The manufacture of fast, processed and convenient products especially food and beverage industries by indigenous manufacturers have increased over the past decade. These increases have made the role of food additives (as preservative, colorant, sweetener amongst others) in modern food and beverage industries become important [1]. Despite their benefits, several studies have reported the existence of several adverse effects. When certain types of component additives are used together, they could react pharmaceutically thereby reducing or enhancing some specific systemic effects making them important risk factor and thus predisposing consumers to some health problems [2]. As a consequence, many questions about the necessity and safety of these chemical additives (used as preservatives) have been raised [2]. One of the most common grounds for concern is the concomitant use of benzoic acid (sodium benzoate) as a preservative and ascorbic acid which is an antioxidant in carbonated soft drinks. These two additives have been postulated to   react together to produce benzene, a human carcinogen and genetic mutants [3]. This reaction is said to be accentuated in the presence of certain extrinsic and intrinsic factors some of which retailed beverages in Nigeria are subjected to, such as; exposure to heat (30°C and above) and ultraviolet (UV) radiation, leaving soft drinks in warm conditions (as in a car boot, garage or the open place under the sun), long storage time and other places [3]. Joint FAO/WHO expert committee on food additives (JECFA) have maintained an average daily intake (ADI) of 0 - 5 mg/kg body weight for benzoic acid and its salts, while the maximum acceptable concentration (MAC) of benzoic acid and benzoates in beverages as stipulated by Codex Alimentarius standards is set at 250 ppm (mg/L) [4]. However, no maximum amount has been defined for ascorbic acid and thus there is no specified limit on the level of its use [5]. To this effect, many countries have conducted surveys on the availability and concentration of benzoic acid, its salts and benzene in soft drinks available in their countries in recent time, but this regulation is not enforced in some developing countries of the world. This laxity raises substantial doubt about the MAC set for benzoic acid (and salts) by the regulating authorities [17]. Thus, this study was conducted to experimentally evaluate the effect of the simultaneous use of sodium benzoic acid and ascorbic acid additives in orange juice in other to justify the authorization framework of their usage and their most likely safe combined dosages.

Chemicals

Sodium benzoate was acquired from NAAFCO^© scientific supplies Limited (Nigeria) and ascorbic acid was acquired from JOPAN^© Pharmaceutical Limited (Nigeria).

Experimental Treatment Preparation

Experimental treatment was prepared in an orange fruit juice at 15%, 20%, 25%, 30% weight by volume percentage concentration of sodium benzoate for groups 2 and 3; groups 4 and 5; groups 6 and 7; groups 8 and 9 respectively and ascorbic acid at 2.5% for groups 3, 5, 7 and 9 in the orange fruit juice. Treatment preparations were packaged in non-transparent 100ml polypropylene (PP) bottles and were exposed to ultraviolent (UV) light (UV intensity 28 µW/cm²) (SG603EN, Baker SterilGARD^©III, Sanford) over a period of 7 days prior to start of administration and then subsequently for the 28 days of administration [6]. (The treatment preparations had been exposed to about 600 – 840 hours of 28 µW/cm² UV light intensity at the end of the sub-chronic exposure).

Animal Grouping

Thirty-six Wistar rats of different sex (150 – 250 g body weight) reared under an intensive care management in a box deep litter system at the Department of Veterinary Pharmacology and Toxicology, University of Ilorin. The animals were acclimatized for two weeks before the start of the experiment. The experimental animals were divided into nine groups of four rats per group. Each group was allocated to different treatments for four weeks in a completely randomized design as indicated in (Table 1).

Table 1: Division of experimental rats into various groups.

Ethical Consideration

Ethical approval was sought from the Ethical Review Committee of the Faculty Veterinary Medicine, University of Ilorin, Nigeria. The ethical approval number was FVMERC/1/2019. The standard for management and welfare of the animals was ensured to concur to international regulation. The experiment took place at the Department of Veterinary Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Ilorin, Nigeria.

Sample Preparation

Daily observation for morbidity and mortality, weekly detailed physical examination, weekly body weight and feed consumption estimation were all carried out. At four weeks of administration (twenty-eight day). All rats were sacrificed and blood sample was collected from the medial canthus into EDTA, fluoride citrate and plain vacutainer sample bottles for whole blood and for serum analysis respectively. Packed cell volume (PCV) was determined by the micro-haematocrit centrifugation method. Haemoglobin (Hb) concentration was determined by the cyanmethaemoglobin method. Red blood cell (RBC) count and white blood cell (WBC) count were determined using the haemocytometer method. Mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were extrapolated from Hb, PCV and RBC. All procedures were undertaken as described [7]. Serum glucose, alanine transaminase (ALT), aspartate transaminase (AST), creatinine, urea, Na²⁺ and K^- electrolytes levels were analysed using a UV spectrophotometer analyzer (UV 752 PEC MEDICAL^©, USA) and total serum protein with an ion selective electrode.

