Skin sensitisation prediction using read-across, an illustrative next generation risk assessment (NGRA) case study for vanillin.

Skin sensitisation is a key adverse human health effect to be addressed in the safety assessment of cosmetic ingredients. Regulatory demands and scientific progress have led to the development of a Next Generation Risk Assessment (NGRA) framework, relying on the use of New Approach Methodologies (NAM) Defined Approaches (DA) and read-across instead of generating animal data. This case study illustrates the application of read-across for the prediction of the skin sensitisation potential of vanillin at the hypothetical use concentration of 0.5% in a shower gel and face cream. A three-step process was applied to select the most suitable analogues based on their protein reactivity, structural characteristics, physicochemical properties, skin metabolism profile and availability of skin sensitisation data. The applied read-across approach predicted a weak skin sensitiser potential for vanillin corresponding with a Local Lymph Node Assay EC3 value of 10%. Based on this EC3 value a point of departure of 2500 µg/cm2 was derived, resulting in an acceptable exposure level (AEL) of 25 µg/cm2. Because the consumer exposure levels (CEL) for the face cream (13.5 µg/cm2) and shower gel (0.05 µg/cm2) scenarios were lower than the AEL, the NGRA concluded both uses as safe.

In vivo toxicity assessment of eugenol and vanillin-functionalised silica particles using Caenorhabditis elegans.

The toxicological properties of different silica particles functionalised with essential oil components (EOCs) were herein assessed using the in vivo model C. elegans. In particular, the effects of the acute and long-term exposure to three silica particle types (SAS, MCM-41 micro, MCM-41 nano), either bare or functionalised with eugenol or vanillin, were evaluated on different biological parameters of nematodes. Acute exposure to the different particles did not reduce nematodes survival, brood growth or locomotion, but reproduction was impaired by all the materials, except for vanillin-functionalised MCM-41 nano. Moreover, long-term exposure to particles led to strongly inhibited nematodes growth and reproduction. The eugenol-functionalised particles exhibited higher functionalisation yields and had the strongest effects during acute and long-term exposures. Overall, the vanillin-functionalised particles displayed milder acute toxic effects on reproduction than pristine materials, but severer toxicological responses for the 96-hour exposure assays. Our findings suggest that the EOC type anchored to silica surfaces and functionalisation yield are crucial for determining the toxicological effects of particles on C. elegans. The results obtained with this alternative in vivo model can help to anticipate potential toxic responses to these new materials for human health and the environment.

A benzaldehyde-indole fused chromophore-based fluorescent probe for double-response to cyanide and hypochlorite in living cells.

With the rapid development of various industries, cyanide (CN-) and hypochlorite (ClO-) have a tremendously adverse effect on the health of humans and animals. In this study, a fluorescent probe HHTB based on a benzaldehyde-indole fused chromophore was designed to detect cyanide and hypochlorite simultaneously. The synthesized probe was found to have strong anti-interference ability. In addition, the designed probe could respond rapidly to ClO- in just 80 s, while the color changed visibly from red to colorless. Moreover, the response time to CN- was longer (about 160 s), with the apparent color change from red to light red. The ratiometric and colorimetric absorbance variation of HHTB was due to the nucleophilic attack of CN- on the indole CN functional group and the strong oxidization of ClO- which destroyed the CC bonds and the conjugation systems. Furthermore, the probe HHTB responding to ClO- and CN- presented high sensitivity, as the calculated detection limits were 1.18 nM and 1.40 nM, respectively. The probe was also found to have low biological toxicity and was used in living cells successfully. Therefore, it has good application prospect in the field of cell imaging and biomedicine. The binding mechanism of HHTB-CN and the reaction mechanism of HHTB and ClO- were further elucidated by a series of experiments.

Dihydropyriculol produced by Pyricularia oryzae inhibits the growth of Streptomyces griseus.

