Triclocarban, Triclosan, Bromochlorophene, Chlorophene, and Climbazole Effects on Nuclear Receptors: An in Silico and in Vitro Study.

BACKGROUND: Endocrine-disrupting chemicals can interfere with hormonal homeostasis and have adverse effects for both humans and the environment. Their identification is increasingly difficult due to lack of adequate toxicological tests. This difficulty is particularly problematic for cosmetic ingredients, because in vivo testing is now banned completely in the European Union. OBJECTIVES: The aim was to identify candidate preservatives as endocrine disruptors by in silico methods and to confirm endocrine receptors' activities through nuclear receptors in vitro. METHODS: We screened preservatives listed in Annex V in the European Union Regulation on cosmetic products to predict their binding to nuclear receptors using the Endocrine Disruptome and VirtualToxLab™ version 5.8 in silico tools. Five candidate preservatives were further evaluated for androgen receptor (AR), estrogen receptor (ERα), glucocorticoid receptor (GR), and thyroid receptor (TR) agonist and antagonist activities in cell-based luciferase reporter assays in vitro in AR-EcoScreen, hERα-HeLa-9903, MDA-kb2, and GH3.TRE-Luc cell lines. Additionally, assays to test for false positives were used (nonspecific luciferase gene induction and luciferase inhibition). RESULTS: Triclocarban had agonist activity on AR and ERα at 1µM and antagonist activity on GR at 5µM and TR at 1µM. Triclosan showed antagonist effects on AR, ERα, GR at 10µM and TR at 5µM, and bromochlorophene at 1µM (AR and TR) and at 10µM (ERα and GR). AR antagonist activity of chlorophene was observed [inhibitory concentration at 50% (IC50) IC50=2.4µM], as for its substantial ERα agonist at >5µM and TR antagonist activity at 10µM. Climbazole showed AR antagonist (IC50=13.6µM), ERα agonist at >10µM, and TR antagonist activity at 10µM. DISCUSSION: These data support the concerns of regulatory authorities about the endocrine-disrupting potential of preservatives. These data also define the need to further determine their effects on the endocrine system and the need to reassess the risks they pose to human health and the environment. https://doi.org/10.1289/EHP6596.

Degradation kinetics and transformation products of chlorophene by aqueous permanganate.

This paper evaluates the oxidation of an antibacterial agent, chlorophene (4-chloro-2-(phenylmethyl)phenol, CP), by permanganate (Mn(VII)) in water. Second-order rate constant (k) for the reaction between Mn(VII) and CP was measured as (2.05 ±â€¯0.05) × 101 M-1 s-1 at pH 7.0 for an initial CP concentration of 20.0 µM and Mn(VII) concentration of 60.0 µM. The value of k decreased with increasing pH in the pH range of 5.0-7.0, and then increased with an increase in solution pH from 7.0 to 10.0. The presence of MnO2 and Fe3+ in water generally enhanced the removal of CP, while the effect of humic acid was not obvious. Fourteen oxidation products of CP were identified by an electrospray time-of-flight mass spectrometer, and direct oxidation, ring-opening, and decarboxylation were mainly observed in the reaction process. The initial reaction sites of CP by Mn(VII) oxidation were rationalized by density functional theory calculations. Toxicity changes of the reaction solutions were assessed by the luminescent bacteria P. phosphoreum, and the intermediate products posed a relatively low ecological risk during the degradation process. The efficient removal of CP in secondary clarifier effluent and river water demonstrated the potential application of this Mn(VII) oxidation method in water treatment.

Biomonitoring of estrogenic exposure and identification of responsible compounds in bream from Dutch surface waters.

The exposure to and effects of estrogenic compounds in male breams from Dutch freshwater locations were investigated. Ovotestis was observed infrequently (maximum frequency 16%). However, plasma vitellogenin (VTG) concentration was elevated highly at some locations. Estrogenic activities in male bream plasma, liver, and in gastrointestinal content were measured in the estrogen-responsive chemical-activated luciferase gene expression (ER-CALUX) assay. Plasma concentrations of vitellogenin correlated very well with the estrogenic activities in gastrointestinal content. The ER-CALUX activity in gastrointestinal content thus could provide a biomarker for recent exposure to estrogenic compounds, and the gastrointestinal content was chosen as investigative matrix for the toxicity identification and evaluation ([TIE]; bioassay-directed fractionation) of estrogenic compounds in bream. The approach consisted of a reversed-phase high-performance liquid chromatography fractionation of gastrointestinal content extract, directed by ER-CALUX and followed by gas chromatography analysis. The estrogenic hormones 17beta-estradiol and its metabolite estrone were identified as major contributors to the activity at all locations (except the reference location), independent of the presence or absence of a known source of estrogenic activity, such as a sewage treatment plant. Chemical screening showed the presence of other pollutants, such as a lower chlorinated dioxin and the disinfectants clorophene and triclosan. However, these compounds did not have high estrogenic potencies and their concentrations were not high enough to contribute significantly to the observed estrogenic activity.

