Prior Exposure to Ortho-Phthalaldehyde Augments IgE-Mediated Immune Responses to Didecyldimethylammonium Chloride: Potential for 2 Commonly Used Antimicrobials to Synergistically Enhance Allergic Disease.

Health-care workers have an increased incidence of allergic disease compared with the general public and are exposed to a variety of high-level disinfectants. Although exposure to these agents has been associated with allergic disease, findings between epidemiology and animal studies often conflict respecting immunological mechanisms. Therefore, we hypothesized that previous exposure to a representative IgE-mediated sensitizer (ortho-phthalaldehyde [OPA]) alters immune responses to a representative T-cell-mediated sensitizer (didecyldimethlyammonium chloride [DDAC]). Here, BALB/c mice were topically exposed to OPA (0.5%) for 3 days, rested, then topically exposed to DDAC (0.0625%, 0.125%, and 0.25%) for 14 days. Coexposure resulted in phenotypic changes in draining lymph node (dLN) cells, including a decreased frequency of CD8+ T cells and increased frequency and number of B cells compared with DDAC-only treated mice. The coexposed mice also had enhanced Th2 responses, including significant alterations in: dLN Il4 (increased), B-cell activation (increased), CD8+ T-cell activation (decreased), and local and systemic IgE production (increased). These changes were not observed if mice were exposed to DDAC prior to OPA. Exposure to OPA alone shows Th2 skewing, indicated by increased activation of skin type 2 innate lymphoid cells, increased frequency and activation of draining lymph node B cells, and increased levels of type 2 cytokines. These findings suggest that the OPA-induced immune environment may alter the response to DDAC, resulting in increased IgE-mediated immune responses. This data may partially explain the discordance between epidemiological and laboratory studies regarding disinfectants and provide insight into the potential immunological implications of mixed chemical exposures.

Differentiating between Testicular Toxicity and Sexual Immaturity in Ortho-phthalaldehyde Inhalation Toxicity Studies in Rats and Mice.

Early deaths of young or juvenile animals (before sexual maturation is achieved) in routine regulatory safety studies present pathologists and toxicologists with the challenge of interpreting findings in the male reproductive tract. Additionally, the advent of toxicity testing regulations has resulted in a growing need for the use of juvenile animals in toxicology studies. Here, we present the reproductive toxicity findings from a 13-week inhalation toxicity study with ortho-phthalaldehyde (OPA) in male rats and mice as a case example for working through this challenging task. In this study with OPA, survival was significantly reduced in the two highest exposure concentrations of OPA tested. Early deaths and histopathological lesions in the testes and epididymides were generally also limited to these two highest exposure groups. Therefore, there was concern that peripubertal morphological features could be a confounding factor for the histopathological evaluation of exposure-related testicular and epididymal findings. Although it can be difficult to differentiate exposure-related effects from the normal morphological features defining peripubertal changes in the testes and epididymides in animals that die early in a toxicity study, the use of age-matched controls in this case study with OPA provided a reference and aided in the differentiation of these effects.

Evaluation of the respiratory tract toxicity of ortho-phthalaldehyde, a proposed alternative for the chemical disinfectant glutaraldehyde.

ortho-Phthalaldehyde (OPA) is a high-level chemical disinfectant that is commonly used for chemical sterilization of dental and medical instruments as an alternative to glutaraldehyde, a known skin and respiratory sensitizer. Concern for safe levels of human exposure remains due to a lack of toxicity data as well as human case reports of skin and respiratory sensitization following OPA exposure. The present study evaluated the inhalational toxicity of OPA in Harlan Sprague-Dawley rats and B6C3F1/N mice. Groups of 10 male and female rats and mice were exposed to OPA by whole-body inhalation for 3 months at concentrations of 0 (control), 0.44, 0.88, 1.75, 3.5, or 7.0 ppm. Rats and mice developed a spectrum of lesions at sites of contact throughout the respiratory tract (nose, larynx, trachea, lung), as well as in the skin and eye, consistent with a severe irritant response. In general, histologic lesions (necrosis, inflammation, regeneration, hyperplasia and metaplasia) occurred at deeper sites within the respiratory tract with increasing exposure concentration. As a first site of contact, the nose exhibited the greatest response to OPA exposure and resulted in an increased incidence, severity and variety of lesions compared to a previous study of glutaraldehyde exposure at similar exposure concentrations. This increased response in the nasal cavity, combined with extensive lesions throughout the respiratory tract, provides concern for use of OPA as a replacement for glutaraldehyde as a high-level disinfectant.

