Toxicology of Ngirimbo Samples across Chitipa District

Introduction: Smokeless tobacco describes a wide variety of tobacco products that do not require combustion, and is typically used either orally or nasally. Ngirimbo is a form of oral smokeless tobacco used by smokers in Malawi for tobacco harm reduction. The aim of this study was to determine the acidity (pH), nicotine content, mineral content (iron, zinc, calcium, magnesium and copper), heavy metal content (lead, chromium and cadmium), and presence of other volatile compounds in ngirimbo across Chitipa District in Malawi . Methods: Atomic absorption spectrophotometry spectrometry were used to estimate nicotine content, concentration of toxic heavy metals, minerals of potential toxicity and other harmful chemicals in the samples. Results: Samples were found to contain harmful chemicals, high pH and nicotine [2-(1-methyl-2-pyrolidinyl)-pyridine, (S)- and (S)-3-(1-methyl-2-pyrolidinyl)-pyridine] levels. Mineral concentrations were found to be much higher than typical safety limits. Conversely, samples were not found to contain lead, and had low concentrations of chromium and cadmium. Conclusions: These findings suggest that prolonged use of ngirimbo is a significant health risk to people with chronic diseases. Nonetheless, ngirimbo provides a valid method of tobacco harm reduction and a potential smoking cessation tool. Therefore, further analytical toxicological studies are needed to fully characterize variations in the quality of the product.


Introduction
The term smokeless tobacco describes a wide variety of tobacco products which do not require combustion. These products are typically used orally or nasally. When used orally, tobacco is predominantly sucked (as dry or moist snuff), or chewed (chewing tobacco), whereas tobacco for nasal use is sniffed (dry snuff). Ngirimbo is a form of smokeless tobacco used orally in Malawi. The product is considered to be a method of tobacco harm reduction as it is safer in comparison to cigarettes due to the absence of combustion/burning during consumption. Ngirimbo is made from a mixture of ilambo (a crystalline solid comprised of plant remains such as stalk or husks), cold water, and local tobacco.

According to the Malawi National STEPwise Survey for Non-Communicable Diseases
Risk Factors 2017 Report, tobacco use is increasingly becoming a significant cause of mental health problems, morbidity, and mortality among adults and young people in Malawi. There is no current infrastructure in Malawi providing assistance for tobacco cessation, nor a widespread availability of safer nicotine products or nicotine replacement therapies such as patches or gum at affordable prices. The general population favors cheaper local smokeless tobacco products to reduce the harm caused by smoking and nicotine addiction. Reportedly, 0.4% of Malawians use smokeless tobacco products 1 . Despite this relatively low popularity of smokeless tobacco use, it represents an ongoing public health challenge that is amplifying the levels of drug and substance exploitation and mental illness in Malawi. Furthermore, very little is known about the potential toxicity or use of locally-made tobacco harm reduction products. This study aims to provide an understanding of ngirimbo use, characterizing the properties of the product, while assessing the prospect of utilizing ngirimbo in more wide-reaching tobacco harm reduction programs.

Materials and Methods
Samples of ngirimbo were collected from producers in twelve different areas of Chitipa district. Sources were selected based on the popularity of the producers in terms of the number of local markets per area, thereby representing a large and uniform sample group. Samples were personally collected from the producers in their original vacuum-packed containers, and labeled with unique identification codes to signify the ngirimbo production area (GPA), and labeled GPA1-12 for ease of reference.

Determination of sample pH
The pH of each powdered sample of ngirimbo was measured using a pH meter (Denver Instrument, Basic pH meter, 10487; 12V, 500mA, USA) that had been previously calibrated using two buffer solutions (at pH 4.00 and 7.00) prior to testing. A 10.0 g sample of each powder was weighed using an analytical balance (AE ADAM, PW 214, max wt. 210 g) in triplicate. Distilled water (100 mL) was added to the sample and stirred using a glass rod for about 5 minutes prior to being introduced to the pH meter, and the pH value of the sample being read from the screen. Statistical analyses were conducted using Statistical Package for the Social Sciences (SPSS) to compare the mean pH values across samples.

Assessment of mineral and heavy metal content
Ngirimbo samples underwent analysis using atomic absorption spectrophotometry (AAS) for the presence of iron (Fe), zinc (Zn), calcium (Ca), magnesium (Mg), copper (Cu), and heavy metals, lead (Pb), chromium (Cr), and cadmium (Cd). Each sample was treated with 6 mL of 6 M hydrochloric acid, HCl (aq) and dried on a hot plate. After cooling, 10 mL of 6 M HCl (aq) was added to the residue in the crucible and heated to boiling using a hot plate. The sample was cooled at ambient temperature and filtered quantitatively into a flask. The solution was diluted to the 100 mL mark with distilled water. A control sample was also treated in the same way as the samples, but contained only the reagents added to the sample and no ngirimbo.

