|Year : 2015 | Volume
| Issue : 2 | Page : 167-172
Acute and sub-chronic oral toxicity study of black tea in rodents
Tapas Kumar Sur1, Suparna Chatterjee1, Alok Kumar Hazra2, Richeek Pradhan1, Supriyo Chowdhury3
1 Department of Pharmacology, Institute of Postgraduate Medical Education and Research, Kolkata, West Bengal, India
2 Scientist, Chemistry Division, Quality Testing Laboratory (AYUSH), Ramakrishna Mission Ashrama, Vivekananda University, Narendrapur, West Bengal, India
3 Department of Pharmacology, College of Medicine and Sagore Dutta Hospital, Kolkata, West Bengal, India
|Date of Submission||24-Jul-2014|
|Date of Decision||19-Feb-2014|
|Date of Acceptance||21-Feb-2015|
|Date of Web Publication||17-Mar-2015|
Department of Pharmacology, Institute of Postgraduate Medical Education and Research, Kolkata, West Bengal
Source of Support: Tea Board of India (Grant number
RL/33(167)2010/Part-V) (Project protocol ID: SC/TEA
BOARD/2011-2012). We gratefully acknowledge Dr. Uttara Chatterjee,
Department of Pathology, IPGME&R, Kolkata, India, for her assistance
in histopathological examinations and Dr. Surupa Basu, Biochemist,
Institute of Child Health, Kolkata, India for her assistance in special
biochemical estimations., Conflict of Interest: None
Objectives: Systematic oral toxicity study for black tea (Camellia sinensis), the most commonly consumed variety of tea, is lacking. The present study was undertaken to assess the iron load on black tea (Camellia sinensis) and its safety aspects in animals.
Materials and Methods: The analysis of iron was done in six tea samples as per American Public Health Association method using flame atomic absorption spectrophotometer. Maximum physical iron-loaded tea sample was identified on black tea sample 2 (BTS-2), and this was further studied for acute and 90-day sub-chronic toxicity following Organisation for Economic Co-operation and Development guidelines.
Results: Black tea sample 2 did not show any signs of toxicity or mortality at up to 2 g/kg per oral dose in Swiss albino mice. 90-day toxicity studies in Wistar rats did not reveal any evidence of toxicity at up to 250 mg/kg/day (2.5% infusion of BTS-2) oral dose as exhibited by regular observations, body weight, food consumption, hematology, serum chemistry, organ weights, and histopathology. Further, serum iron, total iron binding capacity, unsaturated iron binding capacity, and ferritin were not altered after 90 days of treatment. Masson trichrome staining and Perls' staining did not reveal any abnormalities in hepatic tissue following 90-day treatment of high iron-loaded BTS-2.
Conclusions: This safety study provides evidence that BTSs, in spite of relatively high iron content, show no significant iron-related toxicity on acute or sub-chronic oral administration in animals.
Keywords: Atomic absorption spectrophotometer, black tea, iron, oral toxicity, rodent
|How to cite this article:|
Sur TK, Chatterjee S, Hazra AK, Pradhan R, Chowdhury S. Acute and sub-chronic oral toxicity study of black tea in rodents. Indian J Pharmacol 2015;47:167-72
|How to cite this URL:|
Sur TK, Chatterjee S, Hazra AK, Pradhan R, Chowdhury S. Acute and sub-chronic oral toxicity study of black tea in rodents. Indian J Pharmacol [serial online] 2015 [cited 2020 Feb 16];47:167-72. Available from: http://www.ijp-online.com/text.asp?2015/47/2/167/153423
| » Introduction|| |
Tea, prepared from the leaves of the shrub Camellia sinensis (Theaceae family), is one of the most popular beverages worldwide, with an estimated 20 billion cups being consumed daily.  Of the many varieties, black and green teas are the most popular types. The differences in chemical constituents of black and green teas result from their distinct postharvesting processes.  Studies have shown that green tea extracts contain polyphenols like gallocatechins (epigallocatechin-3-gallate) while oxidized gallocatechins such as thearubigins and theaflavins are present in black tea.  The diverse health benefits of tea are often attributed to the presence of these antioxidants chemicals.  However, safety concerns pertaining to the presence of potentially harmful metals in tea samples are recently emerging amidst widespread reports of detection and quantification of these elements from samples obtained from various parts of the world.  Traces of heavy metals contaminate tea samples not only because of the tendency of these elements to bioaccumulate,  but also the due to the manufacturing and processing methods used before tea is made commercially available.  Published reports have observed that certain tea samples contain quantities of iron sufficient to cause deleterious effects in human beings.  This is compounded by the fact that a significant negative correlation has been reported between the iron content of the soil and polyphenol levels in the tea leaves,  implying that higher iron content of the soil might not only result in higher iron levels in tea samples, but also might decrease levels of beneficial antioxidants. Thus, the presence of iron in black tea samples (BTSs) has drawn the attention of regulatory bodies (Food Safety and Standards Authority of India, 2012). 