Dihydropyriculol is a major secondary metabolite of Pyricularia oryzae. However, the biological activity of dihydropyriculol has not been reported. Here, we showed that dihydropyriculol has inhibitory activity against Streptomyces griseus. Localization analysis of dihydropyriculol revealed that dihydropyriculol could reach to S. griseus under confrontation culture. These results suggest that dihydropyriculol can be used as a chemical weapon against S. griseus.

2-Hydroxy-4-methoxybenzaldehyde from Hemidesmus indicus is antagonistic to Staphylococcus epidermidis biofilm formation.

Staphylococcus epidermidis (SE) is an opportunistic nosocomial pathogen that accounts for recalcitrant device-related infections worldwide. Owing to the growing interest in plants and their secondary metabolites targeting bacterial adhesion, this study was intended to uncover the anti-biofilm potential of Hemidesmus indicus and its major constituent 2-hydroxy-4-methoxybenzaldehyde (HMB) against SE. The minimum biofilm inhibitory concentration (MBIC) of H. indicus root extract and HMB were found to be 500 and 250 µg ml-1, respectively. The results of time-dependent biofilm inhibition and mature biofilm disruption assays confirmed that HMB targets initial cell adhesion. Furthermore, interference by HMB in the expression of adhesin genes (icaA, aap and bhp) and biofilm components was associated with an increased susceptibility of SE to oxidative stress and antibiotics. To conclude, this study reports for the first time HMB as a potential drug against SE biofilms.

Comparison of the metabolism of 10 cosmetics-relevant chemicals in EpiSkin™ S9 subcellular fractions and in vitro human skin explants.

An understanding of the bioavailability of topically applied cosmetics ingredients is key to predicting their local skin and systemic toxicity and making a safety assessment. We investigated whether short-term incubations with S9 from the reconstructed epidermal skin model, EpiSkin™, would give an indication of the rate of chemical metabolism and produce similar metabolites to those formed in incubations with human skin explants. Both have advantages: EpiSkin™ S9 is a higher-throughput assay, while the human skin explant model represents a longer incubation duration (24 hours) model integrating cutaneous distribution with metabolite formation. Here, we compared the metabolism of 10 chemicals (caffeine, vanillin, cinnamyl alcohol, propylparaben, 4-amino-3-nitrophenol, resorcinol, 4-chloroaniline, 2-amino-3-methyl-3H-imidazo[4,5-F]quinoline and 2-acetyl aminofluorene) in both models. Both models were shown to have functional Phase 1 and 2 enzymes, including cytochrome P450 activities. There was a good concordance between the models with respect to the level of metabolism (stable vs. slowly vs. extensively metabolized chemicals) and major early metabolites produced for eight chemicals. Discordant results for two chemicals were attributed to a lack of the appropriate cofactor (NADP+ ) in S9 incubations (cinnamyl alcohol) and protein binding influencing chemical uptake in skin explants (4-chloroaniline). These data support the use of EpiSkin™ S9 as a screening assay to provide an initial indication of the metabolic stability of a chemical applied topically. If required, chemicals that are not metabolized by EpiSkin™ S9 can be tested in longer-term incubations with in vitro human explant skin to determine whether it is slowly metabolized or not metabolized at all.

Bioactivity of some Apiaceae essential oils and their constituents against Sitophilus zeamais (Coleoptera: Curculionidae).

