Food additive “lauric acid” possess non-toxic profile on biochemical, haematological and histopathological studies in female Sprague Dawley (SD) rats

Ethical consideration

The acute toxicity study of LA was accomplished in accordance with the guidelines stipulated by the Institutional Animal Care and Use Committee (IACUC), Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia with animal ethics approval no: 2018-210605/TAY/R/AA (2018295).

Reagents

Lauric acid (MKCF8837, purity more than 98%) Sigma-Aldrich (St Louis, MO 63103 USA 314-77-5765), Tween-20 (C01-TW121-00, Systerm, UK), normal saline 0.9% (B Braun),10% neutral buffered formalin solution pH 7.4 (R&M, UK), Haematoxylin (Merck, USA) and Eosin (Systerm, UK), Masson trichrome (Abcam150686), Periodic Acid Schiff (Abcam150680) and Picro Sirius Red stain kit (Abcam 150681).

All other chemicals and solvents were of analytical grade.

Experimental animals

In this study, eighteen (18) healthy specific pathogens-free (SPF) female SD rats, weighing 240–250 gram (g), age 8–10 weeks old were procured from the Animal Experimental Unit (AEU), Faculty of Medicine, University of Malaya. The AEU facility at the University of Malaya is certified by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). All the animals were randomly segregated in three different groups (six animals in each group) and housed in polypropylene cages for seven days to acclimatise laboratory environment prior to the experiments. A consistent environment of 12-hour light/dark cycle, temperature 22 ± 2 °C, and humidity of 50 ± 10% were maintained. The animals were allowed a standard rodent pellet diet and reverse osmosis (RO) water ad libitum throughout the experimental period.

Acute oral toxicity test

Acute toxicity study was conducted to assess whether LA produce any mortality or other adverse reactions upon oral administration of dose (300 and 2,000 mg/kg body weight), according to guideline 423 provided by the OECD for the acute toxicity class method procedure (OECD, 2001) with minor amendments (OECD, 2002). Before the experiment, all the animals were carefully weighed, marked, and grouped accordingly:

Group I (n = 6): Vehicle (Tween 20/normal saline)-treated

Group II (n = 6): Lauric acid-treated, dose 300 mg/kg body weight

Group III (n = 6): Lauric acid-treated, dose 2,000 mg/kg body weight.

The LA was dissolved in Tween 20 and diluted with normal saline 0.9% to get the desired dose (Alves et al., 2017). Animals fasted overnight before dosing, and treatment groups received freshly prepared single doses of LA suspension at 300 and 2,000 mg/kg body weight, while animals in the control group received Tween 20 or normal saline 0.9% suspension equally based on individual body weight. Each gavage volume was 10 mL/kg body weight. All efforts were made to lessen experimental animals’ suffering, especially in handling and oral gavage.

Daily observations

After oral gavage, diet was withheld except water for 2–3 h while animals were monitored individually with special attention in the first half-hour till completion of first 24 h followed by daily cage-side examination for fourteen days for any signs of toxicity including general behavioural in skin, eyes, touch, activeness, and movement changes. All the changes observed were well documented before and after the gavage.

Body weight, food, and water consumption

The body weight of the individual animal was carefully noted before and after oral administration of the LA or vehicle, then weekly and lastly on day fifteen (euthanized day). Daily food (g/day), and water (ml/ day) intake was noted on daily basis in all the three groups for fourteen days.

Haematology and biochemical analysis

On day fourteen, all the animals fasted overnight for 12 h, only water was allowed. All the animals were euthanized in the morning by an intraperitoneal (IP) injection of ketamine (80 mg/kg) & xylazine (7 mg/kg), and blood was collected via micro-capillary tubes from the retro-orbital plexus region into two different sets of test tubes, one containing anticoagulant, ethylene diaminotetraacetic acid (EDTA) for haematological tests including red blood cells (RBC), white blood cells (WBC), platelets count, haemoglobin (Hb), packed cell volumes (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), neutrophil, lymphocytes, monocytes, and eosinophils and the other without additives for clinical biochemistry tests like urea, creatinine, calcium, inorganic phosphate, uric acid, sodium, potassium, chloride, total cholesterol (TC), high-density lipoproteins (HDL), low-density lipoproteins (LDL), triglycerides (TG), total (HDL) ratio, albumin, globulin, albumin-globulin (A/G) ratio, ALP, AST and ALT levels. The collected blood samples were then sent to an ISO-certified blood-testing laboratory.

Measurement of relative organ weight

Internal organs such as adrenal glands, brain, eyes, heart, kidneys, liver, lungs, ovaries, spleen, stomach, urinary bladder, uterus, and cervix/vagina were isolated through a fine incision in the abdomen at the midline of the euthanized animals. All the harvested tissues were cleaned with phosphate buffer saline (PBS), wiped with clean blotting paper and weighed on a calibrated weighing balance. Relative organ weight (ROW) of all the animals was noted in proportion to body weight according to the following equation:

ROW = $\frac{\text{absolute organ weight}}{\text{body weight at the time of sacrifice}}\times 100$

Histopathological analysis

Histopathological examination was conducted on the harvested liver, kidney, heart, lungs, brain, spleen, and pancreas in all the animals. Tissues were preserved in 10% neutral, buffered formalin solution for 48 h at 4 °C for histopathological study. Paraffin-embedded specimens of selected tissues were cut into 4–5 µm thick samples and stained with haematoxylin and eosin (H&E), Masson trichrome, Periodic Acid Schiff (PAS) and Picro Sirius Red staining as per the manufacturer protocol detailed in the kits. Later, all the stained tissues were sectioned and observed under the fluorescence compound microscope (40X) (Eclipse Ni-U, Nikon Corporation, Japan).