Comparative Cytotoxicity and Sperm Motility Using a Computer-Aided Sperm Analysis System (CASA) for Isomers of Phthalic Acid, a Common Final Metabolite of Phthalates.

The general population is exposed to phthalates through consumer products, diet, and medical devices. Phthalic acid (PA) is a common final metabolite of phthalates, and its isomers include isophthalic acid (IPA), terephthalic acid (TPA), and phthalaldehyde (o-phthalic acid, OPA). The purpose of this study was to investigate whether PA and PA isomers exert reproductive toxicity, including altered sperm movement. In vitro cell viability assays were comparatively performed using Sertoli and liver cell lines. In animal experiments, PA or PA isomers (10, 100, or 1000 mg/kg) were administered orally to Sprague-Dawley (SD) rats, and semen samples were analyzed by computer-aided sperm analysis (CASA). PA treatment produced a significant effect on curvilinear velocity (VCL), straight-line velocity (VSL), mean velocity or average path velocity (VAP), amplitude of lateral head displacement (ALH), and frequency of head displacement or beat cross-frequency (BCF), whereas IPA, TPA, and OPA induced no marked effects. In vitro cell viability assays showed that mouse normal testis cells (TM4) and human testis cancer cells (NTERA 2 cl. D1) were more sensitive to PA and OPA than mouse liver normal cells (NCTC clone 1469) and human fetal liver cells (FL 62891). Our study suggests that PA and PA isomers specifically produced significant in vitro and in vivo reproductive toxicity, particularly sperm toxicity and testis cell cytotoxicity. Of the isomers examined, PA appeared to be the most toxic and may serve as a surrogate biomarker for reproductive toxicity following mixed exposure to phthalates.

Developmental neurotoxicity of ortho-phthalate diesters: review of human and experimental evidence.

Ortho-phthalate diesters, or phthalates, are widely used synthetic chemicals found primarily in consumer products and polyvinyl chloride plastics. Experimental evidence suggests that several phthalates possess antiandrogenic properties and may disrupt endocrine pathways resulting in abnormal reproductive outcomes. Low-level exposure to phthalates has been well documented in humans, with higher levels found in children and women of childbearing age. Recent epidemiologic studies postulate that prenatal exposure to measurable urine phthalate concentrations may be associated with altered genital and pubertal development in infants and children. This review addresses the emerging evidence that some phthalates may have an adverse impact on the developing brain. The supporting animal studies and proposed mechanisms underlying the deleterious properties of phthalates in relation to neurodevelopmental outcomes are also discussed. While the observed associations are based on limited studies with a broad range of endpoints, the implications of such outcomes are of concern from a public health standpoint and merit further investigation given the widespread nature of the exposure.

Cytotoxicity assessment of residual high-level disinfectants.

Some studies show the uptake of disinfectants on medical devices but no studies on their cytotoxicity have been reported. This study aimed to assess that cytotoxicity in a 3-dimensional culture system using HeLa cells grown in matrices composed of collagen. Plastic materials were soaked in the use solutions of the widely used high-level disinfectants, glutaraldehyde (GA), ortho-phthalaldehyde (OPA) and peracetic acid (PAA). After being rinsed, they were allowed to dry and were embedded into the cell medium to investigate the cytotoxicity of the residual disinfectants. Cytotoxicity was observed with the polyvinyl chloride, polyurethane and silicon tubes soaked in GA and OPA, indicating that both disinfectants were absorbed in the test pieces, whereas for PAA, none was observed. As for the polytetrafluoroethylene (PTFE) tubes, no disinfectant displayed cytotoxicity. GA and OPA are primary irritants, having a potential to cause anaphylaxis and other forms of allergic reactions. There should be consideration not only about the toxicity of the residual disinfectant from poor rinsing, but also about the toxicity that would result from the disinfectants that were absorbed and consequently released from the medical devices or materials.

