes and constituent fragrances identified commonly as allergens. A brief insight is also provided for the diagnosis of allergic contact sensitivity from fragrances and present safety regulatory status.

Weight of Evidence Approach for Skin Sensitization Potency Categorization of Fragrance Ingredients.

BackgroundReliable human potency data are necessary for conducting quantitative risk assessments, as well as development and validation of new nonanimal methods for skin sensitization assessments. Previously, human skin sensitization potency of fragrance materials was derived primarily from human data or the local lymph node assay.ObjectivesThis study aimed to define skin sensitization potency of fragrance materials via weight of evidence approach, incorporating all available human, animal, in vitro, in chemico, and in silico data.MethodsAll available data on 106 fragrance materials were considered to assign each material into 1 of the 6 defined potency categories (extreme, strong, moderate, weak, very weak, and nonsensitizer).ResultsNone of the 106 materials were considered an extreme sensitizer, whereas a total of 6, 23, 41, and 26 materials were categorized as strong, moderate, weak, and very weak sensitizers, respectively. Ten materials lacked evidence for the induction of skin sensitization.ConclusionsSkin sensitization potency categorization of the 106 fragrance materials based on the described weight of evidence approach can serve as a useful resource in evaluation of nonanimal methods, as well as in risk assessment.

Supercritical CO2 Extraction of Narcissus poeticus L. Flowers for the Isolation of Volatile Fragrance Compounds.

The flowers of Narcissus poeticus are used for the isolation of valuable fragrance substances. So far, as the majority of these substances consist of volatile and sensitive to heat compounds, there is a need of developing effective methods for their recovery. In this study, freeze-dried N. poeticus inflorescences were extracted with pure supercritical CO2 (SFE-CO2) and its mixture with 5% co-solvent ethanol (EtOH) at 40 °C. Extract yields varied from 1.63% (12 MPa) to 3.12% (48 MPa, 5% EtOH). In total, 116 volatile compounds were identified by GC-TOF/MS in the extracts, which were divided into 20 different groups. Benzyl benzoate (9.44–10.22%), benzyl linoleate (1.72–2.17%) and benzyl alcohol (0.18–1.00%) were the major volatiles among aromatic compounds. The amount of the recovered benzyl benzoate in N. poeticus SFE-CO2 extracts varied from 58.98 ± 2.61 (24 MPa) to 91.52 ± 1.36 (48 MPa) mg/kg plant dry weight (pdw). α-Terpineol dominated among oxygenated monoterpenes (1.08–3.42%); its yield was from 9.25 ± 0.63 (12 MPa) to 29.88 ± 1.25 (48 MPa/EtOH) mg/kg pdw. Limonene was the major monoterpene hydrocarbon; (3E)-hexenol and heneicosanol dominated among alcohols and phenols; dihydroactinidiolide and 4,8,12,16-tetramethyl heptadecan-4-olide were the most abundant lactones; heptanal, nonanal, (2E,4E)-decadienal and octadecanal were the most abundant aldehydes. The most important prenol lipids were triterpenoid squalene, from 0.86 ± 0.10 (24 MPa) to 7.73 ± 0.18 (48 MPa/EtOH) mg/kg pdw and D-α-tocopherol, from 1.20 ± 0.04 (12 MPa) to 15.39 ± 0.31 (48 MPa/EtOH) mg/kg pdw. Aliphatic hydrocarbons (waxes) constituted the main part (41.47 to 54.93%) in the extracts; while in case of a 5% EtOH the percentage of alkanes was the lowest. The fraction of waxes may be removed for the separation of higher value fragrance materials. In general, the results obtained are promising for a wider application of SFE-CO2 for the recovery of fragrance substances from N. poeticus flowers.

Use of alternative test methods in a tiered testing approach to address photoirritation potential of fragrance materials

The safety assessment of fragrance materials for photoirritation utilized by The Research Institute for Fragrance Materials has recently been modified and is described in detail. Materials demonstrating significant absorbance in the ultraviolet and visible light (UV/VIS) range (290–700 nm) may present a concern for photoirritation and require further investigation. If there are no photoirritation data or data are insufficient, then data on read-across materials are considered before a tiered approach for testing begins. The hazard-based 3T3-Neutral Red Uptake (NRU) Phototoxicity Test (OECD TG 432) is used as a first-tier assay; if it predicts photoirritation, it is followed by the reconstructed human epidermis (RhE) phototoxicity assay (OECD TG 498). The RhE phototoxicity assay is used to determine a No Observed Effect Level (NOEL) for photoirritation that is used in a confirmatory human photoirritation test. Data are presented on 108 fragrance materials exhibiting significant UV/VIS absorbance and evaluated in the 3T3-NRU Phototoxicity Assay. Twenty-one materials were predicted to be phototoxic; twenty were evaluated in the RhE Phototoxicity Assay to establish a NOEL. Fourteen materials were then evaluated in a confirmatory human phototoxicity test. The tiered testing approach presented represents a scientifically pragmatic method to minimize the likelihood of photoirritation from fragrance materials.

