Advancing ocular safety research: A comprehensive examination of benzocaine acute exposure without animal testing.

Benzocaine is a widely employed local anaesthetic; however, there is a notable dearth of preclinical and clinical evidence regarding its safety in ophthalmological products. To address this, a comprehensive strategy incorporating in silico and in vitro methodologies was proposed for assessing benzocaine's ocular toxicity without animal testing. To collect the in silico evidence, the QSAR Toolbox (v4.5) was used. A single exposure to two benzocaine concentrations (2% and 20%) was evaluated by in vitro methods. Hen's Egg Chorioallantoic Membrane Test (HET-CAM) was performed to evaluate the effects on the conjunctiva. To study corneal integrity, Short Time Exposure test (STE) and Bovine Corneal Opacity and Permeability (BCOP) assay, followed by histopathological analysis, were carried out. Results from both in silico and in vitro methodologies categorize benzocaine as non-irritating. The histopathological analysis further affirms the safety of using benzocaine in eye drops, as no alterations were observed in evaluated corneal strata. This research proposes a useful combined strategy to provide evidence on the safety of local anaesthetics and particularly show that 2% and 20% benzocaine solutions do not induce eye irritation or corneal damage, supporting the potential use of benzocaine in the development of ophthalmic anesthetic products.

Accuracy of the First Stage of Hepatotoxicity Testing with the Use of Drosophila melanogaster Test System.

The genotoxic effects of heterocyclic compounds were evaluated on the basis of genetic and toxicological characteristics of a biological model of Drosophila melanogaster. Analysis of the viability parameters (fertility, progeny mortality) showed that of 6 tested substance, substance No. 3 exhibited minimum toxicity. After application of substances No. 1 and No. 5 in the studied concentrations, the number of survived flies was insufficient for further analysis, which attested to high toxicity of these substances. The intensity of apoptosis was studied in response to substances Nos. 2, 4, and 6. Substance No. 4 proved to be optimal by the parameter toxicity/apoptosis (low toxicity/high apoptosis), while substance No. 3 exhibited low toxicity, which manifested in low apoptosis intensity.

Benzocaine-induced stress in the euryhaline teleost, Centropomus parallelus and its implications for anesthesia protocols.

The use of anesthetic in fish farming is a traditional practice which aims to reduce the stress caused by transport and handling. However, anesthesia-induction protocols are commonly established and implemented without proper physiological/behavioral evaluation. Additionally, concentration and time of exposure to the anesthetic are often set without considering species-specific responses. The fat snook (Centropomus parallelus) is a fish with great potential for aquaculture. Given its remarkable euryhalinity, it can grow in fresh- or seawater. Most studies on fat snook anesthesia tested natural compounds (essential oils) instead of traditional anesthetics. However, the use of benzocaine is much more common in the commercial sector, as it is easy to obtain and of relatively low cost. The present study aimed at analyzing the effects benzocaine exposure on glucose and cortisol plasma levels (two traditional stress markers in teleost fish), as well as on plasma osmolality, chloride and magnesium, (indicators of osmo-ionic allostasis) in animals acclimated to different salinities. Results showed that while osmo-ionic allostasis was strictly maintained across the treatments, time of anesthesia had a strong positive relationship to plasma cortisol and glucose, regardless the salinity of exposure and acclimation. The results are discussed as they relate to anesthesia protocols and how stress response generated by time of anesthesia may challenge farming flexibility.

Environmental behavior of 12 UV filters and photocatalytic profile of ethyl-4-aminobenzoate.