Statistical analysis

All statistical results were expressed as the mean ± standard deviation (SD). The comparison between the treated and control group was performed by a one-way analysis of variance (ANOVA) using statistical software, SPSS package version 22 (IBM, SPSS Statistics, Inc., Chicago, IL, USA) to evaluate the significant differences. The (p < 0.05) compared to the control group was considered statistically significant.

Acute toxicological evaluation and daily observation

Animals in LA (300 and 2,000 mg/kg body weight) treated group were normal and healthy during fourteen days study period. No signs of mortality or sudden death were observed compared to the untreated group animals. No significant changes in the temperature, skin, eye colour, food and water intake were observed. Furthermore, no behavioural changes or signs of diarrhoea, coma, sedation and tremors were noted as compared to the control group Table 1. Experimental results suggest that LD₅₀ of LA should be higher than 2,000 mg/kg body weight.

Effect of LA on body weight, food and water consumption

Body weight, food & water intake in the LA treated groups (300 and 2,000 mg/kg body weight) were noted to be non-significant compared to the control group. However, LA treated groups showed slight changes in the body weight, food and water consumption on day fourteen, compared to the untreated group, as shown in Tables 2 and 3. The mean body weight of the control group was noted as 244.71 ± 3.13 g, whereas treatment groups showed 247.53 ± 2.96, and 240.63 ± 4.76 g, respectively. On day fourteen, the mean body weight gained by the control group was 14.81 ± 0.064 g, while LA treated animals (300 and 2,000 mg/kg body weight) displayed non-significant gain in the body weight (14.93 ± 0.68 and 15.36 ± 0.76 g), respectively. The percentage mean in the body weight of all the treated animals was non-significant in comparison to the control group (p > 0.05), as shown in Table 2.

Moreover, LA administration (300 and 2,000 mg/kg) showed non-significant changes (p > 0.05) in the food and water consumption, compared to the control group, as shown in Table 3.

Effect of LA on relative organ weight

The relative organ weight of the harvested tissues—adrenal glands, brain, eyes, heart, kidney, liver, lungs, ovaries, spleen, stomach, urinary bladder, uterus and cervix/vagina, revealed non-significant changes post-LA treatment (300 and 2,000 mg/kg body weight) compared with the control group animals (p > 0.05), as shown in Table 4.

Effect of LA on haematology and clinical biochemistry

The effect on haematological analysis is summarised in Table 5. Parameters including RBC, WBC, platelets count, Hb, PCV, MCV, MCH, MCHC, neutrophil, lymphocytes, monocytes, and eosinophils shows non-significant changes (p > 0.05) after oral administration of LA (300 and 2,000 mg/kg body weight) when compared with the control animals. Importantly, all the values were within the reference range, compared to the control group.

Similarly, the biochemical analysis of liver and renal function tests, including urea, creatinine, calcium, inorganic phosphate, uric acid, sodium, potassium, chloride, TC, HDL, LDL, TG, total HDL ratio, albumin, globulin, A/G ratio, ALP, AST and ALT levels for LA treatment groups were not affected and observed to be normal (non-significant (p > 0.05)), when compared with the control group as presented in Table 6.

Effects of LA on Histopathology of internal organs

The histopathological analysis involves macroscopic and microscopic examination of processed and fixed tissues to visualise various components and structural alterations in the tissues (Greaves, 2011; Khoo et al., 2018). H&E staining is the most common and widely used technique to essentially identify morphological changes during diagnosis and screening of any malignancies in the tissues (Fischer et al., 2008). However, Masson trichrome staining remains reliable for the cytoplasm, which gives in-depth and selective detail about the connective tissue (Tsounapi et al., 2017). PAS staining assesses the changes in glycogen content within muscle fibre sections (Arya et al., 2015; Fairchild & Fournier, 2004). Moreover, the collagen content is analysed with the help of Picro Sirius Red staining, which provides a clear understanding of collagen accumulation in the tissues (Lattouf et al., 2014).

In brief, macroscopic analysis of liver sections (Fig. 2) in LA-treated rats (300 and 2,000 mg/kg body weight) displays no alterations in the colour or texture, importantly no changes to hepatocytes, hepatic sinusoids, central vein, and portal triad were observed when compared with the control group (Fig. 3). Kidney showed no structural degeneration, and colour changes in the renal cortex and tubules, without any tubular necrosis and inflammation in LA-treated animals compared to untreated animals, as displayed (Figs. 4 and 5). The photomicrographs of the heart (Figs. 6 and 7) tissue showed a normal size of myocardial fibre and filaments when compared with the control group. Lung tissues (Figs. 8 and 9) appeared to be normal, with no abnormal changes noted in colour. Moreover, microscopic observation displayed thin inter-alveolar septa, clear alveoli and alveolar sacs with the absence of inflammatory cells, including neutrophils, or thickening of alveolar septa, when compared with the untreated group. Furthermore, brain (Figs. 10 and 11) tissues showed normal morphology with the regular shape of the pyramidal neuron and uniform distribution of glial cells in LA-treated and control animals. Macroscopic texture (Figs. 12 and 13) exhibited normal and regular appearance with white and red pulp without any sign of apoptotic, necrotic lesions or megakaryocytosis compared with the control group. Microscopic appearance of the pancreas (Figs. 14 and 15) appeared with healthy acinar, clear round or oval-shaped Islets cells in cluster form, with lightly stained cytoplasm in the LA-treatment and control animals. Thus, the study results suggest that treatment with minimum and maximum doses of LA (300 & 2,000 mg/kg body weight) did not cause any tissue damage or morphological alteration at the cellular level.