Toxicogenetic evaluation of dichlorophene in peripheral blood and in the cells of the immune system using molecular and flow cytometric approaches.

Dichlorophene; a halogenated phenolic compound with wide applications as a fungicide, bactericide and antiprotozoan. Dichlorophene spray also has therapeutic use in the disease digital dermatitis. In guinea pigs, a few studies obtained mixed results in dicholorophene sensitization tests. In consideration of the fact, that the mechanism of its genotoxicity has not been adequately elucidated lead to present study assessing the acute in vivo toxicological impact in Rattus norvegicus. A systematic research has been made encompassing the use of molecular and flow cytometric approaches. The study was designed on blood cells for comet assay which revealed dichlorophene induced DNA damage in all exposures understandable in time dependent manner. The feasibility of this assay was also established as an effective, fast and accurate method with a great potential in biomonitoring. Contemporary molecular techniques were further engaged using leukocytes for the cell apoptosis/cycle and mitochondrial membrane potential employing propidium iodide staining and rhodamine 123 respectively. The effect on cell cycle phases and mitochondrial membrane permeability was analyzed through flow cytometry. These indicators exposed that dichlorophene decreased the mitochondrial membrane potential, altered the cell cycle and confirmed the DNA damage leading to apoptosis of the cells of the immune system accountable for immunotoxic effects of dichlorophene on rat leukocytes.

In vitro cytotoxicity assessment of the biocidal agents sodium o-phenylphenol, sodium o-benzyl-p-chlorophenol, and sodium p-tertiary amylphenol using established fish cell lines.

The cytotoxicity of three biocidal agents frequently employed as active ingredients in phenolic-based disinfectants, were evaluated in three established fish cell lines (EPC, CHSE and RTG-2). Cell viability was assessed using two fluorescent indicator dyes, Alamar Blue for metabolism and neutral red for lysosomal activity. Total protein content was also quantified as a measure of cell detachment. In order to evaluate the sensitivity of the cell cultures, the results obtained were compared with toxicity data obtained from a previous study with the same three compounds and the in vivo lethality test with rainbow trout. Results from this study established that each of the three cell lines ranked the tested chemicals in the same order of toxicity as the in vivo test; however, the cell cultures were found to be an order of magnitude less sensitive than whole fish studies with the same compounds. The chemical sodium o-benzyl-p-chlorophenol was consistently ranked the most toxic of the tested compounds with each cell line and the endpoints employed. The rank order of toxicity was always sodium o-benzyl-p-chlorophenol > sodium p-tertiary amylphenol > sodium o-phenylphenol. The EPC cells were found to be the most sensitive cell line tested based on Alamar Blue IC(50) data, and the Alamar Blue assay was consistently found to be the most sensitive endpoint of the three cytotoxicity assays employed.

Laccase-catalyzed removal of the antimicrobials chlorophene and dichlorophen from water: Reaction kinetics, pathway and toxicity evaluation.

As active agents in cleaning and disinfecting products, antimicrobials have been widely spread in the environment and have drawn extensive attention as potential threats to the ecological system and human health. In this study, the laccase-catalyzed removal of two emerging antimicrobials, chlorophene (CP) and dichlorophen (DCP), was investigated under simulated environmental conditions. Intrinsic reaction kinetics showed that the removal of CP and DCP followed second-order reaction kinetics, first-order with respect to both the enzyme and the substrate concentration. It was also found that fulvic acid could suppress the transformation of CP and DCP by reversing the oxidation reactions through its action as a scavenger of the free radical intermediates produced from reactions between laccase and the substrates. Several reaction products were identified by a quadrupole time-of-flight mass spectrometer, and detailed reaction pathways were proposed. For both CP and DCP, direct polymerization was the principal pathway, and the coupling patterns were further corroborated based on molecular modeling. The nucleophilic substitution of chlorine by the hydroxyl group was observed, and further oxidation products capable of coupling with each other were also found. Additionally, toxicity evaluation tests using Scenedesmus obliquus confirmed that the toxicity of CP and DCP was effectively eliminated during the reaction processes.

A growth inhibition test with sewage bacteria--results of an international ring test 1995.