Sitophilus zeamais is a key pest of stored grains. Its control is made, usually, using synthetic insecticides, despite their negative impacts. Botanical insecticides with fumigant/repellent properties may offer an alternative solution. This work describes the effects of Anethum graveolens, Petroselinum crispum, Foeniculum vulgare and Cuminum cyminum essential oils (EOs) and (S)-carvone, cuminaldehyde, estragole and (+)-fenchone towards adults of S. zeamais. Acute toxicity was assessed by fumigation and topical application. Repellence was evaluated by an area preference bioassay and two-choice test, using maize grains. LC50 determined by fumigation ranged from 51.8 to 535.8 mg L-1 air, with (S)-carvone being the most active. LD50 values for topical applications varied from 23 to 128 µg per adult for (S)-carvone > cuminaldehyde > A. graveolens > C. cyminum > P. crispum. All EOs/standard compounds reduced significantly the percentage of insects attracted to maize grains (65-80%) in the two-choice repellence test, whereas in the area preference bioassay RD50 varied from 1.4 to 45.2 µg cm-2, with cuminaldehyde, (S)-carvone and estragole being strongly repellents. Petroselinum crispum EO and cuminaldehyde affected the nutritional parameters relative growth rate, efficiency conversion index of ingested food and antifeeding effect, displaying antinutritional effects toward S. zeamais. In addition, P. crispum and C. cyminum EOs, as well as cuminaldehyde, showed the highest acetylcholinesterase inhibitory activity in vitro (IC50 = 185, 235 and 214.5 µg mL-1, respectively). EOs/standard compounds exhibited acute toxicity, and some treatments showed antinutritional effects towards S. zeamais. Therefore, the tested plant products might be good candidates to be considered to prevent damages caused by this pest.

Quantification of selected aroma compounds in e-cigarette products and toxicity evaluation in HUVEC/Tert2 cells.

During recent years, the number of consumers using so-called e-cigarettes, which are electrical devices to aerosolize a liquid consisting of propylene glycol, glycerol, optional nicotine and flavoring chemicals, has been increasing. Aromas vary from common flavors such as mint to more unusual flavors such as buttermilk or pepperoni pizza. Consumers today can buy e-concentrates that consist of propylene glycol and aroma to blend their own desired flavor at home. Little is known about the composition and concentration of various aroma molecules in the different e-liquids and e-concentrates. In addition, the process of EU-wide regulation is still ongoing. The aim of this research study was to identify and quantify possible undesirable aroma compounds in e-liquids and e-concentrates. Flavoring chemicals such as estragole, benzaldehyde and cinnamaldehyde were quantified. The measurements were carried out on a GC-MS system. The results show the presence of highly concentrated flavoring compounds and limonene oxide in lemon-flavored e-concentrates. In the final step, samples and single-aroma standards were tested for their toxicity to HUVEC/Tert2 cells, where some single-flavoring chemicals such as cinnamic aldehyde revealed significant toxic effects.

A comparison of endocrine disruption potential of nonylphenol ethoxylate, vanillin ethoxylate, 4-n-nonylphenol and vanillin in vitro.

The widely used surfactant nonylphenol ethoxylate (NPEO) and its raw material 4-n-nonylphenol (4-n-NP), as well as its degradation products, are recognized as endocrine disrupting chemicals. The USA Environmental Protection Agency (EPA) released an assessment that looked for safe alternatives to NPEO. Vanillin ethoxylate (VAEO) is a novel substitute for NPEO and is quite similar to NPEO in structure; there is a risk that it has similar endocrine disrupting effects to NPEO. However, their effects on various nuclear hormone receptors have not been thoroughly examined. In this study, the effects of NPEO, VAEO, 4-n-NP and Vanillin on the estrogen receptor α (ERα), androgen receptor (AR), thyroid hormone receptor (TR), retinoic X receptor ß (RXRß) and estrogen-related receptor γ (ERRγ) were determined and compared using a battery of recombined yeast strains expressing ß-galactosidase. The results showed that NPEO and 4-n-NP acted as significant antagonists of ER, AR, TR and ERRγ. In addition, 4-n-NP also had antagonistic activity toward RXRß. Moreover, VAEO was shown to be a very weak antagonist of TR and ERRγ, and Vanillin had no interaction with any nuclear receptors. For the first time, it was found that NPEO had AR, TR and ERRγ antagonistic effects and that 4-n-NP was an antagonist of RXRß. The in vitro data indicated that NPEO, 4-n-NP and VAEO have the potential to act as endocrine disruptors involving more than one nuclear hormone receptor, but VAEO has much lower endocrine disrupting potential than NPEO. Thus, it is critical to find safe substitutes for NPEO and a substitute of NPEO with structural analogues should be carried out with caution. Furthermore, to look for preferable alternatives for NPEO, more in vivo and in vitro studies of the alternatives concerning endocrine disruption are needed, especially in vitro studies need to involve various target points, not only focus on their effects on ER but also take other nuclear hormone receptor pathways into consideration.