Ethyl-4-aminobenzoate (Et-PABA) is currently used as a substitute for 4-aminobenzoate (PABA) in sunscreens and anesthetic ointments. Despite its widespread use and hydrophilicity, Et-PABA has never been found in environmental waters. This study, probed the occurrence of Et-PABA in both seawater and drinking water sources in Hong Kong, and evaluated its transformation products (TPs) and environmental fate via cumulative potency and photocatalytic profile analyses. Another 11 UV filters used in skin-care products were also studied. Et-PABA was not detected in any water sample. Four other UV filters were dominant at ng/L level in both seawater and drinking water sources. UHPLC-QTOF-MS was used to elucidate the structure of TPs. With high resolution accurate mass data and fragment rationalization, 11 Et-PABA TPs were characterized, including seven intermediates firstly proposed as TPs; two compounds were reported for the first time. It is proposed that photocatalysis induces transformation pathways of (de)hydroxylation, demethylation and molecular rearrangement. Luminescent bacteria tests showed decreasing toxicity with increasing irradiation of Et-PABA, suggesting that irradiation TPs are less toxic than the parent compound. Transformation of Et-PABA appears to explain why Et-PABA has not been detected in the natural environment.

Liposomal butamben gel formulations: toxicity assays and topical anesthesia in an animal model.

The aim of this study was to evaluate the in vitro cytotoxicity and the in vivo analgesic effect and local toxicity of the local anesthetic butamben (BTB) encapsulated in conventional or elastic liposomes incorporated in gel formulations. The results showed that both gel formulations of liposomal BTB reduced the cytotoxicity (p < 0.001; one-way ANOVA/Tukey's test) and increased the topical analgesic effect (p < 0.05; one-way ANOVA/Tukey's test) of butamben, compared to plain BTB gel. The gel formulations presented good rheological properties, and stability assays detected no differences in physicochemical stability up to 30 d after preparation. Moreover, histological assessment revealed no morphological changes in rat skin after application of any of the gel formulations tested.

Identification of N-Hydroxy-para-aminobenzoic acid in a cyanotic child after benzocaine exposure.

CONTEXT: Methemoglobinemia (MetHb) after exposure to benzocaine (BZC) has been reported for more than 50 years, however the pathophysiologic mechanism has not been previously established. Direct administration of BZC to blood does not produce MetHb. After topical use, due to the lipophilicity and rapid acetylation in the tissue, little BZC reaches the liver for hepatic biotransformation. However, isolated human livers have been shown to produce MetHb forming N-hydroxyl metabolites from BZC. We report a case of BZC-induced MetHb with the first identification and quantification of the reactive metabolite responsible for the oxidative stress: N-Hydroxy-Para-amino benzoic acid (N-OH-PABA). CASE DETAILS: An 8 year old male was admitted to a hospital for an appendectomy. Several applications of BZC spray were used during multiple attempts at nasogastric tube placement. In various attempts to achieve local anesthesia, benzocaine spray was used in both nares and through the mouth aimed at the posterior oropharynx. The patient subsequently became cyanotic with an initial MetHb level of 32.9 %. Methylene blue was administered and the patient promptly responded with resolution of cyanosis. Blood taken within 20 min of the initial symptoms contained benzocaine (5.2ug/mL), bupivacaine (740ng/mL), lidocaine (530ng/mL), acetaminophen (12ug/mL), midazolam (60ng/mL), PABA and N-OH-PABA (35ng/mL). Serum was analyzed using Liquid Chromatography- Quadrupole Time-of-Flight Mass Spectrometry. Mass spectrometry was done using an electrospray ionization source run in negative and positive polarities. A reference standard for N-OH-PABA was synthesized for confirmation and quantification. DISCUSSION: The rare and idiopathic nature of methemoglobinemia after benzocaine use has made study of the pathophysiologic mechanism in humans difficult. Lack of understanding has brought calls for restriction of use of the widely used medication that may not be based on evidence. Our case presents several unique features: 1) benzocaine absorption after topical administration was documented with serum concentrations 2) confirmation of an in vivo formation of MetHb-forming n-hydroxyl-metabolite after benzocaine use and 3) the documentation of N-OH-PABA in humans within 20 min of MetHb post-benzocaine administration.

More methemoglobin is produced by benzocaine treatment than lidocaine treatment in human in vitro systems.

