The P.campestris is used as a traditional medicine in curing erectile dysfunction without any scientific data on its toxicity profile to date. Since toxicological screening is a crucial tool to evaluate the safety of drugs or plants , acute and sub-acute oral toxicity studies of P.campestris extract were conducted. The rats are one of the most important animal models in toxicology. Hematological, respiratory, and cardiovascular adverse effects have the greatest cumulative comparability of toxicity in animals and humans . However, the toxicity observed in animals is poorly correlated with certain adverse effects like hypersensitivity and idiosyncratic reactions in humans. Besides, side effects such as headache, stomach pain, dizziness, and visual hallucinations are also difficult to detect in animals. Moreover, it is difficult to extrapolate certain adverse effects between the species due to the pharmacokinetic variations between the species. However, rats are the primary predictive models for human effects in toxicity assessments  and the study was done on rats as per OECD guidelines [11, 13].
The phytochemical screening is usually done for the aphrodisiac plants to detect the phytoconstituents like carbohydrates, alkaloids, proteins, amino acids, tannins, phenolics, saponins, flavonoids, triterpenoids, steroids, glycosides, fixed oils, gums, and mucilages. So far no studies were conducted on P.campestris except a study that identified a new kaurene diterpene dimer . However, the Parinari species showed a predominance of flavonoids glycosides based on myricetin, quercetin, and kaempferol . The present study focuses only on the toxicological evaluation and the second phase of this ongoing research is aimed to carry out the phytochemical analysis and evaluation of the aphrodisiac potential of P.campestris.
The acute toxicity tests did not exhibit any mortality, morbidity, unusual behavior, and adverse clinical signs in all the animals. The P.campestris extract can therefore be considered non-toxic up to 2000 mg/kg single oral administration and can be classified as a Class 4 drug as per the acute toxicity classification criteria for substances [13, 19].
The P.campestris stem bark is consumed as an herbal brew (approximately 20–50 g of bark infused into alcohol for a week) or tea (a spoon of stem bark powder, roughly 5–8 g, added to one liter of boiling water). It can be interpreted that the LD50 dose was much higher than the amount consumed in conventional medicine by considering the yield percentage (3.2 % w/w) and the assumed LD50 (2000 mg/kg) of the P.campestris extract in the present study. It should also be remembered that, relative to humans, rodents are more vulnerable to oral toxicity .
Toxicological assessments provide a dose-response relationship on potential health risks after repeated administrations. Thus, three different doses (100, 300, and 1000 mg/kg/day) were tested in both sexes for 28 days in a subacute oral toxicity study. The non-significant increase in body weight every week indicated that the feed and water intake was proportionate to the normal growth and physiology. However, a significant increase in body weight in the 100 mg /kg group on week 3 and the 100 and 300 mg/kg groups on week 4 and in the 100, 300, and 1000 mg/kg groups on the day of sacrifice (fasted body weight) in males indicated the treatment-related effect. This increase was not considered as an adverse effect since it could be due to body fat accumulation  or muscular development during the treatment due to the possible aphrodisiac effect. In the females, the treatment-related effect was seen as a decrease in body weight and body weight gain in the high dose (1000 mg/kg) group which could be a dose-dependent effect. It needs further investigation as body weight decrease was seen only in the 1000 mg/kg group So, the above results indicate that P.campestris extract increases the weight in males.
The detrimental effect of the test drug on metabolism is demonstrated by any major changes in food and water consumption . No treatment-related effect was noticed in the feed ad water intake in the present study. Therefore, the P.campestris extract did not cause any change in appetite or thirst and alter the metabolic system on long-term administration.
The relative organ weight is considered one of the primary indicators of organ toxicity. The relative organ weights did not differ significantly except for the ovaries. The increase in relative ovaries weights was not dose-dependent as the increase was seen only in 100 and 1000 mg/kg groups. Therefore, it is unlikely that P.campestris extract resulted in organ-level toxicity in rats.
The hematopoietic system is the common and sensitive target for toxic substances. It serves as a broad indicator of the overall physiological and pathological status of the body . When translating data from animal research, the hematological parameters provide a higher level of predictability of toxicity in humans . All the hematological parameters studied in the present study were within the reference range for rats  and the values of treated animals were comparable to control except for an increase in PLT in the 300 mg/kg group. The increased PLT value in the 300 mg/kg treated animals was also within the normal range and was not dose-dependent, hence it was considered as an incidental finding. As a result, the P.campestris extract was found to have no harmful effects on the hemopoietic system. However, the present study did not evaluate all hematological parameters including Complete Blood Count (CBC), clotting-related parameters, which must be further studied.
The liver function can be assessed by measuring the levels of protein, bilirubin, and liver enzymes . The elevated levels of AST and ALT are usually associated with liver damage . All the biochemical parameters studied in the present study showed no significant changes except a significant decrease in AST levels in the 300 mg/kg group. The most sensitive marker for hepatocyte damage is ALT, which is found mainly in the liver whereas AST is also found in red blood cells, cardiac and skeletal muscles, and kidneys apart from the liver. Further, the mean values of AST were low at the 300 mg/kg group whereas, at the 1000 mg/kg group, it was increased and levels were comparable with control. So, it can be considered biologically not significant and could be an incidental finding. This statement was further confirmed with normal liver histology in 300 mg/kg treated rats. So, it can be concluded that the P.campestris extract is not toxic to the liver.
The increased levels of blood urea nitrogen and creatinine can indicate the impaired kidney function . In the present study, the values of urea, creatinine, sodium and potassium ions, glucose, and other parameters related to kidney function were within the normal range and did not differ significantly in all the animals. So, the P.campestris extract did not harm the kidney.
The histopathological examination is used to back up the hematological and biochemical findings . All the organs showed normal architecture comparable to control in the histopathological study. Although some variations were noticed, they were minimal and matched the control group observations, and were not dose-dependent. The lungs showed a thickened alveolar wall with mild congestion in all the groups including control animals, so it was not considered as a treatment-related effect. The results of the histopathological study validate the claim of P.campestris extract to be non-toxic.
Based on the above findings, the No Observed Adverse Effect Level (NOAEL) for P.campestris extract is considered as 1000 mg/kg/day. According to the FDA  guideline, the human equivalent dose (HED) based on the body surface area was computed as 1000 mg/kg divided by 6.2, which is equal to 161 mg/kg body weight in humans, and 16.1 mg/kg body weight in humans is a safe dose of P.campestris extract by oral route in humans, recognizing the safety factor of 10 to HED. Therefore, for an average body weight of 60 kg, the safe human dosage is 966 mg (16.1 × 60) or approximately 1 g, which can be consumed orally for less than 28 days without any adverse effects in males.
Additional studies of genotoxicity, carcinogenicity, and teratogenicity  are also required to be done as they will strengthen the safety profile of P.campestris extract. This limitation, however, is suggested to be the potential course of our future research.