Toxicological Assessment of Intermediates in Natural Attenuation of p-Xylene to Marine Microalgae.

The toxic effects of p-xylene, 4-methylbenzyl alcohol, p-methyl benzaldehyde, and p-toluic acid on two marine microalgae (Phaeodactylum tricornutum Bohlin, and Skeletonema costatum) were investigated. p-Xylene was the most toxic to Pha. tricornutum with a 96 h EC50 value of 6.76 mg L-1. Based on the 96 h EC50 values for two microalgae, the toxicity of the four chemicals, in descending order, was: p-xylene, p-methyl benzaldehyde, 4-methylbenzyl alcohol, then p-toluic acid. The results showed that the toxicity of the transformed products of p-xylene was lower than that of p-xylene.

Comparative toxicity study of a novel non-ionic surfactant, vanillin ethoxylates, and nonylphenol ethoxylates in Chinese hamster ovary cells in vitro.

Surfactants such as alkylphenol polyethoxylates (APEOs) and nonylphenol ethoxylates (NPEOs) are commonly used worldwide, but the majority of these compounds, together with their metabolites, have been reported to induce severe biological toxicity. Here, we evaluated for the first time the cytotoxicity, genotoxicity and mitochondrial damage in Chinese hamster ovary (CHO-K1) cells caused by a novel non-ionic surfactant, vanillin ethoxylates (VAEOs), an alternative to APEOs. In parallel, the same in vitro bioassays were conducted on NPEOs along with their metabolic byproducts 4-nonylphenol (4-NP) and vanillin. The results showed that the cytotoxic potency order was NPEOs > 4-NP > VAEOs>vanillin using CCK-8 assays. Also, 4-NP showed potential direct DNA damage in SOS/umu tests, whereas NPEOs, VAEOs and vanillin showed no positive result with and without S9 addition. In addition, none of the test compounds showed obvious genotoxic effects with low olive tail moment value using comet assays. However, all test compounds were shown to cause mitochondrial impairment by increasing mitochondrial mass and decreasing mitochondrial membrane potential in a concentration-dependent manner. And further analysis of reactive oxygen species (ROS) and mitochondrial superoxide (MNSOD) measurement showed that mitochondrial impairment was induced by oxidative stress with intracellular ROS and MNSOD overproduction. It's worth noting that VAEOs and vanillin cause relative lower cytotoxic, genotoxic and mitochondrial damage effects than NPEOs and 4-NP, indicating that VAEOs have the potential to substitute NPEOs as suitable surfactants. Take together, this study elucidates the toxicity profiles of VAEOs and NPEOs relatively comprehensively, and further toxicity analyses are suggested in the population, community and ecosystem.

Genotoxicity of waterpipe smoke in buccal cells and peripheral blood leukocytes as determined by comet assay.