The clinical use of local anesthetic products to anesthetize mucous membranes has been associated with methemoglobinemia (MetHba), a serious condition in which the blood has reduced capacity to carry oxygen. An evaluation of spontaneous adverse event reporting of MetHba submitted to FDA through 2013 identified 375 reports associated with benzocaine and 16 reports associated with lidocaine. The current study was performed to determine the relative ability of benzocaine and lidocaine to produce methemoglobin (MetHb) in vitro. Incubation of 500µM benzocaine with whole human blood and pooled human liver S9 over 5h resulted in MetHb levels equaling 39.8±1.2% of the total hemoglobin. No MetHb formation was detected for 500µM lidocaine under the same conditions. Because liver S9 does not readily form lidocaine hydrolytic metabolites based on xylidine, a primary metabolic pathway, 500µM xylidine was directly incubated with whole blood and S9. Under these conditions MetHb levels of 4.4±0.4% were reached by 5h. Studies with recombinant cytochrome P450 revealed benzocaine to be extensively metabolized by CYP 1A2, with 2B6, 2C19, 2D6, and 2E1 also having activity. We conclude that benzocaine produces much more MetHb in in vitro systems than lidocaine or xylidine and that benzocaine should be more likely to cause MetHba in vivo as well.

The in vitro and in vivo genotoxicity of benzocaine: a brief communication.

Benzocaine has a long history of use in human medicine. However, benzocaine also has been used in aquaculture with finfish for more than 40 years for sedating fish for marking, transport, surgery, and so on, although benzocaine does not have a current Food and Drug Administration (FDA) approval for this application in the United States. As part of a FDA approval for use as an animal drug, the genotoxicity of benzocaine was evaluated in the in vitro bacterial reverse mutation assay and the forward mutation assay and in vivo in the mouse micronucleus assay. These studies were conducted in compliance with Good Laboratory Practice regulations and according to Veterinary International Conference on Harmonisation guidelines. Based on the results of these studies, benzocaine was determined not to be genotoxic.

Benzocaine-induced methemoglobinemia in an acute-exposure rat model.

Tricaine methanesulfonate, a sedative for temporarily immobilizing fish, has a 21-day withdrawal time. Benzocaine has been proposed as an alternative sedative because a withdrawal period may not be required. Since benzocaine is known to induce methemoglobinemia, the potential for orally administered benzocaine to induce methemoglobin was assessed in rats. Sprague-Dawley rats were given a single gavage administration of 64mg benzocaine hydrochloride per kg bw and then euthanized at intervals up to 120min. Plasma levels of benzocaine were relatively low at all times, whereas methemoglobin peaked at 24min. Additional rats were orally gavaged with 0-1024mg benzocaine hydrochloride per kg bw and euthanized after 24min. Plasma levels of benzocaine increased from 0.01µM at 2mg per kg bw to 2.9µM at 1024mg per kg bw. Methemoglobin levels did not differ from controls at doses up to 32mg per kg bw in females and 64mg per kg bw in males, whereupon the value increased to ∼80% at 1024mg per kg bw. These data were used to estimate the potential impact of benzocaine residues in fish and suggest that the consumption of fish treated with benzocaine hydrochloride will not cause methemoglobinemia in humans.

Estrogenic activity of UV filter mixtures.

UV-absorbing chemicals (UV filters) are widely used for protection against UV radiation in sunscreens and in a variety of cosmetic products and materials. Depending on the breadth and factor of UV protection, they are added as single compounds or as a combination thereof. Some UV filters have estrogenic activity, but their activity and interactions in mixtures are largely unknown. In this work, we analyzed 8 commonly used UV filters, which are pure or partial hERalpha agonists, for their estrogenic activity in equieffective mixtures in a recombinant yeast assay carrying the human estrogen receptor alpha (hERalpha). Mixtures of two, four and eight UV filters alone, or in combination with 17 beta estradiol (E2), were assessed at different effect levels and no-observed-effect-concentrations (NOEC). Predictions of the joint effects of these mixtures were calculated by employing the concentration addition (CA) and independent action (IA) model. Most binary mixtures comprising of pure hERalpha agonists showed a synergistic activity at all mixture combinations. Only in combination with benzophenone-1, antagonistic activity was observed at some effect levels. All mixtures of four or eight, pure or pure and partial hERalpha agonists, alone or including E2, showed synergistic activity at concentrations giving an increase of 10% of basal activity (BC10). This occurred even at concentrations that were at the NOEC level of each single compound. Hence, there were substantial mixture effects even though each UV filter was present at its NOEC level. These results show that significant interactions occur in UV filter mixtures, which is important for the hazard and risk assessments of these personal care products.