Comparison of the cytotoxicity of high-level disinfectants by the MTT assay and direct contact assay.

Most critical instruments are not designed for heat sterilization and autoclaving. These items are usually treated with chemical agents such as peracetic acid(PAA), glutaraldehyde (GA) and ortho-phthalaldehyde (OPA). MTT assay is often used to evaluate the in vitro cytotoxicity of these chemical agents. In this study, disinfectants were allowed to come in direct contact with cells. Their cytotoxicity was evaluated based on cell viability and adhesive properties. The results obtained from the direct contact method were compared with those obtained from the conventional MTT assay wherein the disinfectants were added into a nutrient medium. It was found that the two methods yielded very different results, especially when aldehyde- and halogen-containing disinfectants were tested, and that toxicity may be underestimated in the MTT assay. Hence, it can be assumed that the direct contact assay is more accurate when evaluating the cytotoxicity of residual chemicals. It was also observed that the cytotoxicity of PAA was lower than that of GA and OPA.

Protective effects of dispersive viscoelastics on corneal endothelial damage in a toxic anterior segment syndrome animal model.

PURPOSE: We evaluated whether viscoelastics have protective effects on the corneal endothelial cell damage in a toxic anterior segment syndrome (TASS) animal model depending on the types of viscoelastics. METHODS: A TASS animal model was established with an injection of 0.1 mL o-phthaldehyde solution (0.14%) into the anterior chamber of New Zealand white rabbits. One of two different viscoelastics, 1% sodium hyaluronate (cohesive group) or a 1:3 mixture of 4% chondroitin sulfate and 3% sodium hyaluronate (dispersive group), was injected into the anterior chamber. After five minutes, it was removed using a manual I/A instrument, and then 0.1 mL of o-phthaldehyde solution (0.14%) was injected into the anterior chamber. Damage to corneal endothelial cells was compared between the two groups. RESULTS: The corneal thickness increased quickly in both groups after the disinfectant injection. However, the dispersive group showed relatively mild corneal edema compared to the cohesive group. The mean corneal haze score in the dispersive group also was lower than that of the cohesive group. These partial protective effects of the dispersive viscoelastic were demonstrated by the different findings of a live/dead cell assay, TUNEL staining, and scanning electron microscopy between the two groups. CONCLUSIONS: The TASS animal model seems to be a useful means to evaluate corneal endothelial cell damage caused by toxic substances to find ways to protect or reduce endothelial cell damage. Dispersive viscoelastics were shown to have partial protective effects against corneal endothelial cell damage caused by a toxic disinfectant.

The alterations in neurotransmitters and their metabolites in discrete brain regions in the rats after inhalation of disinfectant, glutaraldehyde or ortho-phthalaldehyde for 4 weeks.