A bacterial toxicity test method using the determination of growth inhibition of sewage bacteria within an incubation period of 6 hours will be standardized at ISO. A ring test was performed with 24 participants using 3.5-dichlorophenol and potassium cyanide as test substances. The analysis of the test results showed that the method is very accurate and appropriate for determining the bacterial toxicity of chemical compounds. Due to the results obtained 3.5-dichlorophenol was proposed as a reference substance.

Ecotoxicological evaluation of the biocidal agents sodium o-phenylphenol, sodium o-benzyl-p-chlorophenol, and sodium p-tertiary amylphenol.

The ecotoxicological effects of three biocidal agents frequently employed as active ingredients in phenolic-based disinfectants were evaluated using a test battery comprising of organisms representing three aquatic trophic levels. Phenolic-based disinfectants are commonly used by mushroom growers to disinfect spent mushroom compost. In general, the most sensitive assay used in this study was the Microtox test. In the case of the fish lethality assay, sodium o-benzyl-p-chlorophenol was found to be slightly more sensitive than the bacterial test system. The freshwater alga and invertebrate tests were also among the most sensitive test species employed. The active ingredient, sodium o-benzyl-p-chlorophenol (with the exception of the Microtox assay), was the most toxic chemical tested on each species. The majority of ecotoxicity data obtained in this research has not been previously reported and may therefore assist in the management and planning decisions regarding the application of pesticides and utilisation of SMC.

Contact hypersensitivity response to o-benzyl-p-chlorophenol in mice.

o-Benzyl-p-chlorophenol was evaluated for its potential as a sensitizing agent for allergic contact hypersensitivity in mice. Female B6C3F1 mice were sensitized with 1.0, 3.0, and 10.0% o-benzyl-p-chlorophenol and challenged with 20.0% o-benzyl-p-chlorophenol. Doses of o-benzyl-p-chlorophenol were selected from assays for primary irritancy. Mice received 20 microliters by direct dermal application, for 5 days, to sites prepared by shaving, dermabrading and, in some mice, with intra dermal injection of Freund's complete adjuvant. The rest period was 7 days. Measurement of the contact hypersensitivity response in mice was by radioisotopic assay two days after challenge and mouse ear swelling test one and two days after challenge. Mice demonstrated statistically significant dose-dependent contact hypersensitivity response to o-benzyl-p-chlorophenol with or without adjuvant pretreatment.

Effects of chlorinated bisphenols on torula yeast glucose-6-phosphate dehydrogenase.

Chlorinated bisphenol antibacterial and antifungal agents are potent inhibitors of torula yeast glucose-6-phosphate dehydrogenase (G6PD). Several compounds were tested, including hexachlorophene [HCP; 2,2'-methylenebis(3,4,6-trichlorophenol)]; 2,2'-oxybis(tetrachlorophenol); 2',4-dihydroxy-2,3,3',5,5',6-hexachlorodiphenylmethane; 2,2'-methylenebis(3,4-dichlorophenol) (3,4-TCP); bithionol [2,2'-thiobis(4,6-dichlorophenol)]; 2,2'-methylenebis(3,5-dichlorophenol); 2,2'-dihydroxy-3,3',5,6,6'-pentachlorodiphenylmethane; 2,2'-methylenebis(4-chlorophenol) (DCP); 2,2'-methylenebis(4,6-dichlorophenol); and the related uncoupler 2,4-dinitrophenol. The relative inhibitory activity of the chlorinated bisphenols tended to increase with degree of chlorination of the aromatic rings. the concentrations of the bisphenols that caused 50% inhibition ranged from 2.5 micrometers for 2,2'-oxybis(tetrachlorophenol) to 40 micrometers for 2,2'-methylenebis(4,6-dichlorophenol) under comparable assay conditions. More detailed kinetic analysis showed that, as with HCP, the inhibition of G6PD by 3,4-TCP and DCP followed noncompetitive kinetics. Calculations from the kinetic data gave apparent inhibition constant (Ki) values for 3,4-TCP of 267 micrometers with G6P and 308 micrometers with NADP, and for DCP of 697 micrometers with both G6P and NADP.

Bayesian methods for a three-state model for rodent carcinogenicity studies.

The objective of a chronic rodent bioassay is to assess the impact of a chemical compound on the development of tumors. However, most tumor types are not observable prior to necropsy, making direct estimation of the tumor incidence rate problematic. In such cases, estimation can proceed only if the study incorporates multiple interim sacrifices or we make use of simplified parametric or nonparametric models. In addition, it is widely accepted that other factors, such as weight, can be related to both dose level and tumor onset, confounding the association of interest. However, there is not typically enough information in the current study to assess such effects. The addition of historical data can help alleviate this problem. In this article, we propose a novel Bayesian semiparametric model for the analysis of data from rodent carcinogenicity studies. We develop informative prior distributions for covariate effects through the use of historical control data and outline a Gibbs sampling scheme. We implement the model by analyzing data from a National Toxicology Program chronic rodent bioassay.