CONTEXT: Waterpipe smoke causes DNA damage in peripheral blood leukocytes and in buccal cells of smokers. OBJECTIVE: To determine the exposure effect of waterpipe smoke on buccal cells and peripheral blood leukocytes in regard to DNA damage using comet assay. MATERIALS AND METHODS: The waterpipe smoke condensates were analyzed by gas chromatography-mass spectrometry (GC-MS). The study was performed on 20 waterpipe smokers. To perform comet assay on bucaal cells of smokers, 10 µl of cell suspension was mixed with 85 µl of pre-warmed 1% low melting agarose, applied to comet slide and electrophoresed. To analyze the effect of smoke condensate in vitro, 1 ml of peripheral blood was mixed with 10 µl of smoke condensate and subjected for comet assay. RESULTS: The GC-MS analysis revealed the presence of 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4on, nicotine, hydroxymethyl furancarboxaldehyde and 3-ethoxy-4-hydroxybenzaldehyde in the smoke condensates. Waterpipe smoking caused DNA damage in vivo in buccal cells of smokers. The tail moment and tail length in buccal cells of smokers were 186 ± 26 and 456 ± 71, respectively, which are higher than control. The jurak and moassel smoke condensates were found to cause DNA damage in peripheral blood leukocytes. The moassel smoke condensate was more damaging. DISCUSSION: There is wide misconception that waterpipe smoking is not as harmful as cigarette smoking. This study demonstrated that waterpipe smoke induced DNA damage in exposed cells. CONCLUSION: Waterpipe smokes cause DNA damage in buccal cells. The smoke condensate of both jurak and moassel caused comet formation suggesting DNA damage in peripheral blood leukocytes.

High vanillin tolerance of an evolved Saccharomyces cerevisiae strain owing to its enhanced vanillin reduction and antioxidative capacity.

The phenolic compounds present in hydrolysates pose significant challenges for the sustainable lignocellulosic materials refining industry. Three Saccharomyces cerevisiae strains with high tolerance to lignocellulose hydrolysate were obtained through ethyl methanesulfonate mutation and adaptive evolution. Among them, strain EMV-8 exhibits specific tolerance to vanillin, a phenolic compound common in lignocellulose hydrolysate. The EMV-8 maintains a specific growth rate of 0.104 h(-1) in 2 g L(-1) vanillin, whereas the reference strain cannot grow. Physiological studies revealed that the vanillin reduction rate of EMV-8 is 1.92-fold higher than its parent strain, and the Trolox equivalent antioxidant capacity of EMV-8 is 15 % higher than its parent strain. Transcriptional analysis results confirmed an up-regulated oxidoreductase activity and antioxidant activity in this strain. Our results suggest that enhancing the antioxidant capacity and oxidoreductase activity could be a strategy to engineer S. cerevisiae for improved vanillin tolerance.

Toxic effects of some synthetic food colorants and/or flavor additives on male rats.

The objective of the present work was to evaluate the broadest toxic effect of some synthetic additives of colorants and/or flavors on different body organs and metabolic aspects in rats. A number of chemical food color and flavor additives are routinely added during processing to improve the aesthetic appearance of the dietary items. However, many of them are toxic after prolonged use. In this experiment, a total of 100 male albino rats of Spargue Dawley strain were divided into 10 groups: G(1) was fed basal diet and served as control, G(2): basal diet + Brilliant blue (blue dye, No. 2, 124 mg/kg diet), G(3): basal diet + carmoisine (red dye, No. 3, 70 mg/kg diet), G(4): basal diet + tartrazine (yellow dye, FD & C yellow No. 5, 75 mg/kg diet), G(5): basal diet + trans-anethole (4.5 g/kg diet) G(6): basal diet + propylene glycol (0.25 g/kg diet), G(7): basal diet + vanillin(1.25 g/kg diet), G(8): basal diet + Brilliant blue + propylene glycol, G(9): basal diet + carmoisine + trans-anethole, G(10): basal diet + tartrazine + vanillin for 42 successive days. All food colorants mixed with or without flavor additives induced a significant decrease in body weight, hemoglobin concentration and red blood cell count. Also there was a significant decrease in reduced glutathione content; glutathione-S-transferase and superoxide dismutase activities in both blood and liver compared to control group. On the other hand, a significant increase in serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase activities, bilirubin, urea, creatinine, total protein and albumin were observed in all test groups when compared to control group. Finally, it is advisable to limit the uses of these food colorants and/or food flavor additives especially those used by children.

