Palm oil, derived from the fruit of the palm plant (Elaeis guineensis), is among the most widely consumed edible oils (Edem, 2002). Palm oil contains the highest concentration of vitamin E, which is defined as a lipid-soluble antioxidant that acts as a reactive oxygen species (ROS) scavenger, preventing tissue damage caused by free radicals (Naomi et al., 2021). Tocopherols and tocotrienols are classified into alpha (α), beta (β), gamma (γ), and delta (δ) isomers based on the number and position of methyl groups on the chromanol ring structure. The palm oil-derived tocotrienol-rich fraction (TRF) is a high-quality natural source of vitamin E, containing 75 % tocotrienols and 25 % tocopherol (Sen et al., 2006; Aggarwal et al., 2010). It is noteworthy that TRF cannot be produced by the human body, so it should be obtained through external supplementation (Zainal et al., 2022). Tocotrienols are less well characterised than tocopherols but have attracted the interest of researchers due to their numerous benefits, such as anti-inflammatory, antioxidant and neuroprotective effects (Hamdo et al., 2014; Shen et al., 2018; Mohamed et al., 2018; Shaikh and Muthuraman, 2023).

TRF has been shown to provide significant protection against oxidative stress-induced cytotoxicity in rat striatal cells (Osakada et al., 2004). Nor Azman et al. (2018) found that TRF had a comparable effect to α-tocopherol in modulating antioxidant enzyme activities in healthy individuals aged 50–55 years (Nor Azman et al., 2018). Moreover, TRF has also been shown to have anti-inflammatory properties as it downregulates pro-inflammatory genes in macrophage-like cells such as tumor necrosis factor (TNF), nuclear factor kappa B (NF-B) and interleukin-1 (IL-1) (Abd Hafid et al., 2021). According to recent proteomic studies, TRF from palm oil is a potent natural neuroprotective agent in Parkinson's disease (PD), comparable to levodopa in terms of its ability to act on the ubiquitin proteasome, calcium signalling, protein processing in the endoplasmic reticulum, the mitochondrial pathway and the axonal transport system (Magalingam et al., 2022). Recently, there have been studies reporting that TRF was able to improve neuronal functions through the activation of platelet-derived growth factor-C (PDGF-C) in the animal model of type 2 diabetes mellitus-induced vascular dementia in rats (Shaikh et al., 2022; Shaikh and Muthuraman, 2023). TRF's therapeutic potential is well recognized, but a thorough toxicity assessment is essential to ensure its safety for future medical use.

The close relationship between chemical structure and toxicity highlights the significant number of drug candidates that fail in clinical trials due to ADMET-related safety and efficacy concerns. In response, in silico ADMET analysis has become an essential tool for predicting the physicochemical and pharmacokinetic properties of candidate compounds. The pkCSM is specifically designed to predict the pharmacokinetic properties of small molecules. It focuses on parameters critical for drug development, such as volume of distribution, drug bioavailability, and ADMET (Pires et al., 2015; Dulsat et al., 2023). Considering in vivo toxicity, zebrafish (Danio rerio) embryo is well-established model to investigate developmental toxicity of drugs or natural products (Yang et al., 2016; Nöth et al., 2024). The characteristics of zebrafish embryo, such as external fertilization, rapid development, transparency and ease of care and handling, have added advantages compared to tests on adult fish (Yang et al., 2016). In addition, the cell structure and vital systems such as the cardiovascular, nervous and digestive systems are anatomically and physiologically similar to those of (Hsu et al., 2007). Importantly, zebrafish possess approximately 70 % of the genes found in humans and have orthologs to 86 % of the drug targets (Gunnarsson et al., 2008; Howe et al., 2013).

Despite growing interest in palm oil-derived TRFs, limited preclinical studies prevent definitive conclusions about their safety. It has been shown that rats given tocotrienols up to 120 mg/kg (males) and 130 mg/kg (females) exhibited no clear adverse effects, with a no observed adverse effect level (NOAEL) of 0.19 % (Nakamura et al., 2001). In addition, Ima-Nirwana et al. (2011) found that TRF above 1000 mg/day caused bleeding tendency and renal impairment in rats, while lower doses were safe (Ima-Nirwana et al., 2011). In silico modeling to predict individual compound toxicity and in vivo zebrafish embryo studies are essential for understanding TRF's toxicity mechanisms and ensuring safe applications. Therefore, this study aims to investigates TRF's toxicological effects using both methods.