*3.2. Subacute 28-Day and Chronic 6-Month Oral Toxicity Studies*

### 3.2.1. Clinical Signs and Mortality

The TS did not produce any mortality or toxicology-related clinical signs due to the consumption of the test item in either study. However, in the subacute study, one female TS in the medium dosage group on day 18 died due to a gavage accident. While in the chronic study, five females given 0 g/kg BW (day 56), 0.14 g/kg BW (day 121), 0.29 g/kg BW (two TS; day 44 and day 126) and 1 g/kg BW (day 158) and 2 males given 0 g/kg BW (day 158) and 1 g/kg BW (day 76) died also due to gavage accidents. Autopsy revealed no treatment-related alteration in the dead TS.

### 3.2.2. Body Weights, Food and Water Intake

The subacute study showed that the mean body weights for both males and females did not depict any significant difference between the control and the treated groups. The weekly mean body weight also increased gradually over the dosing period (Figure 1a,b). The food consumption (Table A2) was noted to be normal for growing TS and no significant increase or decrease was evident.

**Figure 1.** In-life parameters monitored in male and female test systems: (**a**) Mean body weight (BW) of male test systems in the 28-day subacute toxicity study (red line) and the 6-month chronic toxicity study (dotted blue line). (**b**) Mean body weight of female test systems in the 28-day subacute toxicity study (red line) and the 6-month chronic toxicity study (dotted blue line). (**c**) No difference in the body weight (BW) gain in male and female test systems of the 6-month chronic toxicity study. (Group 0.29 g/kg BW female (S): n = 5, week 1–2; n = 4, week 3–4. Group 0 g/kg BW male (C): n = 20, week 1–22; n = 19, week 23–24. Group 1 g/kg BW male (C): n = 20, week 1–11; n = 19, week 12–24. Group 0 g/kg BW female (C): n = 20, week 1–8; n = 19, week 9–24. Group 0.14 g/kg BW female (C): n = 20, week 1–17; n = 19, week 18–24. Group 0.29 g/kg BW female (C): n = 20, week 1–6; n = 19, week 7–18; n = 18, week 19–24. Group 1 g/kg BW female (C): n = 20, week 1–22; n = 19, week 23–24). S = subacute, C = chronic.

**Figure 2.** In-life parameters monitored in male and female test systems: (**a**) Mean food consumption of male test systems in the 6-month chronic toxicity study. (**b**) Mean food consumption of female test systems in the 6-month chronic toxicity study. (**c**) Mean water intake of male test systems in the 6-month chronic toxicity study. (**d**) Mean water intake of female test systems in the 6-month chronic toxicity study. Each graph is expressed as mean ± SD (Group 0 g/kg BW male: n = 20, week 1–22; n = 19, week 23–24. Group 1 g/kg BW male: n = 20, week 1–11; n = 19, week 12–24. Group 0 g/kg BW female: n = 20, week 1–8; n = 19, week 9–24. Group 0.14 g/kg BW female: n = 20, week 1–17; n = 19, week 18–24. Group 0.29 g/kg BW female: n = 20, week 1–6; n = 19, week 7–18; n = 18, week 19–24. Group 1 g/kg BW female: n = 20, week 1–22; n = 19, week 23–24).

In the 6 months chronic study, female rats administered 1 g/kg BW dosage of the test item had a significant (*p* < 0.05) increase in body weight each week (Figure 1a,b). However, there was no significant difference in the percentage body weight gain throughout the study of TS administered 1 g/kg BW as compared to the control group in either gender (Figure 1c). Overall, elevating trends in body weight was observed weekly. Looking at food consumption (Figure 2a) in males, a significant increase was observed in weeks 15, 21 and 22 in the 0.29 g/kg BW dosage group when compared to the control group. A significant increase in food intake was seen in females (Figure 2b) given 1 g/kg BW dosage in weeks 11 and 15 when compared to the control group. In week 17, a significant increase in food intake was recorded for all the treated groups. Female TS given 1 g/kg BW showed a significant decrease in food consumption in week 3.

The water consumption trend (Figure 2c) in male TS showed that TS administered 1 g/kg BW consumed less water in weeks 7, 8, 12 and 13, while those given 0.29 g/kg BW only showed a decrease in week 8. Female TS given high doses of the test item (1 g/kg BW dosage) had a significant increase in water intake in weeks 1, 8–10 and 18 (Figure 2d). A significant increase in water intake of female rats given low and medium dosage test item occurred in week 8 and week 22, respectively.

#### 3.2.3. Hematology Analysis

No abnormal levels were detected in the hematological analysis of the blood samples for the subacute study (Table 1). After the 6-month prolonged exposure, the hematologic profile (Table 2) showed a decrease in the platelet (PLT) count in all treated male dosage groups, while other parameters did not show any significant difference. Females given 1 g/kg showed a significant increase in HGB levels when compared to the control group.


