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.

Effects of Carbon Chain Length on the Perfluoroalkyl Acids-Induced Oxidative Stress of Erythrocytes in Vitro.

Perfluoroalkyl acids (PFAAs) have been found extensively in wildlife and human bodies by sources of drinking water and food. In this study, we investigated the effects of three PFAAs, perfluoropentanoic acid (PFPA), perfluorooctanoic acid (PFOA), and perfluorodecanoic acid (PFDA), on the antioxidative defense system and lipid peroxidation in erythrocytes separately. The results demonstrated that they could lead to significant decline trends in the glutathione (GSH) levels together with increases of malondialdehyde (MDA) content, suggesting that three PFAAs induced oxidative stress to erythrocytes. Also PFDA with a longer carbon chain length posed more of a threat than other two PFAAs. Furthermore, the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were also altered in the presence of PFAAs upon erythrocytes. The changes of oxidative stress markers and the concomitant alterations of antioxidant enzymes suggest the role of oxidative stress in PFAA-induced damage upon erythrocytes.

RIFM fragrance ingredient safety assessment, Linalyl isovalerate, CAS Registry Number 1118-27-0.

The use of this material under current use conditions is supported by the existing information. This material was evaluated for genotoxicity, repeated dose toxicity, developmental toxicity, reproductive toxicity, local respiratory toxicity, phototoxicity, skin sensitization potential, as well as, environmental safety. Reproductive toxicity was based on the Threshold of Toxicological Concern (TTC) of 0.03 mg/kg/day for a Cramer Class I material. The estimated systemic exposure is determined to be equal to this value while assuming 100% absorption from skin contact and inhalation. A systemic exposure at or below the TTC value is acceptable.

Carcinogenicity assessment of the pan-caspase inhibitor, emricasan, in Tg.rasH2 mice.

Emricasan, formerly IDN-6556, is a small molecule currently being evaluated in clinical trials to reduce hepatic injury and liver fibrosis. Since emricasan is an irreversible pan-caspase inhibitor that potently inhibits caspase-mediated apoptosis and inflammation, its carcinogenic potential was evaluated in a humanized mouse model. Tg.rasH2 mice received LabDiet formulated with 0, 10, 25, and 75mg/kg/day of emricasan, for 26weeks. At terminal sacrifice, blood was collected for clinical pathology analysis and tissues were collected, processed, and evaluated microscopically. There were no treatment related deaths or overt signs of toxicity for the duration of the study. There was no evidence of a carcinogenic effect in the peripheral blood leukocyte counts. Liver microgranulomas, which are background lesions, were slightly increased, especially in males. Increases in the incidence of the activated germinal centers were seen in the spleens and mesenteric lymph nodes of males and females, and in the mandibular lymph nodes of male mice. Atrophy of ovaries and testicular degeneration were also seen in emricasan treated animals. Although several non-neoplastic lesions were observed, there was no evidence of emricasan-related tumor formation in any tissue. In addition, the non-neoplastic lesions were not considered pre-neoplastic. Thus, emricasan is not considered carcinogenic.

Evaluation of stereoselective anticonvulsant, teratogenic, and pharmacokinetic profile of valnoctylurea (capuride): a chiral stereoisomer of valproic acid urea derivative.

PURPOSE: The purpose of this study was to evaluate the stereoselective anticonvulsant activity, neurotoxicity, pharmacokinetics, and teratogenic potential of two stereoisomers of valnoctylurea (VCU), a central nervous system (CNS)-active urea derivative of valnoctic acid, which is a constitutional isomer of valproic acid (VPA). METHODS: VCU stereoisomers (2S,3S)-VCU and (2R,3S)-VCU were synthesized. Their anticonvulsant activity was determined and compared to VPA and racemic-VCU in rats utilizing the maximal electroshock seizure (MES) and the subcutaneous pentylenetetrazole (scMet) tests. Neurotoxicity was determined in rats using the positional sense test, muscle tone test, and gait and stance test. The induction of neural tube defects (NTDs) by VCU stereoisomers was evaluated in a mouse strain highly susceptible to VPA-induced teratogenicity. The pharmacokinetics of VCU was studied in a stereoselective manner following intraperitoneal (i.p.) administration to rats. RESULTS: (2S,3S)-VCU and (2R,3S)-VCU median effective dose ED(50) values were 29 mg/kg [95% confidence interval (CI) = 8-60 mg/kg] and 42 mg/kg (95% CI = 36-51 mg/kg) (MES) and 22 mg/kg (95% CI = 13-51 mg/kg) and 12 mg/kg (95%CI = 7-21 mg/kg) (scMet), respectively. (2S,3S)-VCU was more potent and had a wider safety margin (p < 0.05), defined as the protective index (PI = TD(50)/ED(50)), at both the MES (PI > 17) and scMet (PI > 23) tests than racemic-VCU or (R,S)-VCU (PI = 2.8 and 9.9, respectively). VCU stereoisomers caused NTDs at doses >4 times that of their anticonvulsant ED(50) values. At a dose of 112 mg/kg, (2R,3S)-VCU was nonteratogenic and less embryotoxic than its stereoisomer (2S,3S)-VCU. No stereoselective pharmacokinetics was observed following intraperitoneal dosing of racemic-VCU to rats. VCU was mainly eliminated by metabolism with a half-life of 2 h. CONCLUSIONS: VCU anticonvulsant activity and wide PI values make it a potential candidate for development as a new, potent antiepileptic drug (AED).