Calculation of calibration curves
In order to calculate the calibration curves for each element of interest, standard samples were prepared for Fe, Zn, Ca, Mg, Cu, Pb, Cr, and Cd. This was conducted using a 1000 mg/L stock solution of each metal solution, whereby standards were prepared at serial dilutions of 0 (control), 0.1, 0.2, 0.5, 1.0, 1.5, 2.0, 2.5 and 5.0 mg/L for each element.

Analysis of ngirimbo samples
Each standard and sample (including the control sample) underwent AAS following the manufacturer's protocol. The results are presented in mg/100g sample which was obtained using the following formula:

Assessment of volatile compound content
Samples underwent gas chromatography-mass spectrometry (GC-MS) analysis to determine the presence and concentration of volatile compounds. 1.5 g of each powdered sample was accurately weighed using an analytical balance (AE ADAM, PW 214, max wt. 210 g), 2 . Distilled water (20 mL) was added to each sample, followed by 40 mL of n-Hexane, and finally 10 mL of 2 M sodium hydroxide, NaOH (aq). The mixture was stirred for about 5 minutes using a glass rod. The sample was transferred into a separating funnel and shaken thoroughly for about 5 minutes, before being left to stand for about 20 minutes until two distinct layers formed. The aqueous (bottom) layer was removed and the organic n-hexane (top) layer was transferred into a stoppered conical flask. Anhydrous sodium sulphate, Na 2 SO 4 (s) was added as an inert drying agent. The sample was filtered into a 2 mL vial using a 0.45µm filter membrane (nylon syringe filter).

Sample pH
The pH level of the 12 samples was found to range from 7.5 to 10.7 with a mean of 9.2. Sample GPA6 had the highest pH of 10.7 and sample GPA7 gave the lowest value of 7.5.

Concentration of metallic ions in ngirimbo.
The concentrations of metallic ions, Fe, Zn, Ca, Mg, and Cu, within ngirimbo samples are expressed in Table 2, in units of mg/100g.

Concentrations of heavy metals
Various heavy metals are categorized by the International Agency for Research on Cancer (IARC) as group 1 and 2 carcinogenic metals, signifying that they are known and probable human carcinogens. Cadmium and chromium are classified as group 1, and lead as group 2. The presence of these metals in ngirimbo was of particular interest in this study as it poses a potential threat to humans. Cadmium and chromium levels varied from 0.00mg/100g to 0.89gm/100g in all samples while lead was not detected in any of the samples.

Nicotine concentration
Nicotine concentration varied from 0.97% to 2.79% across all samples, above the standard sample (for comparison, nicotine gum typically reports a concentration of 0.32%).

Volatile compounds in ngirimbo
Ngirimbo powder is a mixture of substances, containing a variety of organic volatile compounds as presented in Tables 1 to 12 in supplementary material. Ngirimbo was found to contain high levels of