Histopathological Procedure

After harvesting the liver and the kidney from the rats, the organ was promptly and adequately treated with 10% formaldehyde (fixation) in order to preserve its structure and molecular composition. After fixation, the piece of organ was dehydrated by bathing it successively in graded mixture of ethanol and water (70 – 100%). The ethanol was then replaced with a solvent miscible with the embedding medium. As the tissues were infiltrated with xylene, it became transparent (clearing). The impregnated tissue by xylene was placed in melted paraffin in an oven, maintained at 58 – 60^oC (embedding). The heat caused the solvent to evaporate and the spaces within the tissues became filled with paraffin. The tissue together with its impregnating paraffin hardened after removal from the oven. The sections (5 μm) were then floated on water and transferred to a glass slide, and stained with haematoxylin and eosin stains. The slides were viewed under light microscope with magnification X400.

Statistical Analysis

All data generated were expressed as mean ± SD. The differences between the groups were analysed by one-way analysis of variance (ANOVA) followed by Dunnet’s post-hoc multiple comparison test using GraphPad PRISM 5^© for Windows, Version 5.03, 2010. The level of significance was P≤ 0.05.

Morbidity and Mortality

Following oral administration of sodium benzoate only and sodium benzoate with ascorbic acid in graded doses, the result showed that the combination had no effect on morbidity and mortality as shown in (Table 2).

Table 2:  Observations for Mortalities on Sub-chronic Exposure with Sodium Benzoate alone and on Combination with Ascorbic acid as Additive in Orange Juice.

The fractions denotes numerator as number dead and denominator denotes total number in group.

Weight Gain

There was a significant (p<0.05) decrease in percentage weight gain, especially in the high dose treatment as observed in group 7, (200 mg/kg of sodium benzoate, with 25mg ascorbic acid), group 8 (250 mg/kg of sodium benzoate) and group 9 (300 mg/kg of sodium benzoate with 25 mg ascorbic acid) respectively, indicating a decreased body weight as seen in (Table 3).

Table 3:  Effects of Sodium Benzoate and Ascorbic acid as Orange juice additives on percentage weight Gain.

All values are express in mean ± standard deviation of mean.

   ^a Significantly lower (P<0.05).

Sodium benzoate and ascorbic acid combination at 250 and 300 mg/kg affected the feeding habit of the animals as the difference in the average quantity of feed consumed in each group (especially at high dose) were significant (P <0.05) as indicated in (Table  4).

Table 4: Effects of Sodium Benzoate and Ascorbic Acid as Orange Juice Additives on Feed consumption pattern.

All values are express in mean ± standard deviation of mean.

 ^a Significantly lower (P<0.05).

Hematological and Serum Biochemical Parameters

The treatment also resulted in no significant difference (P >0.05) for all haematologic parameters. However, there was a consistent non-significant increase in leukocytic parameters of most groups treated with sodium benzoate only in comparison with those treated with sodium benzoate and ascorbic acid combination as stated in (Table 5). The serum biochemical results also followed the same trend as heamatology parameters with all treated groups showing a non-significant difference when compared to the control as indicated in (Table 6).

Table 5: Effects of Sodium Benzoate and Ascorbic acid On the Heamatological Parameters.

All values are express in mean ± standard deviation of mean.

      ^a Significantly lower (P<0.05).

Table 6: Effects of Sodium Benzoate and Ascorbic Acid as Orange Juice Additives on some Serum Biochemical Parameters.

All values are express in mean ± standard deviation of mean

^aSignificantly lower (P<0.05).

Histopathology Result

Kidney

Figure 1: Plate i: Photomicrograph of Kidney (rat Group 1); non exposed (negative control), no visible lesion 100X H&E stain).

Plate ii: Photomicrograph of Kidney (rat, Group 2); there is increase in tubular luminal diameter and tubular atrophy (Arrow), 100X H&E stain).

Figure 2: Plate iii: Photomicrograph of Kidney, (rat, Group 2); there is toxic tubular epithelial cell necrosis (Arrow) and tubular atrophy (Star) , 400X H&E stain.

Plate iv: Photomicrograph of Kidney, (rat, Group 3); there is toxic tubular necrosis. 400X H&E stain.

Figure 3: Plate v: Photomicrograph of Kidney (rat, Group 5); there is tubular epithelial necrosis with intraluminal cast (Star) and severe diffuse papillary haemorrhage (arrow) 400X H&E stain.