Prechronic toxicity of o-benzyl-p-chlorophenol in rats and mice.

o-Benzyl-p-chlorophenol (BCP) is a major household and industrial germicide. Its prechronic toxicity was evaluated in male and female F344 rats and B6C3F1 mice. Treatment was by gavage in corn oil. BCP was slightly toxic after acute oral exposure, with high mortality at 4000 and 2000 mg/kg in rats and mice. Exposure to 12 oral doses of BCP at 1000, 500, 250, 125, 62.5, or 0 mg/kg body wt resulted in dose-related cecal dilatation and nephrosis in both rats and mice. Doses for the subchronic studies were based on the results of the 2-week studies. Ten animals were treated per dose per sex. Rats received 480, 240, 120, 60, 30, or 0 mg BCP/kg. Mice were treated with 1000, 800, 650, 500, or 0 mg BCP/kg. Animals were dosed 5 days per week for 13 weeks. Clinical signs related to dosing included urogenital staining in rats and rough/oily haircoats in mice. No effects on growth rate were seen in rats, but growth was retarded at the higher doses used in mice. Kidney weights increased in rats, and liver weights increased in mice. A decrease in thymus weight accompanied by a depletion in thymic lymphocytes (rats) or thymic necrosis (mice) occurred only in a high-dose animals. In both species, the kidney was the major target organ. In rats, there was an increase in incidence and severity of nephropathy and renal tubule regeneration. The lesions in mice primarily involved the renal cortex and included necrosis, casts, chronic inflammation, and regeneration of the renal tubules. No effects of BCP exposure in rats were seen in a broad spectrum of hematology or urinalysis parameters. Minor decreases in blood urea nitrogen, creatinine, and alanine amino transferase were detected in male and female rats. There were no biologically significant neurobehavioral effects in rats or immunotoxic effects in mice due to exposure to BCP. Thus, the kidney is the major target organ for BCP.

Toxicological profile for o-benzyl-p-chlorophenol.

As part of a health-hazard survey on the health risk of hospital cleaning workers from exposure to Lyorthol, a hazard assessment of o-benzo-p-chlorophenol, one of the constituents of Lyorthol, has been prepared. In this paper, the physical and chemical characteristics, kinetics and effects of o-benzochlorophenol are described and discussed, and an overall, summarizing hazard evaluation is presented.

Safety assessment of dichlorophene and chlorophene.

Dichlorophene is a halogenated phenolic compound that functions as a bacteriocide and fungicide in cosmetics. Chlorophene is a halogenated phenolic compound that functions as a biocide and preservative in cosmetics. Dichlorophene was reported to be used in a total of five cosmetic formulations at concentrations of 0% to 1.0%, but Chlorophene was not reported to be used. Dichlorophene is prohibited for use in cosmetic ingredients in Japan. In Europe, the maximum authorized concentration allowed for Dichlorophene is 0.5% and for Chlorophene is 0.2%. The major impurity of Dichlorophene is the trimer 4-chloro-2,6-bis(5-chloro-2-hydroxybenzyl)phenol. In rats, Dichlorophene sulfate, Dichlorophene monoglucuronide, and Dichlorophene diglucuronide were the major metabolites of Dichlorophene and were excreted, mainly in the urine. The glucuronic acid conjugate, the sulfate ester conjugate, and two minor metabolites of Chlorophene were the metabolites found in rat urine. Chlorophene was incompletely absorbed through the rat skin. These chemicals exhibited low toxicity in acute oral toxicity studies in several animal species. Some evidence of toxicity with both chemicals was found in short-term oral toxicity studies in mice and rats; nephropathy was the principal finding. Chronic toxicity data were not available for Dichlorophene. Rats and mice dosed with Chlorophene for 2 years had a dose-related and sex-related increase in the severity of nephropathy. In animal tests, Dichlorophene and Chlorophene were ocular irritants. No inhalation toxicity data were available for these ingredients. Dichlorophene up to 10% concentration resulted in no to minimal irritation when applied to the intact and abraded skin of rabbits. Chlorophene was severely irritating to rabbits in most dermal irritation studies. Studies on guinea pigs gave positive and negative results in sensitization tests of Dichlorophene. A dose-related contact hypersensitivity response to Chlorophene was reported in mice. No reproductive or developmental toxicity data were available for Dichlorophene, but there was some evidence of non-dose-dependent developmental toxicity with Chlorophene in rabbits. Dichlorophene was positive in the Ames mutagenicity assay, but not in mammalian or fruit fly test systems. Chlorophene was mutagenic in four in vitro mammalian test systems. Carcinogenicity studies for Dichlorophene were not found. Neoplasms were not observed in rats treated with Chlorophene for 2 years; however a significant incidence of neoplasms was observed in mice so treated. A 1-year National Toxicology Program (NTP) study concluded that Chlorophene was a cutaneous irritant and a weak skin tumor promoter but had no activity as an initiator or complete carcinogen. Dichlorophene was not a sensitizer in clinical dermal sensitization tests. Some reactions to Chlorophene occurred in some, but not all, clinical dermal sensitization tests. Positive photopatch tests to Dichlorophene were found in 13/469 patients. Although these ingredients were ocular irritants at high concentrations, the risk at concentrations which are actually used in cosmetic formulations was uncertain. Overall, the available data were insufficient to support safety of Dichlorophene or Chlorophene. Additional data needed include (1) method of manufacture and impurities data (especially the trimer in Dichlorophene); (2) photosensitization and photocarcinogenicity data for Dichlorophene; (3) dermal reproductive and developmental toxicity data for Dichlorophene (as a function of dose); and (4) ocular irritation at concentration of use, if available.