Discussion
Previous findings suggest that the total nicotine content in local smokeless tobacco products (LSTP) such as ngirimbo is the primary determinant of product consumption and consumer attractiveness 3 .
The product pH is a significant element to the net nicotine dose obtainable from using these products.
A variety of volatile compounds (Tables 1 -12) found in the ngirimbo samples analyzed in this study show a variation in product acidity, which may in turn modulate levels of nicotine absorption and toxicity. This may create an avenue for manufacturer competition for consumer preference, and also impact the potential for addiction 4 . Studies indicate that smokeless tobacco products contain greater levels of nicotine and carcinogenic compounds 5,6 in comparison to nicotine gum 7 . These findings are in agreement, in it can be concluded that ngirimbo contains harmful chemicals, high pH and nicotine levels as shown in Table 1b, and Table 4, and Tables 1-12  Alpha-nicotine is a nicotine analog that has a high affinity for nicotine receptors. It has the ability to mediate the release of neurotransmitters, which makes it a good candidate for treating psychiatric disorders, such as schizophrenia, and neurodegenerative diseases, such as Alzheimer's disease. In addition, it can be used as a smoking cessation agent 12 . Animal studies have shown that the compound has no toxic effects 11 . Therefore, the use of alpha-nicotine could be used to replace the use of ngirimbo and other conventional nicotine products to reduce the toxicity of substances consumed, depending on type of nicotine product taken and the level of intake.
Furthermore, these analysis found that ngirimbo samples contained high levels of trace metals of clinical importance (see Table 2), as well as low levels of heavy metals (Table 3). Assessing the concentrations of these metals present in ngirimbo is highly relevant for assessing the safety of the product as these metals are involved in regulating normal physiological processes in the human body.
Increasing the concentrations of these metals beyond the physiologically normal range has the capacity to cause harm to human health 13 .
Iron, copper, and zinc are essential minerals. The recommended daily intake of iron is 8-18g, copper 0.9mg, and zinc 8-11g 14 . Excessive iron intake may lead to build up of iron in tissues and organs exacerbating hemochromatosis 15 , while high copper intake can cause functional damage to organs such as the liver 16 . Zinc can suppress the function of the immune system 17 , but conversely can aid in the treatment of depression 17 .
Magnesium plays useful roles in reducing hypertension, and improving insulin sensitivity and lipid profiles in patients at risk of cardiovascular diseases. Nonetheless, a high magnesium level of 2.6 mg/dL or above for a prolonged time can cause hypermagnesemia and associated toxic effects 18 .
Calcium is important in bone development, however, too much calcium can cause hypercalcemia 19 .
In this study, the samples tested showed concentrations exceeding the recommended daily intake levels of copper, magnesium, zinc, calcium, and iron. This is likely to be due to environmental, occupational, biological, and geographic sources of these elements resulting in build-up within the tobacco plants. It is important to note that the range of typical daily ngirimbo intake for consumers has not been established. Further, toxicity levels for ngirimbo and many of its contaminants have not yet been determined.
Tobacco has been identified as a source for many heavy metals such as cadmium, chromium, and lead 20 . Animal studies have shown cadmium to be a cytotoxic agent with the potential to cause cancer and hemolysis even in low availability, 21 . Through animal, toxicological, human, and epidemiological studies, it has been shown that hexavalent chromium is toxic and carcinogenic, with any amount of hexavalent chromium entering cells having the potential to initiate tumor formation 22 .
Conversely, trivalent chromium has been proposed as a dietary supplement, with the ability to induce insulin sensitivity in humans with insulin resistance (pre-diabetes), and improve impaired blood glucose tolerance 23,24 . It is necessary to note that ngirimbo consumption may increase the risk of hexavalent chromium or trivalent chromium intake and toxicity when other contributing sources of chromium are considered. Aside from the heavy metals mentioned above, the present study has detected no presence of lead in ngirimbo (Table 3). This provides some reassurance that LSTP use is unlikely to cause complications associated with lead consumption.
In addition, it should be noted that the extent and degree of metal accumulation in plant products is determined by the geographical region of cultivation, climatic circumstances, soil pH and chemistry 25 .
As such, human exposure to higher volumes of the metallic ions detected in ngirimbo may cause different clinical side effects depending on the baseline levels of the compound, the quantity absorbed or consumed through ngirimbo use, and on the target organ.

Conclusion
The results of this analysis demonstrate that harmful volatile chemical compounds in ngirimbo are catalysts that elevate acidity levels, toxicity, and the carcinogenic capacities of cadmium and nicotine associated with ngirimbo use. Conversely, the analysis suggests that the nicotine antagonist and a potential smoking cessation agent, 2-(1-methyl-2-pyrolidinyl)-pyridine, (S), is available in ngirimbo.
These findings demonstrate the need for an in-depth analysis of the raw materials that are used in the production of ngirimbo. This information will then be critical to inform the widespread use of ngirimbo as a smoking cessation aid. Trace metals and chromium must be minimized to avoid toxicity and risk to human health when dietary and environmental exposures are also taken into consideration.
In view of this evidence, it is recommended that the production of LSTP should be placed under quality control procedures according to national guidelines in Malawi, and that producers be given licenses under government observation. Furthermore, it is necessary to refine production methods to reduce the incidence of potential addictive and toxic compounds in ngirimbo such that it can be recognised as a tobacco harm reduction product. Consequently, future research is needed to determine recommended dosing and precautionary measures, with concrete evidence of the adverse effects on living organisms, such that this information can be published on the label of the product.
Consequently, the findings of this study provide an impetus for further research, as follows:  To determine population behaviour regarding the quantities of ngirimbo consumed, and the concentrations of nicotine obtained from varying levels of use.
 Potential toxicity with varying levels of ngirimbo use.
 Whether there is any relationship between ngirimbo consumption and the absorption of metal ions.
 A reconnaissance survey of ngirimbo use.