Multiple hormonal activities of UV filters and comparison of in vivo and in vitro estrogenic activity of ethyl-4-aminobenzoate in fish.

UV filters have been detected in surface water, wastewater and fish, and some of them are estrogenic in fish. At present, little is known about their additional hormonal activities in different hormonal receptor systems despite their increasing use and environmental persistence. Besides estrogenic activity, UV filters may have additional activities, both agonistic and antagonistic in aquatic organisms. In our study, we investigate a series of UV filters for multiple hormonal activities in vitro in human receptor systems and evaluate the predictive value of these findings for the activity in fish in vitro and in vivo. First we systematically analysed the estrogenic, antiestrogenic, androgenic, and antiandrogenic activity of 18 UV filters and one metabolite in vitro at non-cytotoxic concentrations with recombinant yeast systems carrying either a human estrogen (hERalpha) or androgen receptor (hAR). All 19 compounds elicited hormonal activities, surprisingly most of them multiple activities. We found 10 UV-filters having agonistic effects towards the hERalpha. Surprisingly, we identified for the first time six UV filters with androgenic activities and many of them having pronounced antiestrogenic and antiandrogenic activities. As much as 17 compounds inhibited 4,5-dihydrotestosterone activity in the hAR assay, while 14 compounds inhibited estradiol activity in the hERalpha assay, indicating antiandrogenic and antiestrogenic activity, respectively. In particular, the antiandrogenic activities of phenyl- and benzyl salicylate, benzophenone-1 and -2, and of 4-hydroxybenzophenone were higher than that of flutamide, a known hAR antagonist. In a second series of experiments, we investigated the predictive power of the hERalpha assay for aquatic organisms by further investigating the estrogenic UV filter ethyl 4-aminobenzoate (Et-PABA) in vitro and in vivo in fish. Et-PABA showed estrogenic activity in a recombinant yeast system carrying the rainbow trout estrogen receptor (rtERalpha) with higher activity than in the hERalpha assay. In addition, Et-PABA induced vitellogenin after 14 days of exposure in juvenile fathead minnows at 4394mug/L. Our study shows estrogenic activity of this UV filter in fish both in vitro and in vivo. In conjunction with in vitro human receptor-based systems our results give a more detailed picture about distinct hormonal activities of UV filters occurring in aquatic systems. We conclude that receptor-based assays are important for in vitro assessment of UV-filters prior to or concurrently with in vivo assays, which ultimately provide data for the environmental risk assessment of these important personal care products.

Physico-chemical characterization of benzocaine-beta-cyclodextrin inclusion complexes.

Local anesthetics are able to induce pain relief by binding to the sodium channel of excitable membranes, blocking the influx of sodium ions and the propagation of the nervous impulse. Benzocaine (BZC) is a local anesthetic whose low water-solubility limits its application to topical formulations. The present work focuses on the characterization of inclusion complexes of BZC in beta-cyclodextrin (beta-CD). Differential scanning calorimetry and electron microscopy gave evidences of the formation and the morphology of the complex. Fluorescence spectroscopy showed a BZC/beta-CD 1:1 stoichiometry. Phase-solubility diagrams allowed the determination of the association constants between BZC and beta-CD (549 M(-1)) and revealed that a three-fold increase in BZC solubility can be reached upon complexation with beta-CD. The details of BZC/beta-CD molecular interaction were analyzed by 1H 2D NMR allowing the proposition of an inclusion model for BZC into beta-CD where the aromatic ring of the anesthetic is located near the head of the beta-CD cavity. Moreover, in preliminary toxicity studies, the complex seems to be less toxic than BZC alone, since it induced a decrease in the in vitro oxidation of human hemoglobin. These results suggest that the BZC/beta-CD complex represents an effective novel formulation to enhance BZC solubility in water, turning it promising for use outside its traditional application, i.e., in infiltrative anesthesia.