Pivalate lowers litter sizes and weights in female rats independent of its effect on carnitine status.

The present study investigated whether treatment of female rats with pivalate affects their reproductive function. Therefore, two experiments with female rats were performed. The first experiment included two groups of rats which received drinking water without (control) or with 20 mmol pivalate/L. The second experiment included a control group (which received drinking water without pivalate and a diet without added carnitine) and four groups which received drinking water with 20 mmol/L pivalate and diets without or with 1, 3 or 5 g added carnitine/kg, respectively. In both experiments, rats treated with pivalate had a lower number of pups born alive and, as a consequence of this, lower litter weights than control rats (p<0.05); pup weights were not altered by pivalate treatment. Supplementation of dietary carnitine in Experiment 2 increased plasma and tissue carnitine concentration even in excess of those in control rats but did not restore normal litter sizes. This study shows for the first time that pivalate affects the reproductive function in female rats independent of its effect on the carnitine status.

Isovaleric acid reduces Na+, K+-ATPase activity in synaptic membranes from cerebral cortex of young rats.

1. Patients affected by isovaleric acidemia (IVAcidemia) suffer from acute episodes of encephalopathy. However, the mechanisms underlying the neuropathology of this disease are poorly known. The objective of the present study was to investigate the in vitro effects of the metabolites that predominantly accumulate in IVAcidemia, namely isovaleric acid (IVA), 3-hydroxyisovaleric acid (3-OHIVA) and isovalerylglycine (IVG), on important parameters of energy metabolism, such as (14)CO(2) production from acetate and the activities of the respiratory chain complexes I-IV, creatine kinase and Na(+), K(+)-ATPase in synaptic plasma membranes from cerebral cortex homogenates of 30-day-old rats. 2. We observed that 3-OHIVA acid and IVG did not affect all the parameters analyzed. Similarly, (14)CO(2) production from acetate (Krebs cycle activity), the activities of creatine kinase, and of the respiratory chain complexes was not modified by IVA. In contrast, IVA exposition to cortical homogenates provoked a marked inhibition of Na(+), K(+)-ATPase activity. However, this activity was not changed when IVA was directly exposed to purified synaptic plasma membranes, suggesting an indirect effect of this organic acid on the enzyme. Furthermore, pretreatment of cortical homogenates with alpha-tocopherol and creatine totally prevented IVA-induced inhibition on Na(+), K(+)-ATPase activity from synaptic plasma membranes, whereas glutathione (GSH) and the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) did not alter this inhibition. 3. These data indicate that peroxide radicals were probably involved in this inhibitory effect. Since Na(+), K(+)-ATPase is a critical enzyme for normal brain development and functioning and necessary to maintain neuronal excitability, it is presumed that the inhibitory effect of IVA on this activity may be involved in the pathophysiology of the neurological dysfunction of isovaleric acidemic patients.

Cutoff in detection of eye irritation from vapors of homologous carboxylic acids and aliphatic aldehydes.

Using neat vapors of selected homologous aldehydes (decanal, undecanal, dodecanal) and carboxylic acids (pentanoic, hexanoic, heptanoic, octanoic, nonanoic), we explored the point where a certain homolog (and all larger ones) becomes undetectable by eye irritation (i.e. by ocular chemesthesis). This phenomenon has been observed in other homologous series that also reach a break-point, or cutoff, in chemesthetic detection. Participants (11

In vitro effects of hydrochloric acid and various concentrations of acetic, propionic, butyric, or valeric acids on bioelectric properties of equine gastric squamous mucosa.