Chronic iron toxicity occurs in various genetic and metabolic diseases, repeated blood transfusions, and also due to excessive intake. Again, excess of iron in the body might contribute to the progression of conditions such as Alzheimer's disease, atherosclerosis, and diabetes mellitus probably because of the fact that iron is a heavy metal with well-characterized tissue oxidative properties.  Thus, there is the importance of estimation of iron content of a beverage as commonly consumed as a tea before recommending its safe use on long term basis. While systematic safety studies on green tea  and Pu-erh tea  have been performed, systematic evaluation of oral toxicity of black tea, by far the most commonly consumed variety, is lacking. Therefore, in the current study, we have quantified iron in various BTSs and evaluated the acute and sub-chronic toxicity of black tea in rodents.
| » Materials and Methods|| |
Graded and processed curl, tear and crush BTS were obtained from the commercial production unit and supplied by Tea Board of India, Kolkata. A total of six BTSs were studied.
Determination of Iron Content of the Black Tea Samples and Infusions
Iron was estimated from the six BTSs and tea infusions following American Public Health Association method 3030 (2001)  using flame atomic absorption spectrophotometer (Varian Spectra AA140, The Netherlands). Five grams each of the six BTSs were separately heated to ashes in a muffle furnace at 773.15 K and digested with 10 ml of concentrated nitric acid for 2 h. Thereafter, 5 ml of 70% perchloric acid was mixed and heated for another 1 h. A few drops of hydrogen peroxide were then added to discolor the digested material, and this was now dissolved in deionized water and filtered. The filtrate was transferred to a 50 ml volumetric flask. Three replicate digestions were made for each sample. Tea infusions were prepared according to Wei et al., 1999.  In brief, 1.25 g of each of the six BTSs was transferred into a conical flask, and 25 ml of deionized boiled water was added to it. The tea was allowed to infuse for 15 min and then filtered. The same process was repeated, and the two filtrates were combined. The six separate tea infusions thus prepared were individually heated in a muffle furnace following the same procedure as described earlier. The instrumental working conditions were set as follows: Wavelength =248.3 nm; slit width = 0.2 nm; cathode lamp current = 5.0 mA; flame type = air/acetylene; air flow = 3.5 l/min; acetylene flow =1.5 l/min; integration time =10 s. A blank digest was prepared without any sample. A four-point calibration graph was prepared from a known iron standard (Ferric nitrate, Sigma, USA). The correlation coefficient (r) of the values obtained from the tea samples and infusions with that of the standard ranged between 0.9997 and 0.9999. Out of the six samples, the maximum concentration of iron was present in the BTS-2. This was selected for further toxicological studies.
Experimental Animals and Housing Conditions
Male Swiss albino mice (body weight 25 ± 5 g) and Wistar rats (body weight 150 ± 5 g) of both sex were used in the study and were obtained from registered animal vendor. Animals were housed in suspended polycarbonate cages in groups of 2-3 animals per cage with cutting straw bedding. A 12 h light-dark cycle was maintained while the temperature and relative humidity of the animal rooms were maintained at 22°C ±3°C and 50-60%, respectively. All animals were allowed an acclimatization period of 4 weeks during which the animals were fed standard rodent diet (Provimi Animal Nutrition India Pvt. Ltd. Bangalore, India) and maintained as per National Institutes of Health guidelines (Publication No. 86-23, 1985).  The study was initiated after approval (IAEC/SCH-5/2011/UCM-73) of the plan of research from Institutional Animal Ethics Committee.
Acute Oral Toxicity Study
Single dose toxicity study was done on male Swiss albino mice according to Organisation for Economic Co-operation and Development (OECD) guidelines no. 423  adopted for acute toxicity in animals up to 2 g/kg (limit test). For this purpose, 12 mice were used in total in accordance with the guidelines.
Sub-chronic Oral Toxicity Study
Repeated dose 90-day oral toxicity studies were performed on Wistar strain rats with BTS-2 as per the OECD guidelines no. 408.  From a total of 30 rats, 10 animals (5 female and 5 male) each were randomly assigned to the control and the two treatment groups. The rats in the two treatment groups were administered a 2.5% aqueous solution of BTS-2 by gavage method at the doses of (i) 0.5 × 10−2 ml/g/day (125 mg/kg/day) and (ii) 1 × 10−2 ml/g/day (250 mg/kg/day) for a period of 90-day, respectively. The doses were selected on the basis of physical iron load of the sample and acute toxicity study results. The tea infusion was prepared according to Wei et al. as described earlier.  Control animals received deionized water at the dose of 1 × 10−2 ml/g/day along with standard laboratory diet for 90 days. During the period of tea administration, the animals were observed closely for signs of any toxicity. Cage side behavioral observations, food and water consumption, body weight, and urinalysis were conducted as a part of the regular checkup of the animals. At the end of 90 days, the overnight fasting animals were sacrificed under deep anesthesia (intraperitoneal thiopentone sodium, 40 mg/kg).