Glutaraldehyde (GA) and ortho-phtalaldehyde (OPA) have been widely used as major components of disinfectants in hospitals. We evaluated the alterations in GA or OPA in rats after subacute inhalation exposure by determining levels of neurotransmitters (norepinephrine [NE], dopamine [DA], DA metabolites, dihydroxyphenylacetic acid [DOPAC] and homovanillic acid [HVA], indoleamine serotonin [5-HT] and 5-HT metabolite, 5-hydroxyindoleacetic acid [5-HIAA]) in discrete brain regions using high performance liquid chromatography (HPLC) equipped with an electrochemical detector. Female Wistar rats were exposed to 0, 50, 100, or 200 ppb gaseous GA or OPA by inhalation for 1 h per day, 5 d per week for 4 wk. Following the exposure, the brain of each rat was removed and dissected into cerebrum, cerebellum, medulla oblongata, midbrain, corpus striatum and hypothalamus. The neurotransmitters and their metabolites were extracted from each brain region, and determined by HPLC. Regarding GA, the daily water intake of the 50 or the 200 ppb exposed groups was significantly lower than that of the control. DA and 5-HIAA levels in the medulla oblongata among the GA exposed groups were significantly lower than those of the control. For OPA, the mean final body weight and daily food intake of the 100 or 200 ppb exposed groups were significantly lower than those of the control. The mean DA concentrations in the cerebrum in the groups exposed to OPA were significantly lower than those of the control. OPA may modulate DA metabolism in the cerebrum of female rats. The levels GA or OPA that induced alienations in neurotransmitters were comparable to those levels usually found in hospitals, further studies are warranted to evaluate the of safety of disinfectants containing GA or OPA.

Ortho-phthalaldehyde-induced skin mucous membrane damage from inadequate washing.

Because body fluids and blood have a tendency to adhere to transesophageal echo devices, a high level of sterilisation is required when cleaning them. Ortho-phthalaldehyde (OPA) has been widely used in Japan since being approved as a high-level sterilant. The authors report a patient with widespread, severe skin and mucous membrane damage of the lip, tongue, pharynx and oesophagus areas that was attributed to inadequate washing after the sterilisation of a transesophageal echo device with OPA. This patient experienced sequelae, which did not improve after more than 1 year of continuous treatment. When using medical devices sterilised with OPA, the use of a probe cover, when applicable, is recommended and complete washing prior to use is required.

Irritancy and allergic responses induced by topical application of ortho-phthalaldehyde.

Although ortho-phthalaldehyde (OPA) has been suggested as an alternative to glutaraldehyde for the sterilization and disinfection of hospital equipment, the toxicity has not been thoroughly investigated. The purpose of these studies was to evaluate the irritancy and sensitization potential of OPA. The EpiDerm Skin Irritation Test was used to evaluate in vitro irritancy potential of OPA and glutaraldehyde. Treatment with 0.4125 and 0.55% OPA induced irritation, while glutaraldehyde exposure at these concentrations did not. Consistent with the in vitro results, OPA induced irritancy, evaluated by ear swelling, when mice were treated with 0.75%. Initial evaluation of the sensitization potential was conducted using the local lymph node assay at concentrations ranging from 0.005 to 0.75%. A concentration-dependent increase in lymphocyte proliferation was observed with a calculated EC3 value of 0.051% compared to that of 0.089%, previously determined for glutaraldehyde. Immunoglobulin (Ig) E-inducing potential was evaluated by phenotypic analysis of draining lymph node (DLN) cells and measurement of total and specific serum IgE levels. The 0.1 and 0.75% exposed groups yielded significant increases in the IgE+B220+ cell population in the lymph nodes while the 0.75% treated group demonstrated significant increases in total IgE, OPA-specific IgE, and OPA-specific IgG(1). In addition, significant increases in interleukin-4 messenger RNA and protein expression in the DLNs were observed in OPA-treated groups. The results demonstrate the dermal irritancy and allergic potential of OPA and raise concern about the proposed/intended use of OPA as a safe alternative to glutaraldehyde.

Ortho-phthalaldehyde exposure levels among endoscope disinfection workers.