**Table 1.** Hematology values of male and female test systems in the 28-day subacute study.

Values expressed as mean ± SD (n = 5 for all groups, except n = 4 for group 0.29 g/kg BW female). No significant changes were found (*p* > 0.05). Body weight, BW; white blood cells, WBC; red blood cells, RBC; hemoglobin, HGB; hematocrit, HCT; mean corpuscular hemoglobin concentration, MCHC; platelets, PLT.

**Table 2.** Hematology values of male and female test systems in the 6-month chronic study.


Values expressed as mean ± SD. BW = body weight. \* Significantly different from control group at *p* < 0.05.

#### 3.2.4. Clinical Biochemistry Analysis

The liver and enzyme parameters showed reduced levels of albumin, alkaline phosphatase (ALP), alanine amino-transferase (ALT) and triglyceride in males administered 0.14 g/kg BW (Table 3). Significantly elevated total protein, creatinine and urea levels in males administered 0.29 g/kg BW was observed. There was also a significant increase in creatine kinase level in males given 1.0 g/kg BW dosage. In female test groups, elevated albumin, ALT, aspartate amino-transferase (AST), creatinine, urea, cholesterol and calcium levels were observed in the 0.29 g/kg BW group. The 1 g/kg BW group had a significant increase in lactate dehydrogenase (LDH), cholesterol and calcium levels, while there was a significant decrease in the triglyceride level.

#### 3.2.5. Organ Weights

In the subacute study, there was no significant difference in the relative organ weights (ROW) of any organs in males. Some ROW in females were significantly decreased, including stomachs in the 0.14 g/kg BW group and spleens for the 0.14 and 0.29 g/kg BW groups (Table 4). In the 6-month chronic study, no significant differences in the ROW was observed in any of the collected organs for all the tested groups (Table 5).

#### 3.2.6. Gross Necropsy and Histopathology Examination

No abnormalities were visually detected when gross pathological examination was carried out postmortem. Microscopic histopathology revealed no abnormalities.




 group at *p* < 0.05.



#### **4. Discussion**

Herbal-infused beverages have been introduced into the market where countless other beverages are marketed. With the extra edge of having herbal concoctions incorporated, herbal additives in beverages have gained popularity and are widely consumed in Malaysia. A rat model was used to investigate the acute and prolonged repeated consumption of such product to establish its safety profile and infer its effects at its expected exposure dose in humans.

The test item did not exert any acute toxicity and no mortality was observed at any of the tested doses. In both the subacute and chronic studies, there were no significant clinical signs of toxicity, body weight changes were normal and gross and histopathology investigations did not reveal any significant toxicity effect. There was a total of seven incidental deaths in the repeated dose studies due to technical error by personnel. The gavage error may have led to gavage-assisted reflux in the TS causing these deaths [26]. We report the following findings, namely fluctuations in the food and water intake measurements, the relative organ weights (ROW) in two organs in the subacute study and platelet changes observed in the chronic study with some alterations to the clinical biochemical parameters.

The statistically significant reduction in food consumption which only occurred in the female high dose group in week 3 of the chronic study and the increased water intake in some female treated groups may indicate natural physiological changes. It has been reported that female TS consume less food and become active during their estrus cycle which may incur responses as manifested in the amount of food and water consumed, as seen in this study [27,28]. On the other hand, males in the chronic studies consumed significantly less water, where coffee consumption itself may have caused the TS to drink less water [29]. Despite the fluctuating food and water intake, the body weight measurements showed elevating trends, indicating normal development of the TS. Similar body weight trends were also reported by other authors [30–32]. Hence, it is concluded that the fluctuations observed in the food and water intake did not cause any treatment-related effects in the TS.

In the subacute female group, there was a reduction in ROW for the stomach (0.14 g/kg BW group) and spleen in the 0.14 and 0.29 g/kg BW dosage groups. There was no clear dose-relationship in reduction of ROW as the highest dose group did not produce any changes. Further microscopic examination of these two organs did not show any correlation to the decrease in size. As the changes were only observed in the subacute female group and prolonged exposure of six months revealed no effect on any of the organs inspected in either gender, these two observations are not considered treatment-related.

No significant differences were seen in the hematology profile of the 28-day study of the treated groups when compared to the control group. The hematology parameter findings for the subacute and chronic studies were similar to published control data, except for the WBC, HGB, MCHC and PLT counts, where slight differences were found between the two reference intervals [31,33]. After prolonged exposure of the test item over a substantial lifetime of the TS, some changes in the blood investigations were observed, where platelet numbers of males from all the treated groups significantly decreased. A drop in the platelet reading may be a cause of concern as they are primarily involved in blood coagulation and hemostasis [34]. Coffee by itself has been shown to reduce platelet activation resulting in reduced platelet aggregation though platelet aggregate formation was not evaluated in the current study [35]. The decreased platelet levels recorded were lower than the average platelet levels in males when compared to the reference interval by He et al. (2017) but were comparable to the reference values from Delwatta et al. (2018) [31,36]. However, the decrease was not dose-dependent, as the highest dose did not further reduce the platelet reading compared with the lowest dose.