Anesthesia of fish with benzocaine does not interfere with comet assay results.

Fish blood erythrocytes are frequently used as sentinels in biomonitoring studies. Usually, fish blood is collected by painful cardiac or caudal vein punctures. Previous anesthesia could decrease animal suffering but it is not known at present whether anesthesia can cause confounding effects. Therefore, using the alkaline single cell gel (SCG)/comet assay with blood erythrocytes of the cichlid fish Nile tilapia, we tested for a possible modulation of induced DNA damage (methyl methanesulfonate; MMS) by the anesthetic benzocaine administered by bath exposure (80mg/l for approximately 10min). Furthermore, benzocaine (80-600mg/l) was tested for its genotoxic potential on fish erythrocytes in vitro and for potential interactions with two known genotoxins (MMS and hydrogen peroxide). Our results did neither indicate a significant increase in the amount of DNA damage (even after a 48h follow-up), nor indicated interactions with MMS-induced DNA damage when fish were exposed to benzocaine in vivo. There was also no increase in DNA damage after in vitro exposure of fish erythrocytes to benzocaine. Clear concentration-related effects were observed for the two genotoxins in vitro, which were not significantly altered by the presence of benzocaine. These results suggest that anesthesia of fish does not confound comet assay results and the use of blood samples from anesthetized fish can be recommended with regard to animal welfare.

Effect of prolonged exposure to low antigen concentration for sensitization.

The local lymph node assay (LLNA) is an assay in mice to identify potential allergens. Compounds that do not induce a stimulation index (SI)>or=3 are not considered sensitizers. Of the chemicals that do, the SI of 3 is used as a benchmark, and indicates the sensitizing potency of a chemical. Compared to the exposure duration of the LLNA (3 days), real life exposure often lasts for months or years. We therefore investigated whether prolonged exposure to sensitizers at concentrations that do not induce a SI>or=3 in the LLNA, were able to surpass this threshold. Mice were treated for 2 months at 7-day intervals with a range of concentrations of the known allergens ethyl-p-aminobenzoate (benzocaine, BENZ), 2,4-dinitrochlorobenzene (DNCB), and tetramethyl thiuram disulfide (TMTD). Both proliferative activity and cytokine production were established at day 60. Neither BENZ nor TMTD showed a significant increase in the proliferation rate compared to vehicle controls. Only DNCB at concentrations originally above the EC(3) a significant increase in proliferation was seen after prolonged exposure. No significant effect on IFN-gamma and IL-4 production was observed for all three compounds compared. These findings indicate that for classification of sensitizers the shorter exposure period employed in the standard LLNA is sufficient, and longer periods of exposure have no bearing on this classification.

Local lymph node assay with non-radioisotope alternative endpoints.

The local lymph node assay has recently been accepted by regulatory agencies as a stand-alone alternate method for predicting allergic contact dermatitis. To compare the sensitivity of non-radioisotope methods with that of the standard assay, we determined if these modified methods would affect evaluation of sensitization potency. For this reason, we used 2,4-dinitrochlorobenzene (DNCB) and benzocaine for different sensitizing criteria. Female CBA mice were treated for 3 days with a test compound or vehicle applied to each side of both ears. Bilateral auricular lymph node proliferative activity was assessed by the following endpoints with incorporation of 3H-methyl thymidine (3H-TdR), bromodeoxyuridine (BrdU) in vivo, and BrdU ex vivo, IL-2 production, and proliferating cell nuclear antigen (PCNA) expression. Ear thickness was also tested. The strong sensitizer DNCB was detectable by any of the non-radioisotope endpoints as well as by radioisotope-dependent standard assay. On the other hand, when evaluating the weak sensitizer benzocaine, significant changes were evident in BrdU incorporation ex vivo and in vivo, and IL-2 production. We believe that these non-radioisotope methods can assess allergic contact dermatitis caused by chemicals even in the laboratory, where it can be difficult to handle radioisotopes.