OBJECTIVES AND METHODS: Recently, the use of ortho-phthalaldehyde (OPA) has been increasing as an alternative to glutaraldehyde for endoscope disinfection. To better understand OPA exposure and its health effects among disinfection workers, we conducted environmental monitoring and administered a questionnaire in 17 endoscope disinfection rooms. There were 9 manual disinfection rooms using immersion vats for scope disinfection and 8 automatic rooms using automatic washers. RESULTS: OPA exposure concentration during the disinfection process of scope was significantly higher in the manual group (median: 1.43ppb, range: not detected (ND-5.37ppb) than in the automatic group (median: 0.35 ppb, range: ND-0.69 ppb). Similarly, during charging and discharging the antiseptic solution, OPA levels were significantly higher in the manual group (median: 2.58 ppb, range: 0.92-10.0 ppb) than in the automatic group (median: 0.46ppb, range: ND-1.35 ppb). Time-weighted averages of OPA exposure concentration during work shifts were 0.33 to 1.15 ppb (median 0.66 ppb) in the manual group and 0.13 to 1.28 ppb (median 0.33 ppb) in the automatic group, which suggests that manual workers are exposed to OPA at higher levels. Among 80 female disinfection workers who used only antiseptic solutions containing OPA, the incidence of disinfection-related complaints were 10% skin, 9% eye, and 16% respiratory symptoms. CONCLUSIONS: These findings suggest that it is desirable to introduce automatic washers to decrease OPA exposure levels among disinfection workers.

Acute inflammation and immunoresponses induced by ortho-phthalaldehyde in mice.

ortho-Phthalaldehyde (OPA) has been used as a safe alternative disinfectant instead of glutaraldehyde; however, recently some adverse effects of OPA were reported in patients and medical professions. We examined the acute toxicity of OPA in male ICR mice injected with 0.125-0.5% OPA and killed some animals 1 day after a single OPA injection, and others 1 or 13 days after two OPA injections 5 days apart. Hematology, blood cell counts, specific antibody production, organ weights, hepatic enzymes, hepatic histopathology and gene expression of cytochrome P450 (CYP) mRNA in liver were examined. Single OPA injections elevated leukocyte counts, the proportion of neutrophils, alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Two OPA injections dose-dependently increased leukocyte counts, the proportion of neutrophils, ALT and AST, and decreased alkaline phosphatase. Leukocyte counts and proportions of neutrophils normalized 13 days after the second of two injections. However, both ALT and AST remained high in mice given higher OPA doses. Significant increased liver-to-body weight ratio and mild hepatic lesions were observed. Gene expression of CYP1a1 and CYP2e1 revealed a tendency of up-regulation 1 day after two OPA injections. However, expression of these genes was then down-regulated 13 days after OPA injections. OPA induced specific IgE and IgG significantly in the sera, suggesting that OPA acts as a hapten. Overall, OPA caused acute inflammation and acted as a haptenic allergen, although it caused only mild liver injury. Such evidence suggested that careful washing and prevention of exposure were needed after OPA disinfection of medical instruments.

ortho-Phthalaldehyde enhances allergen-specific IgE production without allergen-specific IgG in ovalbumin-sensitized mice.

ortho-Phthalaldehyde (OPA) is commonly used as a safer and more effective chemical disinfectant for use with medical devices in hospitals. However, the cases of patients with occupational bronchial asthma or contact dermatitis are recently reported among workers in the medical professions who were exposed to OPA disinfectant. Mechanism of allergic reaction associated with OPA is poorly understood. The purpose of this study is that OPA may act as an immunological adjuvant in the allergic reaction accompanied by enhanced specific-IgE production in response to allergen challenge in OVA-sensitized mice. OPA induced increase of total cell numbers, and reflected infiltration of neutrophils in BAL fluid after allergen challenge in sensitized mice, dose-dependently. However, total protein concentration in BAL fluid did not change in the all of groups. The OPA induced up-regulation of eotaxin and monocyte chemotactic protein-1 mRNAs in the lung as well as the increase in OVA-specific IgE in sensitized mice compared with non-sensitized controlled mice without increase in the level of OVA-specific IgG. Cytokines IL-4 and IL-5 mRNA were expressed by allergen (OVA) challenge in both lungs collected from OPA-administrated-sensitized and OPA-administrated-nonsensitized mice. From these data, we concluded that low concentration of OPA that enhanced the OVA-induced recruitment of neutrophils to the lung and the production of allergen-specific IgE, suggesting that OPA acts as an immunological adjuvant.

Corneal and conjunctival toxicity of disinfectants--assessing safety for use with ophthalmic surgical instruments.