Plate vi: Photomicrograph of Kidney, (rat Group 8); there is severe glomeruli and tubular necrosis (Arrow and Star) 400X H&E stain.

Figure 4: Plate i: Photomicrograph of Liver, (rat, Group 1); non exposed (negative control), no visible lesion.400X H&E stain.

Plate ii: Photomicrograph of Liver, (rat, Group 2); there is toxic hepatocellular degeneration and necrosis (Arrow) with kuffper cell hypertrophy, (Star) 400X H&E stain.

Figure 5: Plate iii: Photomicrograph of Liver, (rat, Group 3); there is hepatocellular necrosis and obliteration of sinusoids with nuclear pyknosis and kuffper cell hypertrophy. 400X H&E stain.

Plate iv: Photomicrograph of Liver, (rat Group 4); there is severe diffuse toxic coagulative hepatocellular necrosis 400X H&E stain.

Figure 6: Plate v: Photomicrograph of Liver, (rat, Group 6); there is severe toxic diffuse hepatocellar necrosis (Star) 400X H&E stain.

Plate vi: Photomicrograph of Liver, (rat Group 8); there is severe hepatocellular necrosis with nuclear loss and kuffper cell hypertrophy 400X H&E stain.

(Figure 1-6)Following exposure of benzoate feed additive o rats, there was integumental discoloration at different body regions. This study was in contradiction to previous study which showed no clinical manifestations indicating that the adverse effect of either sodium benzoate was possible but not severe. The reason for this manifestation was likely due to a non-immunologic contact urticaria and ophthalmitis [8] & [9]. Other manifestation like brownish discoloration and fur shedding was observe may be indicative of ageing of hair in the skin coat, skin contamination, adaptive changes and the onset of contact urticarial [10].Sodium benzoate and ascorbic acid combination at 250 and 300 mg/kg affected the feeding habit of the animals as the difference in the average quantity of feed consumed in each group as observed in table 3. The treatments could be said to have caused some biochemical changes which would affected the Para optic nuclei of the hypothalamus through changes of dynamics of mediating neurotransmitters in the higher center and the satiety center [10]. The findings in this study was also in agreement with the previous report of CICAD (2000), a steady decrease in percentage weight gain especially with the high dose group 7 (200 mg/kg of sodium benzoate, with 25mg ascorbic acid), group 8 (250 mg/kg of sodium benzoate) and group 9 (300 mg/kg of sodium benzoate with 25 mg ascorbic acid), was observed from this study indicating a decreased  body  weight gain  which could also pose as one  of adverse effects of  sodium benzoate‘s  at  high dose as stated in (Table 4). The treatment with both benzoic acid and vitamin C resulted in no significant difference for all haematologic parameters. However, there was a consistent non-significant increase in leukocytic parameters of most groups treated with sodium benzoate only in comparison with those treated with the combination. This increase could be attributed to the systemic generation of radicals (neutrophils) and improved activity of the T lymphocytes (cell mediated immunity) while the concurrent decrease in the ascorbic acid treated groups could be associated with the anti-oxidant effects of ascorbic acid as observed in table 5. The anti-oxidative effect of the ascorbic acid would have minimized the generation reactive oxygen species and the detrimental effect of cortisol surge. The cytosolic basis of these is the anti-oxidant would overwhelm the pro-oxidants which would have been cytotoxic to the cells. This would prevent lipid peroxidation and would protect membrane integrity. This cytoprotective mechanism of ascorbic acid also offered cyto-protective effect in the hepatocytes and other parenchymaous tissues. This was in line with previous studies of Latimer, (2011) [11]. The ascorbic acid treated groups occurrence could also be explained as being the effect of a quantity of benzene production and thus points to the onset of lymphocytic leukopaenia and a reducing or defective leucopoiesis [11].In this study, oral administration of sodium benzoate only and sodium benzoate with ascorbic acid in graded doses resulted in no significant difference for serum biochemical parameters analysed. ALT (table 6) was an exception however, with significant difference recorded between groups with ascorbic acid [11]. However, the non-significant decrease in serum protein and glucose levels, non-significant increase in creatinine levels and a significant increase in serum ALT, for most treatment groups when compared with the control group could be an indication of a hepatic effect (as hepatic insufficiency) as seen in table 6. Normonatremia and normophosphotaemia, non-increase in serum urea level (in comparism to control group) that was observed are pointers to the absence of any significant renal insufficiencies, myopathies or any tumour induction [12]. This study revealed that sodium benzoate either alone or in combination with Vitamin C graded dose adversely affected the kidneys of rats or could have nephrotoxic effect (Figure ii-vi). The main reason could be that the toxic irritant brought to the kidney via blood circulation exerts direct toxic effect on tubular epithelium and may cause anoxia as a result of congestion and reduction in blood circulation [13]. Microscopically, the histopathology result of the kidneys of rats treated with sodium benzoate alone or in combination with Vitamin C in graded dose revealed wide spread of degenerative and necrotic changes at both tubules and glomerulus (Figure ii-vi). There were also severe haemorrhages in intertubular spaces especially in groups with higher dose (figure x and xi). This finding was in line with the previous studies of who observed tubular degeneration and proliferative changes in glomeruli as well as in the interstitium in their studies on fluvalinate and benzoate toxicosis in mice [14]. The liver also followed the same trend like the kidneys showing extensive toxic hepatocellular degeneration and necrosis with kuffper cell hypertrophy in rats treated with sodium benzoate alone or in combination with Vitamin C in graded dose (figure vii-xiii). This finding was in agreement with the work of Piramanayagam observed similar lesion in the liver of mice following custard apple seed oil (containing benzoate) toxicity [15] & [16].