OBJECTIVE: To compare the effects of hydrochloric acid (HCl) and various concentrations of volatile fatty acids (VFAs) on tissue bioelectric properties of equine stomach nonglandular (NG) mucosa. SAMPLE POPULATION: Gastric tissues obtained from 48 adult horses. PROCEDURES: NG gastric mucosa was studied by use of Ussing chambers. Short-circuit current (Isc) and potential difference (PD) were measured and electrical resistance (R) and conductance calculated for tissues after addition of HCl and VFAs (5, 10, 20, and 40 mM) in normal Ringer's solution (NRS). RESULTS: Mucosa exposed to HCl in NRS (pH of 1.5 and, to a lesser extent, 4.0) had a significant decrease in Isc, PD, and R, whereas tissues exposed to acetic acid at a pH of < 4.0, propionic and butyric acids at a pH of

Possible mechanism for species difference on the toxicity of pivalic acid between dogs and rats.

In a high dose toxicity study of pivalic acid (PA), PA caused skeletal muscle disorder in dog, and a significant increase of pivaloyl carnitine (PC) was observed in canine muscle, but not in rat muscle. In order to understand species difference of the toxicity of PA, we compared the in vitro metabolism of PA among dog, rat and rabbit, especially focussing on the carnitine conjugate. Canine muscle showed low, but significant carnitine conjugating activity, while that of rat was negligible. Canine kidney mitochondria had significant activity in the pivaloyl CoA synthesis (7 nmol/mg protein/h), but muscle mitochondria showed only trace activity. Both kidney and muscle mitochondria displayed similar carnitine acyltransferase activity (2-3 nmol/mg protein/h) towards pivaloyl CoA. On the other hand, with respect to the activity of carnitine acyltransferase in the reverse direction using PC as substrate, canine muscle mitochondria showed higher activity than that of kidney mitochondria. This means that PC is not the final stable metabolite, but is converted easily to pivaloyl CoA in canine muscle. These results suggest one of the possible mechanisms for canine selective muscle disorder to be as follows. Only canine muscle can metabolize PA to its carnitine conjugate slowly, but significantly. In canine muscle, PC is not the final stable metabolite; it is easily converted to pivaloyl CoA. As carnitine conjugation is thought to be the only detoxification metabolic route in canine muscle, under certain circumstances such as carnitine deficiency, the risk of exposure with toxic pivaloyl CoA might increase and the CoASH pool in canine muscle might be exhausted, resulting in toxicity in canine muscle.

Fragrance material review on linalyl isovalerate.

A toxicologic and dermatologic review of linalyl isovalerate when used as a fragrance ingredient is presented.

Characterization and toxicity of Amanita cokeri extract.

The nonprotein amino acids 2-amino-3-cyclopropylbutanoic acid and 2-amino-5-chloro-4-pentenoic acid were isolated from the mushroom Amanita cokeri. The cyclopropyl amino acid is toxic to the fungus Cercospora kikuchii, the arthropod Oncopeltus fasciatus (milk weed bug), and the bacteria Agrobacterium tumefaciens, Erwinia amylovora, and Xanthomonas campestris. Toxicity to bacteria was reversible by addition of isoleucine to the medium. No toxicity was observed for 2-amino-5-chloro-4-pentenoic acid.

Valnoctamide, valpromide and valnoctic acid are much less teratogenic in mice than valproic acid.

The teratogenic properties of valproic acid (VPA) and its analogues depend to a great extent on their chemical structure. We investigated the structure-teratogenicity relationships of VPA, its structural isomer, valnoctic acid (VCA), and their two amide analogues, valpromide (VPD) and valnoctamide (VCD), respectively. Each substance was injected (3 mmol/kg) in NMRI-mice on the morning of day 8 of gestation. Embryolethality, fetal weight and exencephaly rates were recorded on day 18 of gestation. VPA caused 53% exencephaly, VPD induced 6%, VCA and VCD produced only 1% exencephaly (control values between 0 and 1%). VPA-treated mice also had increased embryolethality rates (52%). There was no significant change of embryolethality in the other treatment groups. Pharmacokinetic studies showed that VCD was eliminated from plasma at a slower rate than VPA. Also, the residual teratogenic activity of VPD was not accounted for by the relatively small amounts of its hydrolysis product VPA. This study indicates that VPD, VCA and VCD were distinctly less teratogenic than VPA. Apparently the amidation of the free carboxylic group and/or methyl-substitution at the beta-position of the carbon chain greatly decreased the teratogenic activity of VPA.

