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Developmental neurobehavioral toxicity of butylated hydroxyanisole (BHA) in rats
Abstract
Butylated hydroxyanisole (BHA) was fed to rats throughout development (from prior to conception through 90 days of postnatal age) in doses of 0, 0.125, 0.25 or 0.5 percent (w/w) of the diet. A fifth group was also prepared as a positive control by administering 50 mg/kg/day of the antimitotic agent hydroxyurea on days 2-10 of postnatal age. Offspring from all groups were reared by their natural dams and were evaluated blind with respect to treatment assignment in a battery of standardized behavioral tests between 3 and 90 days of age. BHA at 0.5% of the diet impaired offspring growth during the last week of preweaning development and increased preweaning mortality (13.5%). No changes in maternal weight, reproductive performance or mortality were observed. No reductions in offspring growth after weaning or changes in day 90 brain weights were found. BHA at 0.25 and 0.125% of the diet had no effect on growth, reproduction or mortality; although a marginal increase was seen in the 0.25% BHA offspring mortality up to 30 days of age (8.3%, p = 0.06). BHA at 0.5 and 0.25% of the diet delayed startle development and showed a marginal trend towards increased diurnal running wheel activity; no other behavioral effects were found. Comparison of the present results to a similar study using BHT clearly indicates that BHA at equivalent dietary doses is considerably less toxic than BHT. The present results also suggest that BHA is not a potent behavioral toxin, although it is developmentally toxic using non-behavioral measures.
... Recently, we have reported the outcome of efforts to translate these recommendations into an actual working test system. The resulting system was the Cincinnati Psychoteratogencity Screening Test Battery [14][15][16][17][18]. Using this system we have recently reported the results of assessments of the food preservatives butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) [17,18] and the food coloring FD and C red dye No. 3 [19]. ...
... Recently, we have reported the outcome of efforts to translate these recommendations into an actual working test system. The resulting system was the Cincinnati Psychoteratogencity Screening Test Battery [14][15][16][17][18]. Using this system we have recently reported the results of assessments of the food preservatives butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) [17,18] and the food coloring FD and C red dye No. 3 [19]. Here we report the results of an experiment on FD and C red dye No. 40 (Allura red AC). ...
... Moreover, the clinical studies have focused exclusively on food colors even though the Feingold hypothesis encompasses more food additives than just colors, including the food preservatives BHT and BHA. We have recently investigated in animals the developmental psychotoxicity of BHT and BHA [17,18] as well as the dye FD and C red dye No. 3 (erythrosine) [19]. We found BHT to possess significantly more psychotoxicity and physical toxicity than BHA, and R3 to be essentially non-toxic using the same test battery as used here. ...
Adult Sprague-Dawley rats were fed diets containing FD and C red dye No. 40 for 2 weeks and were then bred. The diets were continued for the females throughout gestation and lactation and were provided continuously to their offspring thereafter. The treatment groups were: FD and C red dye No. 40 as 0.0, 2.5, 5.0 or 10.0% of the diet, and a positive control group treated with the toxin hydroxyurea on days 2–10 of life with 50 mg/kg/day given s.c. as a positive control group. Parental animals were evaluated for weight and food consumption, and females for reproductive success. The offspring were assessed on a series of tests using the Cincinnati Psychoteratogenicity Screening Test Battery. Additional measures were weight, food consumption, physical landmarks of development, and brain weight. Red-40 significantly reduced reproductive success, parental and offspring weight, brain weight, survival, and female vaginal patency development. Behaviorally, R40 produced substantially decreased running wheel activity, and slightly increased postweaning open-field rearing activity. Overall, R40 produced evidence of both physical and behavioral toxicity in developing rats at doses of up to 10% of the diet.
... food, food contact materials, cosmetics, and pharmaceuticals [61][62][63]. It is an anticipated human carcinogen [64] and is has been noted to have published evidence of developmental neurotoxicity (DNT) in mammals [65,66]. Both in vitro and in vivo published studies indicate that the BHA isomers have endocrine-modulating potential, with most evidence for estrogenic and androgenic effects [63,[67][68][69][70][71][72]. ...
