Introduction

Isothiazolinones are used in cosmetic and as chemical additives for occupational and industrial usage due to their bacteriostatic and fungiostatic activity. Despite their effectiveness as biocides, isothiazolinones are strong sensitizers, producing skin irritations and allergies and may pose ecotoxicological hazards. Therefore, their use is restricted by EU legislation. Considering the relevance and importance of isothiazolinone biocides, the present review describes the state-of-the-art knowledge regarding their synthesis, antibacterial components, toxicity (including structure–activity–toxicity relationships) outlines, and (photo)chemical stability. The isothiazolinones most commonly found in commercial applications, alone or in combination, are methylisothiazolinone (MI), methylchloroisothiazolinone (MCI), benzisothiazolinone (BIT), octylisothiazolione (OIT), and dichlorocthylisothiazolinone (DCOIT). Methylisothiazolinone is commonly used in wastewater treatment processes, cosmetics, paints, and detergents, and in combination with MCI (in proportions of 3:1) as an active ingredient of the commercial biocide, Kathon. Although BIT and OIT are forbidden to be used in cosmetics, they are usually applied in cleaning and leather products, respectively, and as antifouling coating agents. DCOIT, the biocidal ingredient in SeaNine 211, is a widely used antifouling agent to deter the undesirable biofouling phenomenon.[1]

Although there has been an increased use of isothiazolinones over the last years, concerns related to their inherent sensitization potential and allergic contact dermatitis, frequently observed both in consumers as well as workers in various industries, have been reported. Moreover, cross-reactivity between different isothiazolinones has been demonstrated in animal assays and broadens the potential consequences of becoming sensitized to this type of compound. As a result of the widespread use of this type of heterocycles and the renewed interest in this scaffold, this review provides an overview of the most used isothiazolinone ring-containing compounds. In-depth information on the synthesis of the most frequently isothiazolinone biocides used in consumer products and on biological/toxicity profile is presented. Additionally, given the increased concern about the possible unintended side effects that isothiazolinones could have on human health and the environment, this review also covers chemical and photochemical stability issues and the analytical procedures often used for their determination.[2]

Antibacterial Action and Toxicity of Isothiazolinones

As referred previously, isothiazolinone derivatives MI, MCI, BIT, OIT, and DCOIT are powerful biocides that are used as preservatives in a wide range of daily life products, such as detergents, paints, and cosmetic products. These compounds were described to be able to diffuse across the bacterial cell membrane and the cell wall of fungi. In the intracellular media, the electron-deficient sulfur of the N–S bond of these compounds can react with the nucleophilic groups of the cellular components, such as the thiols from cysteines of proteins active sites blocking their enzymatic activity and ultimately causing cellular death

The antibacterial and antifungal activity of isothiazolinones is highly valued by a number of industries. Therefore, over the years, several reports have been prepared illustrating their biocidal activity against a wide spectrum of industrial biological contaminants. For instance, Collier et al. evaluated the biocidal activity of MI, MCI, and BIT against Schizosacchurornyces pombe and Escherichia coli . In this study, MI showed the highest minimum growth inhibitory concentration (MIC) values, 245 μg/mL and 41 μg/mL for S. pombe and E. coli, respectively. On the other hand, MCI displayed the lowest MIC values of the tested compounds, 2.6 μg/mL and 0.5 μg/mL for S. pombe and E. coli, respectively. These results highlighted that the presence of the chlorine in the double bond of the isothiazolinone ring improves the compound’s biocidal activity.

A number of reports linked allergic contact dermatitis derived from isothiazolinones exposure to the activation of inflammatory mediators in skin cells. Conversely, the first studies about the toxicity of isothiazolinones inhalation to human respiratory systems were only released in the last years. In 2019, Yang’s group demonstrated that alveolar epithelial cells (MLE-12 cells) treated with a mixture of MI/MCI presented high levels of pro-apoptotic proteins such as BAX-Bcl-2 and cleaved caspase-3. Furthermore, in the same study, the authors observed that MI/MCI led to the release of pro-inflammatory cytokines such as TNF-α and IL-1β through the upregulation of the mitogen-activated protein kinases (MAPK) signaling pathway. More recently, another group demonstrated that MI was capable of inducing cellular death and the activation of pro-inflammatory responses in bronchial epithelial cells (BEAS-2B cells). Additionally, the authors also signaled the possible carcinogenic effect of MI after gene profile analysis.