Fluorinated analogues as mechanistic probes in valproic acid hepatotoxicity: hepatic microvesicular steatosis and glutathione status.

It is postulated that the hepatotoxicity of valproic acid (VPA) results from the mitochondrial beta-oxidation of its cytochrome P450 metabolite, 2-propyl-4-pentenoic acid (4-ene VPA), to 2-propyl-(E)-2,4-pentadienoic acid ((E)-2,4-diene VPA) which, in the CoA thioester form, either depletes GSH or produces a putative inhibitor of beta-oxidation enzymes. In order to test this hypothesis, 2-fluoro-2-propyl-4-pentenoic acid (alpha-fluoro-4-ene VPA) which was expected to be inert to beta-oxidative metabolism was synthesized and its effect on rat liver studied in comparison with that of 4-ene VPA. Similarly, the known hepatotoxicant 4-pentenoic acid (4-PA) and 2,2-difluoro-4-pentenoic acid (F2-4-PA) were compared. Male Sprague-Dawley rats (150-180 g, 4 rats per group) were dosed ip with 4-ene VPA (0.7 mmol/kg per day), 4-PA (1.0 mmol/kg per day), or equivalent amounts of their alpha-fluorinated analogues for 5 days. Both 4-ene VPA and 4-PA induced severe hepatic microvesicular steatosis ( > 85% affected hepatocytes), and 4-ene VPA produced mitochondrial alterations. By contrast, alpha-fluoro-4-ene VPA and F2-4-PA were not observed to cause morphological changes in the liver. The major metabolite of 4-ene VPA in the rat urine and serum was the beta-oxidation product (E)-2,4-diene VPA. The N-acetylcysteine (NAC) conjugate of (E)-2,4-diene VPA was also found in the urine. Neither (E)-2,4-diene VPA nor the NAC conjugate could be detected in the rats administered alpha-fluoro-4-ene VPA. In a second set of rats (3 rats per group), total liver GSH levels were determined to be depleted to 56% and 72% of control following doses of 4-ene VPA (1.4 mmol/kg) and equivalent alpha-fluoro-4-ene VPA, respectively. Mitochondrial GSH remained unchanged in the alpha-fluoro-4-ene VPA treated group but was reduced to 68% of control in the rats administered 4-ene VPA. These results strongly support the theory that hepatotoxicity of 4-ene VPA, and possibly VPA itself, is mediated largely through beta-oxidation of 4-ene VPA to reactive intermediates that are capable of depleting mitochondrial GSH.

Selective depletion of mitochondrial glutathione concentrations by (R,S)-3-hydroxy-4-pentenoate potentiates oxidative cell death.

The hepatocellular glutathione content is partitioned into a cytosolic pool, which accounts for about 85% of the cellular glutathione content, and a mitochondrial pool, which accounts for about 15% of the cellular glutathione content. Previous studies indicated that the mitochondrial glutathione pool may play a critical role in cytoprotection against xenobiotic-induced cell damage. Tests of the role of mitochondrial glutathione in cytoprotection have been hampered by the lack of agents that selectively deplete the mitochondrial glutathione pool. To test the hypothesis that mitochondrial glutathione plays a critical role in protecting against cytotoxic agents, we developed a method to deplete selectively mitochondrial glutathione concentrations. (R,S)-3-Hydroxy-4-pentenoate, an analog of (R)-3-hydroxybutanoate, caused a rapid and selective depletion of mitochondrial glutathione concentrations. Incubation of (R,S)-3-hydroxy-4-pentenoate with rat liver mitochondria or with 3-hydroxybutyrate dehydrogenase in the presence of glutathione afforded a glutathione conjugate whose chromatographic properties were identical with synthetic S-(3-oxo-4-carboxybutyl)glutathione, indicating that (R,S)-3-hydroxy-4-pentenoate was oxidized to the Michael acceptor 3-oxo-4-pentenoate, which reacts with glutathione. Exposure of rat hepatocytes to (R,S)-3-hydroxy-4-pentenoate, which was not cytotoxic and did not induce mitochondrial dysfunction, potentiated the cytotoxicity of tert-butyl hydroperoxide. These results establish the critical role of mitochondrial glutathione in cytoprotection and demonstrate and (R,S)-3-hydroxy-4-pentenoate may find utility in exploring mitochondrial glutathione homeostasis.