... Both 2-and 3-BHA were predicted active for TPO inhibition by QSAR2, and 3-BHA was included in the QSAR2 training set as a TPO inhibitor. Studies in rats and pigs indicate that exposure to BHA (mixture of the two isomers) in utero can cause effects such as changed thyroxine serum levels, altered thyroid gland function and histology, and altered brain weight and behavior in the offspring [66,67,72]. TPO inhibition is as mentioned above identified to be the MIE in an AOP for thyroidrelated neurodevelopmental adverse effects (under development) [41]. ...
Thyroperoxidase (TPO) is the enzyme that synthesizes thyroid hormones (THs). TPO inhibition by chemicals can result in decreased TH levels and developmental neurotoxicity, and therefore identification of TPO inhibition is of high relevance in safety evaluation of chemicals. In the present study, we developed two global quantitative structure-activity relationship (QSAR) models for TPO inhibition in vitro. Rigorous cross- and blinded external validations demonstrated that the first model, QSAR1, built from a training set of 877 chemicals, was robust and highly predictive with balanced accuracies of 80.6% (SD = 4.6%) and 85.3%, respectively. The external validation test set was subsequently merged with the training set to constitute a larger training set totaling 1,519 chemicals for a second model, QSAR2, which underwent robust cross-validation with a balanced accuracy of 82.7% (SD = 2.2%). An analysis of QSAR2 identified the ten most discriminating structural features for TPO inhibition and non-inhibition, respectively. Both models were used to screen 72,524 REACH substances and 32,197 U.S. EPA substances, and QSAR2 with the expanded training set had an approximately 10% larger coverages compared to QSAR1. Of the substances predicted within QSAR2’s applicability domain, 8,790 (19.3%) REACH substances and 7,166 (19.0%) U.S. EPA substances, respectively, were predicted to be TPO inhibitors. A case study on butyl hydroxyanisole (BHA), which is extensively used as an antioxidant, was included to exemplify how predictions from the developed QSAR2 model may aid in elucidating the modes of action in adverse outcomes of chemicals. Overall, predictions from QSAR2 can for example be used in priority setting of chemicals and in read-across cases or weight-of-evidence assessments.
... 124,125 Moreover, the addition of 0.25% and 0.5% (w/w) BHA to the diet of mice also resulted in developmental neurobehavioral toxicity as evidenced by delayed development of the startle response and increased diurnal running wheel activity. 126 Reproductive system damage BHA has a non-negligible role in reproductive dysfunction, which can lead to hypogonadism and reproductive dysplasia. It was found that BHA caused male reproductive dysfunction in mouse Leydig and Sertoli cells by inducing calcium imbalance and decreasing the mRNA levels of steroidogenic acute regulatory protein (Star), hydroxysteroid (17⊎) dehydrogenase 1 (Hsd17b1), 5⊍-reductase 1 (Srd5a1) and 5⊍-reductase 3 (Srd5a3), which are involved in the process of steroid hormone synthesis. ...
Butylated hydroxyanisole (BHA) is mainly used as a food additive due to its antioxidant properties, which prevent or delay oxidation reactions and extend the storage life of products. The widespread use of BHA has led to its extensive presence in various environmental matrices and human tissues. Food intake is the main route of human exposure to BHA. Under different conditions, BHA can produce different metabolites, with tert‐butyl hydroquinone (TBHQ) being one of the major products. Several studies have shown that BHA could cause thyroid system damage, metabolic and growth disorders, neurotoxicity, and carcinogenesis. Mechanisms such as endocrine disruption, genotoxicity, disturbances of energy metabolism, reactive oxygen species (ROS) production, signaling pathways, and imbalances in calcium homeostasis appear to be associated with the toxic effects of BHA. Avoiding the toxic effects of BHA to the maximum extent possible is a top priority. Finding safe, non‐toxic and environmentally friendly alternatives to BHA should be the focus of subsequent research. In all, this review summarized the current situation related to BHA and might make recommendations for future research directions. © 2023 Society of Chemical Industry.