After euthanizing the animals, blood samples were collected by cardiac puncture for hematological and biochemical examinations. Hematological investigations were performed for the measurement of hemoglobin (Hb) levels, total count of red blood cells (RBC) and white blood cells (WBC), differential count, hematocrit packed cell volume, mean corpuscular volume, and mean corpuscular Hb concentration. Biochemical parameters measured were glucose, total protein, albumin, total cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN), and creatinine in serum. Furthermore, serum iron, total iron binding capacity (TIBC), and unsaturated iron-binding capacity (UIBC) were measured using Ferrozine method while serum ferritin was estimated by electrochemiluminescence immunoassay on cobas e 411 immunassay analyzer (Roche Diagnostics, Germany).
Following euthanasia, organs (liver, kidneys, spleen, stomach, heart, adrenals, testes [male], uterus, cervix, and ovaries [female]) from all the animals were separated and trimmed of any adherent tissue, as appropriate. The wet weight of each of these organs was measured. The liver was fixed in 10% buffered formalin and eventually thin paraffin sections were made for staining with (i) hematoxylin and eosin stain for pathological examination, (ii) Perls' stain for the detection of iron deposition and (iii) Masson trichrome stain for the identification of fibrosis.
The results have been expressed as mean ± standard deviation (SD). Statistical analysis was done using SPSS version 17 (SPSS Sciences, Chicago, USA). For comparing means, P < 0.05 was considered as statistically significant.
| » Results|| |
Iron Content of Black Tea Samples and Infusions
The mean ± SD iron content in dry black tea leaves was 216.65 ± 83.68 μg/g. The lowest concentration was exhibited in BTS-1, and the highest concentration was noted in BTS-2 [Table 1]. Iron concentration in black tea infusion ranged from 3.24 to 5.04 μg/g, and the mean value was 4.33 ± 0.64 μg/g. The highest iron concentration was noted in infusion of BTS-2 and the lowest in infusion of BTS-3 [Table 1].
Acute Oral Toxicity of Black Tea Sample 2 in Rats
Black tea sample 2 did not show any signs and features of toxicity or mortality up to 2.0 g/kg per oral dose in Swiss mice. The results of the present study confirmed no observed adverse effect level (NOAEL) of BTS-2 can be defined as >2 g/kg in Swiss mice and was identified as nontoxic.
Sub-chronic Oral Toxicity of black Tea Sample 2 in Rats
Treated and control rats were examined daily up to 90 days for general appearance, behavior, and signs of toxicity, morbidity, and mortality. No deaths occurred during the study period. Cage side observations were made to detect any changes in the general behavior activity, levels of alertness, skin, body fur, eyes, and mucous membranes of animals. All the animals of the treated and control groups remained active and healthy during the period of the study. All groups gained weight in a regular manner during the study period, and significant differences were not evident in the same between controls and animals on tea diet [Table 2]. Again, average food and water intake were comparable in all groups, and there was no evidence of any adverse effect of BTS-2 intake. Findings of the urinalysis examination showed minimal variations between the groups, well within the limits of normalcy. None of the parameters differed significantly between the male and the female rats [Table 2].
Significant differences were not noted in hematological and biochemical parameters after 90-day oral treatment with BTS-2 between the groups. Hb levels, total RBC, and WBC counts as also the differential counts remained within the normal ranges after 90 days of BTS-2 administration [Table 3]. The values for serum biochemical constituents and enzymes were comparable with those of the control group and were all within the normal limits documented for laboratory rats. Biochemical parameters of liver function, such AST, ALT, and ALP and kidney function, such as BUN and creatinine did not reveal hepatic or renal damage following 90-day repeated administration of BTS-2 [Table 4]. Furthermore, serum iron, TIBC, and UIBC were within normal limits and statistically comparable to control. However, serum ferritin in all three groups remained below detection limits of the instrument giving indirect evidence that there was no iron overload.
|Table 3: Hematological parameters in control and treatment groups at the end of the experiment (90-day)|
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|Table 4: Biochemical parameters in control and treatment groups at the end of the experiment (90-day)|
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There were no marked differences in the weights of various vital organs viz., heart, liver, kidney, spleen, stomach, adrenals, and gonads in the BTS-2 treated for 90 days in male and female rats compared to control [Table 5]. Gross examination or microscopic study of the vital organs on autopsy did not reveal any abnormalities. Neither Perls' stain nor Masson trichrome stain detected any treatment-related changes in the liver sections [Figure 1].