Chronic nephropathy and renal carcinogenicity of o-benzyl-p-chlorophenol in F344/N rats and B6C3F1 mice.

o-Benzyl-p-chlorophenol, an aryl halide biocide, was evaluated in male and female F344/N rats and B6C3F1 mice in a series of subchronic and 2-year toxicity and carcinogenicity studies. Kidney was the primary target of toxicity in the 13-week gavage studies in rats and mice, with increased nephropathy noted as low as 240 mg/kg in male rats. Considering the nephropathy to be doselimiting, the chronic (2-year) study was conducted at lower doses (male rats: 30, 60, or 120 mg/kg; female rats: 60, 120, or 240 mg/kg; male and female mice: 120, 240, or 480 mg/kg; in corn oil; n = 50/group). Survival and body weights of dosed rats were similar to controls in the 2-year study. Survival of high-dose male and female mice, and body weights of all dosed male and mid- and high-dose female mice, were lower than controls. The incidence and severity of nephropathy increased with dose and length of treatment in both rats and mice. There was an increased incidence of renal tubule adenomas or carcinomas in both the mid- and high-dose male mice. Despite similar evidence of nephropathy, however, there were no increased incidences of neoplasms in female mice or in male or female rats. This study suggests therefore that while nephrotoxicity may have been a necessary component, factors other than the marked nephrotoxicity of o-benzyl-p-chlorophenol were critical to the development of renal carcinogenesis induced in only male mice.

Identification of estrogenic compounds in fish bile using bioassay-directed fractionation.

Conjugates of estrogenic chemicals, endogenous as well as xenobiotic, are mainly excreted via bile into the intestine. Therefore, measurement of estrogenic activity in bile yields useful information about an organism's internal exposure to (xeno-)estrogens. Although previous studies in The Netherlands have reported estrogenic activity in male fish bile, the contribution of natural hormones and xenobiotic substances to this activity is unknown. To identify compounds responsible for estrogenic activity in fish bile, we developed a bioassay-directed fractionation method for estrogenic chemicals. In this approach, the in vitro reporter gene assay ER-CALUX (Estrogen Responsive Chemical Activated Luciferase Gene Expression) was used to assess estrogenic activity in deconjugated bile samples and to direct RP-HPLC fractionation and chemical analysis (by GC-MS) of estrogenic compounds. The method was applied to bile from male breams (Abramis brama) collected at three locations in The Netherlands. At one of these locations, the River Dommel, extremely high levels of plasma vitellogenin and a high incidence of intersex gonads in these male breams have previously been observed, indicating the exposure to estrogens. In this study, the natural hormones 17beta-estradiol, estrone, and estriol accounted for the majority of estrogenic activity in male bream bile. At the River Dommel, the synthetic contraceptive pill component ethynylestradiol was found in effective concentrations as well. The detected natural and synthetic hormones may be responsible forthe estrogenic effects observed in wild bream from this location. Furthermore, a large number of xenobiotic chemicals was detected at relatively high levels in bile, including triclosan, chloroxylenol, and clorophene. Although chloroxylenol was shown for the first time to be weakly estrogenic, these compounds did not contribute significantly to the estrogenic activity observed.