Comparison of 4 topical anesthetic agents for effect and corneal toxicity in rabbits.

PURPOSE: To compare the onset time, duration of action, corneal toxicity, and corneal epithelial healing time of 4 topical anesthetic agents in rabbits. SETTING: Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada. METHOD: Fifty-six rabbits were treated with 4 topical anesthetics (bupivacaine, lidocaine, procaine, and benzocaine) at different concentrations and different pH of solutions. Corneal sensation, corneal toxicity, and corneal epithelial healing time were measured. RESULTS: The onset time of all 4 anesthetic agents was within 1 minute; however, bupivacaine and lidocaine produced significantly longer action than procaine or benzocaine (P < .05). Buffered bupivacaine and lidocaine had a significantly longer anesthetic effect than that of the nonbuffered solutions (P < .05). No significant effect on corneal epithelial healing time or corneal toxicity was observed. CONCLUSION: Topical bupivacaine and lidocaine had a longer anesthetic effect, particularly in buffered solutions. No significant corneal toxicity was observed.

Skin sensitization testing: the relevance of rechallenge and pretreatment with sodium lauryl sulfate in the guinea pig maximization test.

The guinea pig maximization test is one of the preferred test methods for the identification of skin sensitizers. The OECD/EC test guidelines allow for the conduct of a rechallenge in case doubtful reactions are obtained after challenge. The relevance of rechallenging was investigated by performing multiple challenges (up to four) in the maximization test with four well-known sensitizers of varying strength: nickel sulfate, sulfathiazole, benzocaine, and 1-chloro-2,4-dinitrobenzene. In addition, the effect of sodium lauryl sulfate (SLS)-pretreatment during topical induction with weak sensitizers on rechallenging was investigated. In contrast to what has frequently been hypothesized, rechallenge did not result in an increase of skin reaction as compared with the reactions observed after the first treatment. SLS pretreatment was very effective in increasing the initial challenge response to weak sensitizers. Subsequent rechallenging in these cases however again showed a decrease in sensitivity of the animals.

Evaluation of lymphocyte proliferation by immunohistochemistry in the local lymph node assay.

A modified version of the local lymph node assay (LLNA) is presented, using bromodeoxyuridine to label proliferating lymphocytes. Cell counting is done on mid-sagittal sections of individual nodes under light microscopy. Two irritants (sodium lauryl sulfate and salicylic acid), four allergens of various sensitizing potential (potassium dichromate, 4-chloroaniline, neomycin sulfate and nickel sulfate) and one chemical of unknown sensitizing potential (ethyl 3-aminobenzoate) were tested either in the short protocol using three daily topical applications and/or in the long protocol with a pre-exposure step under an occluded patch. A weak T cell proliferation was noted with both irritants in the short protocol, but not in the long protocol. Potassium dichromate induced a strong proliferative response in the short protocol. A lesser sensitizing potential was detected for 4-chloroaniline, ethyl 3-aminobenzoate and neomycin sulfate but only in the long protocol. Nickel sulfate was negative in both protocols. The long protocol was the most valuable for weak or moderate sensitizers. Histological examination of nodes ruled out intercurrent processes. The present procedure offers several advantages. The use of a non-isotopic marker enables this test to be run in a routine safety assessment department and allows the preparation of permanent slides. An increased specificity is obtained by restricting cell counting to the paracortex. Moreover, the collection of individual data permits statistical analysis of the results. This method is sensitive and reproducible and may be viewed as a useful adjunct to the LLNA.