Analysis and Determination of Isothiazolinone Biocides

The antimicrobial profile of isothiazolinones makes them highly efficient biocides, even at low concentrations. However, despite its effectiveness, some of them are strong sensitizers, producing skin irritation and allergies, and they could pose ecotoxicological risks. Consequently, its use has been restricted by EU legislation to limited concentrations depending on the product type to be preserved. In order to guarantee consumers’ health and ensure compliance to existing regulations, reliable methodologies must be used to identify and quantify this type of biocides. Most of the commercial products containing isothiazolinone biocides include a broad variety of highly complex matrices that imply serious analytical difficulties. Thus, adequate sample preparation is a key aspect of quantitative analysis of isothiazolinones in environmental samples and human consumer products. The most common and widely applied sample preparation techniques include liquid–liquid extraction (LLE) and ultrasonication or solid-phase extraction (SPE).[3]

Liquid–liquid extraction remains one of the most powerful and versatile techniques in sample preparation for cleanup and enrichment, particularly for the analysis of organic compounds in aqueous solutions. LLE is also less expensive and flexible, as several samples may be prepared in parallel. Actually, many recent developments in LLE have been focused on new techniques, which greatly reduce the amount of solvent used (cost and environmental factors) and facilitate automation of the process in conjunction with chromatographic analysis. In the literature, a number of reports describe the use of LLE during sample preparation of different samples prior to the evaluation of their isothiazolinone content 

Methods based on LC or HPLC coupled to different detectors are the most used for the analysis of isothiazolinone biocides. The identification is carried out using several detectors, such as diode array (DAD), simple quadrupole mass spectrometry (MS), and triple quadrupole mass spectrometry (MS/MS). In general, MS/MS detector allows high sensitivity and selectivity, reducing the matrix interferences and increasing the signal-to-noise ratio. Ultra-performance liquid chromatography (UPLC) takes advantage of technological strides made in particle chemistry performance, system optimization, detector design, and data processing and control enabling dramatic increases in resolution, sensitivity, and speed of analysis, when compared with HPLC. Therefore, UPLC-MS/MS has been used as a powerful analytical technique for the determination of isothiazolinones in different matrices. Wittenberg et al. developed and validated a UPLC–MS/MS method for the determination of MIT and MCI in cosmetic products. The proposed methodology was considered to be the fastest and most sensitive method for identifying and quantifying MI and MCI in cosmetic products. Three of the seven products tested that contained both MI and MCI had MCI/MI ratios similar to that of Kathon CG (approximately 3:1), which seems to indicate that probably Kathon CG was the source of isothiazolinones for the preparation of the cosmetic products tested. The MCI/MI ratios for the other four products were calculated to be 2:1 or lower. The authors concluded that Kathon CG may not be the only source of these two preservatives for cosmetic formulations or that, alternatively, reactions of MCI and/or MI with other cosmetic ingredients within a given product may explain the unexpected ratios of MCI/MI observed.

References

[1] Ram V.J., Sethi A., Nath M., Pratap R. The Chemistry of Heterocycles. Elsevier; Amsterdam, The Netherlands: 2019. pp. 149–478.

[2] Wang Y., Chen M., Wang C., Meng X., Zhang W., Chen Z., Crittenden J. Electrochemical degradation of methylisothiazolinone by using Ti/SnO2-Sb2O3/a, b-PbO2 electrode: Kinetics, energy efficiency, oxidation mechanism and degradation pathway. Chem. Eng. J. 2019;374:626–636.

[3] Aerts O., Goossens A., Lambert J., Lepoittevin J.P. Contact allergy caused by isothiazolinone derivatives: An overview of non-cosmetic and unusual cosmetic sources. Eur. J. Dermatol. 2017;27:115–122.