|Table 5: Absolute wet weights (g) of organs in control and treatment groups at the end of the experiment (90-day)|
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|Figure 1: Microphotographs of liver treated with black tea sample 2 (BTS-2). Liver sections of Wistar rats euthanized after BTS-2 treatment for 90-day stained with H and E, (a-c). Masson trichrome (d-f). and Perls' stain (g-i) shows no lesion, fi brosis or iron depositions in hepatocytes. Magnification of all microphotographs were ×40|
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| » Discussion|| |
The presence of heavy metals like iron in tea is dependent not only on the type of tea and its processing methodology but also on the soil characteristics and geographical region of tea cultivation.  Fernαndez-Cαceres et al. showed iron level to be in the range of 74-1000 μg/g for BTSs having different geographical origins.  In 2008, Ashraf and Mian reported iron concentration ranging from 108.5 to 351.6 μg/g in different tea brands in Saudi Arabia imported from India.  In 2005, Naithani and Kakkar reported South Indian herbal tea products contained a mean iron level of 446.6 μg/g, which was higher than any previous reports.  Recently, Marbaniang et al. studied the trace metals present in branded tea used at Shillong, India.  Our data substantiated the presence of iron in BTSs ranged from 131.5 to 358 μg/g as also in tea infusion ranged from 3.24 to 5.04 μg/g. The mean iron content in the tea samples 216.65 μg/g was higher than the accepted Indian standard of 150 μg/g (Food Safety and Standards Authority of India 2012). 
As a tea is one of the most common beverages consumed in a long-term basis, second only to water, it is important to explore the safety of tea through standard toxicity tests in animals. Several reports have been published in the medical literature describing patients presenting with marked liver toxicity in the form of acute hepatitis, attributable to the consumption of supplements containing green tea extracts.  In spite of these reports, many studies have validated the speculated health benefits of tea. On the other hand, there is evidence that the evidence that long-term black tea supplementation was capable of protecting both plasma proteins and plasma lipids from oxidative injury.  Amidst both kinds of reports claiming benefits and hazardous effects of tea, the need for animal studies evaluating the safety of tea consumption becomes imperative. The toxicity of green tea catechins was evaluated with 90-day dietary administration to F344 rats and NOAEL of up to 764 mg/kg body weight/day for males and 820 mg/kg body weight/day for females were reported.  NOAEL for tea flower extract was reported to be 4.0 g/kg/day daily for 13 weeks for Sprague-Dawley rats.  However, till date there are no such convincing toxicity data on BTSs. This is the foremost report of 90-day sub-chronic toxicity study in BTSs.
For the purpose of acute toxicity testing, we used male Swiss albino mice instead of female. This was because of the fact that serum iron level of female mice is normally lower than that of male mice, due to chronic menstrual blood loss. So, toxicity due to iron overloading, if any, would have likely been more apparent in male mice than in female. Furthermore, there is evidence that male mice might be more predisposed to toxic effects of iron overloading than female mice.  However, in the present study, no acute toxicity effects of black tea were apparent by limit test. The NOAEL of iron-rich black tea (BTS-2) was determined to be >2 g/kg. Again, results from the sub-chronic toxicity study do not provide any evidence of harmful effects of iron-rich black tea at 250 mg/kg/day as demonstrated by the findings in regular examination of the animals, body weight and food consumption measurements, hematology, serum chemistry, organ weights, or histopathology.
Serum Fe, TIBC, UIBC, and ferritin levels remained unchanged after 90 days of consecutive BTS-2 consumption. Most important physiopathological effects of iron overload on hepatic tissues are fibrosis and hepatocellular carcinoma.  Masson trichrome staining was used to identify an increase in collagenous tissue and thus indicate fibrotic change in liver while Perls' staining was used to detect iron deposits in tissues. In the present study, Perls' staining showed no liver iron deposits and Masson trichrome staining could not detect any fibrotic damage with liver sections from rats on a 90-day BTS-2 diet. Long-term black tea consumption did not result in iron deposition or fibrotic damage to the liver. Therefore, it confirms that though BTS-2 contains highest iron load among the sample tested, it does not produce any iron-related adverse reaction in rats.
| » Conclusion|| |
Results of this study provide evidence that black tea, in spite of its relatively high iron content, does not produce acute or sub-chronic toxicity in rodents and is thus safe for human consumption.
| » Acknowledgment|| |
This work was supported by the Tea Board of India (Grant number RL/33(167)2010/Part-V) (Project protocol ID: SC/TEA BOARD/2011-2012). We gratefully acknowledge Dr. Uttara Chatterjee, Department of Pathology, IPGME and R, Kolkata, India, for her assistance in histopathological examinations and Dr Surupa Basu, Biochemist, Institute of Child Health, Kolkata, India for her assistance in special biochemical estimations.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]