Creatinine, urea, cholesterol, triglyceride and glucose levels were similar to published control data [31,36]. It was noted that there were large differences in the control data range published. Hence, the control group data in this study were used for all statistical analysis as the control group TS were subjected to the same environment as the treated groups. Elevated renal profile (creatinine and urea), total protein and creatine kinase in the male treated groups (0.29 and 1 g/kg BW dosage) and elevation of the liver, renal and lipid profiles in the female treated groups (0.29 and 1 g/kg BW dosage) may indicate the TS early response to the test item. A recent study by Riza & Andina Putri et al. (2019) also found an increase in creatinine levels in rats administered coffee [37]. However, in our current study, no further correlation in liver nor kidney histopathology were apparent. No dose-dependent conclusion can be drawn from the changes observed in the 0.29 g/kg BW group, as the highest dose did not exacerbate the parameters measured. Contrarily, the consumption of coffee has been reported to decrease liver enzymes associated with liver damage and has shown protective effects attributed to its polyphenol content [38–43]. There were favorable significant reductions in liver enzymes (ALP and ALT) and lipid profile indicator (triglyceride) in the low male dose group but no trend was evident in the higher dose groups except in the female triglyceride level when given the high dose.

The interactions between the different components present in both coffee and TA and additionally with food may have exerted their effects on the parameters measured in this study. Both coffee and TA are made up of various phytochemicals, such as caffeine and polyphenols, found in coffee, and eurycomanone, a major compound found in TA [1,39,40]. Caffeine has been well studied and is reported to have a high tolerance dose of 400 mg/day in human [44–46]. Although eurycomanone has been reported to have low bioavailability in rats [47,48], its pharmacokinetic behavior may be modified when incorporated with coffee. A recent study reported coffee's potential to alter acetaminophen drug pharmacokinetic profile when consumed together [49]. Due to this multi-component nature and interactions, safety evaluation of herbal incorporated food products is crucial.

In this study, the toxicity was assessed in one mammalian rodent model, whereas regulatory submissions may require the safety tests to be conducted in more than one species, typically in either rats, mice or rabbits and in dogs to compare the effect and rate of severity of the toxicity [50]. Despite this limitation, the study was conducted for three different durations that covers the acute response of the administered product, as well as the repeated exposure. The studies covered a substantial duration of the rodents' lifetimes. Therefore, conclusions drawn from this study are exhaustive.

Considering an average consumption of 2 cups (2.5 g test item per cup) per person per day, an average adult (70 kg body weight), ingests on average 0.07 g/kg body weight of the test item daily. The calculated human equivalent dose (HED) for 1 g/kg BW in rats is 0.16 g/kg BW in humans. As the calculated HED is twice the predicted average daily consumption dosage, the present study suggests that the highest dose tested in the prolonged toxicity study (1 g/kg BW per day) of *E. longifolia* infused coffee, may be well tolerated for human consumption.

### **5. Conclusions**

In conclusion, the highest single dose administration of TA coffee (2 g/kg of body weight) did not show acute oral toxicity in Sprague Dawley rats in the present study. The 28-day subacute toxicity study reported no toxic change in any observed parameters. No dose-dependent nor morphological changes were attributed to the consumption of TA coffee in the 6-month chronic toxicity study and consumption of TA coffee at a dose of 1 g/kg/day in male and female rats was identified as the no observed adverse effect level (NOAEL).

**Author Contributions:** Conceptualization, N.A., B.P.T. and H.M.; Methodology, S.Z.H., N.A.Z. and N.R.; Software, N.A., S.Z.H. and N.R.; Validation, N.A., B.P.T. and S.Z.H.; Formal Analysis, N.A., B.P.T. and S.Z.H.; Investigation, S.Z.H., N.A.Z. and N.R.; Resources, N.A. and H.M.; Data Curation, N.A. and S.Z.H.; Writing—Original Draft Preparation, N.A. and B.P.T.; Writing—Review and Editing, N.A., B.P.T. and H.M.; Visualization, N.A., B.P.T. and H.M.; Supervision, H.M.; Project Administration, N.A. and H.M.; Funding Acquisition, N.A. and H.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Ministry of Agriculture, Malaysia under the ScienceFund Grant (05-03-20-SF1001) and the Ministry of Health, Malaysia.

**Acknowledgments:** We would like to thank the Director General of Health Malaysia for his permission to publish this article. We thank GTHerb Industries Sdn. Bhd. for providing the TA coffee. The authors would also like to thank Zakiah Ismail and Noor Rain Abdullah for their perceptive guidance in this research.

**Conflicts of Interest:** The authors declare no conflict of interest.



**Table A1.** Summary of 14-day acute study findings. The body weight, food intake and percentage body weight gain values are expressed as mean ± SD of 5 test systems given the 2 g/kg dosage in the acute study. No comparison was made as the method used was fixed dose procedure with emphasis on the acute clinical response and no acute death was observed. BW = body weight, NAD = abnormality detected.

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**Table A2.** Food consumption of male and female test systems in the 28-day subacute study.