Waterpipe smoking: the role of humectants in the release of toxic carbonyls.

In recent years, the number of waterpipe smokers has increased substantially worldwide. Here, we present a study on the identification and quantification of seven carbonylic compounds including formaldehyde, acetaldehyde and acrolein in the mainstream smoke of the waterpipe. Smoking was conducted with a smoking machine, and carbonyls were scavenged from the smoke with two impingers containing an acidic solution of 2,4-dinitrophenylhydrazine. The derivatives were then analyzed by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). For instance, during one waterpipe smoking session, up to 111 ± 12 µg formaldehyde could be detected. This value is about 5 times higher when compared to one 2R4F reference cigarette. We also found a distinct filter effect of the bowl water for all carbonyls investigated. Our data further demonstrate that increasing amounts of humectants in the unburned tobacco lowers the temperature in the waterpipe head during smoking, thereby resulting in decreasing levels of carbonyls in the smoke produced. Altogether, considerable amounts of toxic carbonyls are present in the waterpipe smoke, thus conferring a health risk to waterpipe smokers.

Sex-related deposition and metabolism of vanisulfane, a novel vanillin-derived pesticide, in rats and its hepatotoxic and gonadal effects.

A series of vanillin derivatives have recently been synthesized as effective candidate antiviral agents, with vanisulfane exhibiting pronounced curative and protective activities against cucumber mosaic virus and potato virus Y. However, research on some new pesticides usually ignores their various metabolites and sex-related toxicity. Assisted by 14C labeling, a trial was conducted to investigate the tissue distribution, excretion, and metabolism of vanisulfane in male and female rats for the first time. The results showed that 83.30-87.51% of applied 14C activity was excreted in urine and feces within 24 h of oral administration, and 14C was most abundant in the liver and kidney in both sexes. Interestingly, sex differences were observed in the experiment, with lower body clearance in males than in females 24 h after treatment and preferences for biliary and renal excretion of the pesticide in male and female rats, respectively. A high degradation rate was found for vanisulfane in the plasma; thus, the metabolites of vanisulfane were investigated using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) combined with 14C labeling. One glucuronic acid conjugate and two oxidation metabolites were detected, supporting the monitoring of vanisulfane in vivo. Additionally, rats exposed to vanisulfane exhibited hepatic steatosis in both sexes, along with mild gonadal effects in males. This research offers an effective method for conducting environmental behavioral research and provides new insights for evaluating the potential risks of novel pesticides in mammals from a sex perspective.

Effects of essential oil components exposure on biological parameters of Caenorhabditis elegans.

The extensive use of essential oil components in an increasing number of applications can substantially enhance exposure to these compounds, which leads to potential health and environmental hazards. This work aimed to evaluate the toxicity of four widely used essential oil components (carvacrol, eugenol, thymol, vanillin) using the in vivo model Caenorhabditis elegans. For this purpose, the LC50 value of acute exposure to these components was first established; then the effect of sublethal concentrations on nematodes' locomotion behaviour, reproduction, heat and oxidative stress resistance and chemotaxis was evaluated. The results showed that all the components had a concentration-dependent effect on nematode survival at moderate to high concentrations. Carvacrol and thymol were the two most toxic compounds, while vanillin had the mildest toxicological effect. Reproduction resulted in a more sensitive endpoint than lethality to evaluate toxicity. Only pre-exposure to carvacrol and eugenol at the highest tested sublethal concentrations conferred worms oxidative stress resistance. However, at these and lower concentrations, both components induced reproductive toxicity. Our results evidence that these compounds can be toxic at lower doses than those required for their biological action. These findings highlight the need for a specific toxicological assessment of every EOC application.

Specific accumulation of CYP94A1 transcripts after exposure to gaseous benzaldehyde: induction of lauric acid ω-hydroxylase activity in Vicia sativa exposed to atmospheric pollutants.