... While it is not the goal of this manuscript to conduct an in-depth safety assessment of BHA, 179 a short synopsis of major regulatory decisions is helpful as we consider the topic of this manuscript -is 180 BHA a carcinogen to be avoided, or a potentially health-protective antioxidant? reproductive and developmental toxicity in Sprague-Dawley rats exposed to BHA before mating, during 199 gestation and through to 90 days of postnatal age (Vorhees et al., 1981). The ADI was calculated by 200 applying an uncertainty factor of 100 to the NOAEL of 100 mg/kg/day. ...
Tumor data from rodent bioassays are used for cancer hazard classification with wide-ranging consequences. This paper presents a case study of the synthetic antioxidant butylated hydroxyanisole (BHA), which IARC classified as Group 2B (“possibly carcinogenic to humans”) on the basis of forestomach tumors in rodents following chronic dietary exposure to high levels. IARC later determined that the mechanism by which BHA induces forestomach tumors is not relevant to humans; however, the classification has not been revoked. BHA was listed on California Proposition 65 as a direct consequence of the IARC classification, and there is widespread concern among consumers regarding the safety of BHA driven by the perception that it is a carcinogen. While many regulatory agencies have established safe exposure limits for BHA, the IARC classification and Proposition 65 listing resulted in the addition of BHA to lists of substances banned from children’s products and products seeking credentials such as EPA’s Safer Choice program, as well as mandatory product labeling. Classifications have consequences that many times pre-empt the ability to conduct an exposure-based risk-based assessment., It is imperative to consider human relevance of both the endpoint and exposure conditions as fundamental to hazard identification.
... It has been reported that both BHA and BHT are suspected endocrine disruptors with potential health risks. 14 The search for naturally occurring antioxidants to use as healthpromotion supplements or as a food preservatives has currently gained much attention from researchers. 4,5,13 Escherichia coli, Staphylococcus aureus, and Bacillus subtilis are typical bacteria causing food deterioration. ...
BACKGROUND
Newhall navel orange (Citrus sinensis Osbeck cv. Newhall) is the major navel orange cultivar planted in China. Almost all Newhall navel orange peels produced in that country are currently discarded, which is not only harmful to the environment but also a waste of resources. It is therefore necessary to carry out research to explore the utilization potential of this resource.
RESULTS
A 95% ethanol extract (EE) of Newhall orange peel was prepared and fractionated into three subextracts: petroleum ether extract (PEE), ethyl acetate extract (EtOAcE), and water extract (WE) by simple liquid / liquid extractions. These four extracts were then subjected to antioxidant, antibacterial, and tyrosinase inhibition assays. Interestingly, EtOAcE was significantly superior to all other three extracts, exhibiting the best biological effects. The total polyphenol content (TPC), total flavonoid content (TFC), and primary individual flavonoids of these four extracts were analyzed and compared. This was followed by principal component analysis (PCA) and the Pearson's correlation test. The result indicates that the primary bioactive compounds responsible for the biological effects of the EtOAcE are sinensetin, 4′,5,6,7‐tetramethoxyflavone, nobiletin, 3,3′,4′,5,6,7‐hexamethoxyflavone, and narirutin. In view of its easy preparation and potent biological effects, EtOAcE might demonstrate excellent application potential in various industrial areas.