Chemical mixtures in human post-mortem tissues assessed by a combination of chemical analysis and in vitro bioassays after extraction with silicone.

Passive equilibrium sampling of chemical mixtures from different human post-mortem tissues (liver, brain (cerebrum and cerebellum), adipose tissue) and blood was combined with instrumental analysis using direct sample introduction (DSI) GC-MS/MS and bioanalytical profiling using in vitro bioassays targeting the activation of the aryl hydrocarbon receptor (AhR-CALUX), the adaptive stress response (AREc32) and cytotoxicity. The tissues stemmed from pathology samples collected in two German cities and covered males and females aged 21 to 100 with a mean age of 67years. Neutral organic chemicals were extracted using polydimethylsiloxane (PDMS) at mass ratios of tissue to PDMS of approximately 6 for blood, 3 for adipose tissue and 10 for liver and brain. Amounts of chemicals in PDMS were converted to lipid-associated concentrations using previously measured partition constants that were chemical-independent despite covering eight orders of magnitude in hydrophobicity. Up to 35 of 99 targeted chemicals were detected in 6 tissues of 16 individuals (88 samples in total), among them legacy persistent organic pollutants (POP) such as DDT and derivatives and polychlorinated biphenyls (PCB) but also modern pesticides and chemicals present in consumer products. POPs were highest in adipose tissue and lipid-associated concentrations increased with age, while concentrations of fragrance materials such as galaxolide were independent of age. In tissues from the same individual, chemical concentrations mostly increased from similar levels in brain and blood to higher levels in liver and highest in adipose tissue. However, easily degradable chemicals such as phenanthrene were mainly detected in blood and brain, and very hydrophilic chemicals were least abundant in adipose tissue. The passive sampling method allows a direct comparison of chemical burden between different tissues and may have forensic applications, for example to study internal distributions or to use one tissue type as a proxy for others. The sum of concentrations of the detected chemicals was positively correlated with the bioassay responses but mixture modeling showed that the detected chemicals explained less than 2% of the activation of the AhR and less than 0.5% of cytotoxicity. This means that more than 10,000 chemicals would need to be included in an analytical method to capture all the effects with many chemicals potentially being below detection limits but still contributing to mixture effects. Therefore, we propose a smart combination of chemical analysis and bioassays to quantify priority chemicals but use bioassay responses as effect-scaled concentrations to capture the entire exposome in future epidemiological studies.

Use of the EpiDermTM 3D reconstructed skin micronucleus assay for fragrance materials

In order to evaluate the utility of the 3D reconstructed skin micronucleus assay (3DRSMN) to assess clastogenic/aneugenic potential of the fragrance chemicals, a set of 22 fragrance materials were evaluated in 3DRSMN assay. These materials evaluated were also evaluated in an in vitro as well as in vivo micronucleus assay, conducted as per Organisation for Economic Co-operation and Development guidelines. The results of the RSMN assay were in 100% agreement with the in vivo micronucleus assay results. From this dataset, 18 materials were positive in an in vitro micronucleus assay but were negative in an in vivo micronucleus assay. All these 18 materials were also concluded to be negative in 3DRSMN assay, stressing the importance of the assay to help minimize misleading positive outcomes from the in vitro assay. Since the highest exposure for fragrances is through the dermal route, the RSMN assay fits the applicability domain for testing. Thus, RSMN assay is an important alternative to animal testing for characterization of the genotoxicity potential of fragrance materials.

The RIFM approach to evaluating Natural Complex Substances (NCS)

The Research Institute for Fragrance Materials, Inc. (RIFM) has evaluated safety data for fragrance materials for 55 years. The safety assessment of Natural Complex Substances (NCS) is similar to that of discrete fragrance materials; all of the same endpoints are evaluated. A series of decision trees, reflecting advances in risk assessment approaches of mixtures and toxicological methodologies, follows a tiered approach for each endpoint using a 4-step process with testing only as a last resort: 1) evaluate available data on NCS; 2) verify whether the Threshold of Toxicological Concern (TTC) can be applied; 3) verify whether the NCS risk assessment can be achieved on a component basis; and 4) determine whether data must be generated. Using in silico tools, RIFM examined NCS similarities based on the plant part, processing, and composition of materials across 81 plant families to address data gaps. Data generated from the Creme RIFM Aggregate Exposure Model for over 900 fragrance NCS demonstrate that dermal exposure is the primary route of human exposure for NCS fragrance uses. Over a third of materials are below the most conservative TTC limits. This process aims to provide a comprehensive Safety Assessment of NCS used as a fragrance ingredient.