In conclusion, the sodium benzoate additive had effects on hepatic enzymes, feeding and weight gain effects. This was evident by the findings of the histopathology. Ascorbic acid as additives to mitigate detrimental effects of additives should be considered. It is therefore recommended that a nationwide standardization of sodium benzoate and quantification of benzene in common trademarked common beverages should be carried out and that caution should be taken in consuming soft drinks that are being openly hawked, or have been stored in warehouses for a long time with near expiration dates.

1. James KK. Determination of benzoic acid and benzene in soft drinks, fruit juices and herbal products using high performance liquid chromatography. A thesis submitted to the department of chemistry, college of science, Kwame Nkrumah University of Science and Technology, in partial fulfilment of the requirement for the award of master of philosophy (mphil) degree in analytical chemistry.
2. James KK, Samuel OA. Levels of Benzoic Acid in Soft Drinks and Fruit Juices in Ghana. J Env Sci J Tox Fd Tech. 2014; 8: 36-39.
3. Fulop I, Padureanu A, Kincses AM, Croitoru MD. Benzene Determination in Soft Drinks. Acta Medica Marisiensis. 2012; 58: 297-299.
4. National Agency for Food and Drug Administration and Control (NAFDAC). Food Additives Regulations. 2005.
5. Matei N, Birghila S, Popescu V, Dobrinas S, Soceanu1 A, Oprea C. et al. Kinetic study of vitamin C degradation from pharmaceutical products, Rom. Journ Phys. 2008; 53: 343-51.
6. Neal MTP, Nyman PJ, Diachenko GW, Hollifield HC. Survey of Benzene in Foods by using Headspace Concentration Techniques and Capillary Gas Chromatography. J AOAC Intl. 1993; 76: 6:1213-1219.
7. Schalm OW, Jain NC, Carroll EJ. Veterinary Haematology. 3rd ed. Lea and Febiger, Philadelphia. 1975; 471- 538.
8. Tfouni SAV, Toledo MCF. Determination of benzoic and sorbic acids in Brazilian food J Fd Contrl. 2002; 13: 117-23.
9. Xavier R, Sreeramanan S, Diwakar A, Sivagnanam G, Sethuraman KR. Soft Drinks and Hard Facts: A Health Perspective. ASEAN Fd J. 2007; 14: 69-81.
10. Cynthia MK. The Merck Veterinary Manual. Merk publishing group. 2011; 781.
11. Latimer KS. Duncan & Prasse’s Veterinary Laboratory Medicine: Clinical Pathology. Wiley Blackwell Publishing Ltd. 
12. Vaden SL, Knoll JS, Smith FWK, Tilley LP. Blackwell’s Five-Minute Veterinary Consult: Laboratory Tests and Diagnostic Procedures: Canine & Feline. 1st Edition. Blackwell publishing Ltd. 2009.
13. Tamang RK. Comparative pathology of pyrethroid and organophosphate pesticides intoxication in mice and goats. 1997.
14. Garg SK, Rastogi SK, Gupta VK, Varshneya C. Toxicological profile of fluvalinate – a synthetic pyrethroid. Indian J Pharmacol. 1992; 24: 154-1577
15. Piramanayagam, S, Monohar BM. Histopathological changes induced by malathion in rats. Indian Vet J. 2002; 114-117.
16. Diogo, JSG, Oliveira L, Pena S A, Lino CM. Risk assessment of additives through soft drinks and nectars consumption on Portuguese population: a 2010 survey. Fd and Chem Toxicol. 2017.
17. Fried EJ, Nestle. The growing political movement against soft drinks in schools. JAMA. 2012; 288: 2181.