CONCLUSIONS
This study successfully identified EtOAcE as a potent naturally occurring antioxidant, antibacterial, and tyrosinase inhibitory agent, which might add value to the utilization of Newhall navel orange peel in the food, cosmetic, and pharmaceutical industries. © 2020 Society of Chemical Industry
Butylated hydroxyanisole (BHA) and the chemically similar butylated hydroxytoluene (BHT) are widely used as antioxidants. Toxicity of BHA and BHT has been reported under in vitro and in vivo experimental conditions. However, the mechanism of BHA-induced toxic effects in cells is unclear. In this study, the cytotoxic effects of BHA and differences in cell death mechanism for BHA and BHT were investigated in rat thymocytes by flow cytometric analysis using a fluorescent probe. We observed a significant increase in propidium iodide fluorescence in the population of cells treated with 100 μM and 300 μM BHA (dead cells). Thymocytes treated with 100 µM BHA showed increased intracellular Ca2+ and Zn2+ levels and depolarized cell membranes. BHA (30–100 µM) decreased non-protein thiol content of cells, indicating decreased glutathione content. Co-stimulation with 100 µM BHA and 300 µM H2O2 acted synergistically to increase cell lethality. Moreover, BHA significantly increased caspase-3 activity and the number of annexin-V-positive cells in a concentration-dependent manner, indicating apoptosis. However, BHT reduced caspase-3 activity and increased the number of annexin-V-negative dead cells, indicating non-apoptotic cell death. Our results reveal the toxicity of BHA could be attributed to increased levels of intracellular Ca2+ and Zn2+, resulting in an increased vulnerability of rat thymocytes to oxidative stress. In addition, we demonstrate that whereas BHA induced apoptosis, BHT induced non-apoptotic cell death in rat thymocytes. Therefore, these results may support the safety of BHA, but also demonstrate the importance of performing toxicity evaluation at the cellular level besides the tissue level.
High-throughput test methods including molecular, cellular, and alternative species-based assays that examine critical events of normal brain development are being developed for detection of developmental neurotoxicants. As new assays are developed, a "training set" of chemicals is used to evaluate the relevance of individual assays for specific endpoints. Different training sets are necessary for each assay that would comprise a developmental neurotoxicity test battery. In contrast, evaluation of the predictive ability of a comprehensive test battery requires a set of chemicals that have been shown to alter brain development after in vivo exposure ("test set"). Because only a small number of substances have been well documented to alter human neurodevelopment, we have proposed an expanded test set that includes chemicals demonstrated to adversely affect neurodevelopment in animals. To compile a list of potential developmental neurotoxicants, a literature review of compounds that have been examined for effects on the developing nervous system was conducted. The search was limited to mammalian studies published in the peer-reviewed literature and regulatory studies submitted to the U.S. EPA. The definition of developmental neurotoxicity encompassed changes in behavior, brain morphology, and neurochemistry after gestational or lactational exposure. Reports that indicated developmental neurotoxicity was observed only at doses that resulted in significant maternal toxicity or were lethal to the fetus or offspring were not considered. As a basic indication of reproducibility, we only included a chemical if data on its developmental neurotoxicity were available from more than one laboratory (defined as studies originating from laboratories with a different senior investigator). Evidence from human studies was included when available. Approximately 100 developmental neurotoxicity test set chemicals were identified, with 22% having evidence in humans.
In this work, the photo catalytic activity of unmodified titanium dioxide and modified titanium dioxide with copper were evaluated on dibutyl phthalate (DBA) and butylhydroxyanisole (BHA) which were enumerated as endocrine disrupting chemicals (EDC) under UV irradiation at room temperature.
The prepared nanoparticles were characterized using SEM and XRD. EDC concentrations were fixed at 20 ppm while the photocatalyst effect was studied with concentrations ranging from 1 g/L to 3 g/L. Presence of copper as dopant enhanced the photo degradation of DBP and BHA when compared with
titanium dioxide alone. An optimum degradation efficiency rate of around 90% was achieved with catalyst loading of 3 g/L of TiO2 with copper as dopant for DBP, whereas 82% degradation is observed with catalyst loading of 3 g/L of TiO2 with copper as dopant for BHA. A
considerable increase of 20% and 30% was observed in degradation efficiency rate of modified titanium dioxide for DBP and BHA respectively.
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