We investigated the corneal toxicity of ortho-phthalaldehyde (Cidex OPA, Johnson and Johnson K.K.) and its predecessor glutaraldehyde (Cidex, Johnson and Johnson K.K.). We made primary cultures of porcine and human corneal endothelial cells. Commercially available cell lines were also used including human, bovine, and rabbit corneal epithelium and human conjunctival cells. Following incubation for two days, cell survival was measured using a WST-1 assay for endothelia and a MTT assay for the other cells. Test solutions included 2.25% and 3.5% glutaraldehyde and 0.55% ortho-phthalaldehyde. Cell survival was presented as a percentage of the control value. ortho-phthalaldehyde displayed less toxicity than glutaraldehyde for all cell types tested. As expected 3.5% glutaraldehyde was slightly more toxic than 2.25% glutaraldehyde. When primary human corneal endothelial cultures were exposed to ortho-phthalaldehyde, the survival rates were 88% for 100-fold dilutions and 95% for 500-fold dilutions. The survival rates for all cells tested were greater than 90% when dilutions of 1000-fold or more were used. In conclusion, the corneal toxicity of glutaraldehyde and ortho-phthalaldehyde appears to be within safe levels following washing procedures and therefore the use of these disinfectants may be suitable for selected ophthalmic surgical instruments in urgent or under-equipped circumstances.

Evidence-based decision making in an endoscopy nurse with respiratory symptoms exposed to the new ortho-phthalaldehyde (OPA) disinfectant.

BACKGROUND: ortho-Phthalaldehyde (OPA) can cause mucous irritation, respiratory symptoms and IgE-mediated hypersensitivity reactions. Very little information is available about OPA-related effects in health personnel. AIM: To report the decision-making process for the case of an endoscopy nurse complaining of cough and burning of the nose and throat during OPA exposure at work. METHODS: The problem focused on the relationship between OPA exposure and the respiratory symptoms and was investigated using an evidence-based (EB) medicine paradigm. RESULTS: A literature search was performed using the database Medline and the search engine Google. Papers and guidelines were assessed for their suitability in the EB case identification of suspected occupational asthma (OA). A multistep approach suggested by a guideline was considered most appropriate for practical use. The nurse shared the decision-making process and underwent evaluation of the clinical suspicion index and interventions for diagnosis of OA. Despite the high clinical suspicion index, the diagnosis of OA was excluded and any work restriction was avoided. Health surveillance follow-up showed a good clinical outcome and prompt recovery from respiratory symptoms after improvement of environmental control measures. CONCLUSION: The case study shows that the implementation of EB guidelines provides the occupational physician with an appropriate decision-making process for the identification and management of workers with suspected OA. Screening out of OA is highly relevant because diagnosis of disease requires removal from exposure and frequently impacts negatively on worker employment.

Studies on the behaviour of Pseudomonas fluorescens biofilms after Ortho-phthalaldehyde treatment.

A relatively novel biocide, ortho-phthalaldehyde (OPA), was tested to control biofilms formed by Pseudomonas fluorescens on stainless steel surfaces. The toxic action of OPA was assessed in terms of inactivation and removal of the biofilm by means of, respectively, the determination of the respiratory activity and the variation in the dry weight of the biofilms. For comparison, the activity of OPA against suspended bacteria was also evaluated. The results showed that higher concentrations of OPA and longer exposure times are needed to inactivate P. fluorescens biofilms than planktonic populations, thus denoting that sessile bacteria have a reduced susceptibility to OPA. This appears to be associated with the reaction with the proteins of the matrix, as demonstrated by the reduction of the antimicrobial action of OPA in the presence of a protein (bovine serum albumin). The application of OPA appeared to cause little effect in the removal of biofilms from the metal slides since the mass of biofilm that remained on the surfaces, after biocide treatment, was within the same range as those observed in the control tests. These results suggest that, with OPA application, biofilms can be inactivated but stay attached to the surfaces, decreasing thereby the success of the chemical treatment.