The effects of air pollutants such as aldehydes, ozone, nitrogen dioxide and benzene on fatty acid ω-hydroxylase activity in Vicia sativa microsomes have been investigated. Four days old etiolated V. sativa seedlings were exposed to different concentrations of selected pollutants for varying exposure times. Growing etiolated V. sativa seedlings in air containing the gaseous benzaldehyde (150 nM) led to an 8-fold enhancement of lauric acid ω-hydroxylase activity in microsomes of treated plants compared to controls grown in pure air (96 ± 10 versus 12 ± 2 pmol/min/mg protein, respectively). The induction increased with increasing gas phase concentrations (10-1300 nM) and the maximum of activity was measured after 48 h of exposure. Northern blot analysis revealed that this induction occurred via transcriptional activation of the gene coding for CYP94A1. The absence of CYP94A2 and CYP94A3 transcription activation together with the missing effect on epoxide hydrolases activities indicate the specificity of CYP94A1 induction by benzaldehyde. Exposure to nitrogen dioxide, ozone and formaldehyde also stimulated lauric acid ω-hydroxylases activity while exposure to benzene did not show any effect.

Are atranols the only skin sensitizers in oakmoss? A systematic investigation using non-animal methods.

Oakmoss and treemoss absolutes are the major natural extracts of concern as potential sources of skin sensitizers in cosmetics and personal care products (PCP). Two single constituents, atranol and chloroatranol, have been identified as primary culprits in both lichens, and industrial self-regulation has been proposed to limit their contents to less than 100 ppm. Nonetheless, evidence points to the presence of additional candidate skin sensitizers in these multicomponent extracts. These observations, along with a lack of data from non-animal alternative methods and the chemical variability of commercial absolutes, prompted further investigation of oakmoss absolute along with altranol-like compounds in these extracts. The major chemical constituents of a commercial sample were identified by two independent analytical techniques, GC-MS and HPLC-DAD-MS. The crude oakmoss extract and pure compounds were assayed with two in chemico methods (HTS-DCYA and DPRA) to gauge their chemical reactivity. Activation of inflammatory responses in vitro was also investigated by KeratinoSens™ and human cell line activation tests (h-CLAT). Based on weight of evidence, orcinol, ethyl orsellinate, and usnic acid were classified as candidate sensitizers, along with both atranols and oakmoss extract.

RIFM fragrance ingredient safety assessment, p-tolualdehyde, CAS Registry Number 104-87-0.

The existing information supports the use of this material as described in this safety assessment. p-Tolualdehyde was evaluated for genotoxicity, repeated dose toxicity, developmental and reproductive toxicity, local respiratory toxicity, phototoxicity, skin sensitization potential, and environmental safety. Data from read-across analog benzaldehyde (CAS # 100-52-7) show that p-tolualdehyde is not expected to be genotoxic. Data from read-across analog cuminaldehyde (CAS # 122-03-2) provided p-tolualdehyde a No Expected Sensitization Induction Level (NESIL) of 1100 µg/cm2 for the skin sensitization endpoint. The repeated dose toxicity, developmental and reproductive toxicity, and local respiratory toxicity endpoints were completed using the threshold of toxicological concern (TTC) for a Cramer Class I material, and the exposure to p-tolualdehyde is below the TTC (0.03 mg/kg/day, 0.03 mg/kg/day, and 1.4 mg/day, respectively). The phototoxicity/photoallergenicity endpoints were evaluated based on data from read-across analog 4-ethylbenzaldehyde (CAS # 4748-78-1); p-tolualdehyde is not expected to be phototoxic/photoallergenic. The environmental endpoints were evaluated; p-tolualdehyde was found not to be persistent, bioaccumulative, and toxic (PBT) as per the International Fragrance Association (IFRA) Environmental Standards, and its risk quotients, based on its current volume of use in Europe and North America (i.e., Predicted Environmental Concentration/Predicted No Effect Concentration [PEC/PNEC]), are <1.