4.2.2. Animal Studies

## (1) Acute and Subacute Toxicity

Piyachaturawat et al. investigated the acute toxicity of piperine (dissolved in equal volumes of DMSO and 95% ethanol) following a single administration via intragastric (i.g.) gavage to mice and rats [56]. For adult male mice, the resulting calculated LD50 value was 330 mg/kg bw, compared to an LD50 of 15.1 mg/kg bw based on i.v. administration. In the same study, an LD50 value of 514 mg/kg bw was derived for single i.g. administration of piperine to adult female rats, while a higher LD50 value was calculated for young female weanling rats (LD50 > 585 mg/kg bw). Animals receiving lethal doses experienced convulsion and died of respiratory paralysis [56].

In another study with mice, all animals survived oral doses of 143 to 574 mg/kg/day given on two consecutive days, but displayed lethargy ranging from a slight degree in the low-dose group to a severe one in the high-dose group [55].

In a subacute oral toxicity study with female rats receiving 0, 100, 250, 350 or 500 mg piperine/kg bw/day for seven days, the body weight gain of animals receiving 100 mg/kg bw was comparable to that of the control group. Daily doses of 250 mg/kg bw led to reduced body weight gain and caused haemorrhage in the stomach of 3 out of 8 animals, whereas the higher doses of 350 and 500 mg/kg bw caused the death of 2 and 5 out of 8 animals, respectively. Animals receiving 500 mg piperine/kg bw/day displayed histopathological changes of different types and degrees in the stomach, urinary bladder, adrenal glands and small intestine. In addition, luteal cells in the central portion of the corpora lutea were degenerated in this dosage group [56].

#### (2) Subchronic Toxicity Studies

In a study with groups of young male rats that received 100 mg piperine/kg feed for 56 days or 110, 220 or 440 mg pepper oleoresin/kg feed (equivalent to 50–200 mg piperine/kg feed), or 2 g pepper/kg feed, no adverse effects on growth, food efficacy, organ weights, blood count and investigated clinical chemistry parameters were observed compared to the control group [50,57]. In its evaluation of piperine as a flavouring agent, JECFA (2006) based its NOEL for piperine of 20 mg/kg bw/day on this animal study, but

EFSA (2008) considered this study inadequate for NOAEL identification due to studydesign limitations [48,50]. It should be mentioned that this animal study reported increased haemoglobin values for the piperine group (242 g/L) compared to the control and to the pepper/pepper oleoresin groups (~140 g/L); however, the red blood cell count was similar to the other groups, making a typing error highly possible [57].

The subchronic toxicity study (90-day study) in rats that was finalised in 2013 and used by EFSA (2015) [32] for the assessment of piperine as a flavouring agent has been published meanwhile (Bastaki et al. (2018) [29]). In this study, 0, 5, 15 or 50 mg piperine/kg bw/day were administered via feed for 90 days. According to the EFSA assessment, the reduced weight gain observed in the highest male dose group was due to reduced feed intake (possibly related to food palatability). There were no mortalities, no gross and microscopic changes nor clinical pathology or organ weight changes attributable to piperine. Some statistically significant changes in haematology, coagulation or clinical chemistry parameters were considered by EFSA as not dose-dependent, small in magnitude and within the range of historical values. However, statistically significant dose-dependent increases in cholesterol levels were observed in male animals receiving 15 and 50 mg piperine/kg bw/day (approximately by 30 and 55%, respectively), which was used by EFSA for NOAEL identification.

According to EFSA, reduced relative epididymides weights were observed in male animals administered 5 and 50 mg/kg bw/day, respectively, but these changes were considered small and not dose-dependent and therefore of limited toxicological relevance [32]. Regarding this finding, it is noted on the one hand that reduced relative (to-brain weight) epididymis weights (17–23%) were observed in all three piperine dosage groups without displaying dose dependence, with reductions being statistically significant only in the 5 and 50 mg/kg bw dosage groups. On the other hand, it must be mentioned that these changes were without histopathological findings. From this study, EFSA identified a NOAEL of 5 mg piperine/kg bw/day due to the dose-dependent elevated cholesterol plasma levels in male animals at the mid and high dose (15 and 50 mg/kg bw/day) [32].

Contrary to the EFSA (2015) assessment, Bastaki et al. (2018) ascribed no toxicological relevance to the observed increases in cholesterol levels described above because, in their opinion, the cholesterol levels were within the historical control range for male animals and because of corroborating evidence from other studies showing an absence of a cholesterol increase. Rather, they identified a NOAEL of 50 mg piperine/kg bw/day (the highest dose tested) from this study [29].

#### (3) Paternal Reproductive Toxicological Effects

In 1999, as a consequence of administration of 0, 5 or 10 mg piperine/kg bw/day (suspended in 0.9% saline) via a gastric catheter to young adult male rats for 30 days (n = 10 per group), Malini et al. [58,59] reported in the high-dose group statistically significantly (*p* < 0.05) reduced sperm concentrations in caput and cauda epididymides, statistically significantly reduced relative weight of testis (relative to body weight) and reduced absolute weight of the cauda epididymides, vas deferens, seminal vesicle and ventral prostate. The relative organ weights were also reduced, according to the authors' own calculations based on the mean body and organ weights stated in one of the two publications by Malini et al. [59]; however, no information on statistical significance is available for these calculations. Furthermore, histopathological changes in the testis, increased serum gonadotropins (FSH, LH) and reduced the intra-testicular testosterone concentration, as well as reduced the testicular lipid content, changes in the testicular lipid profile and reduced activity of some testicular lipogenic enzymes were observed (for details, see Table 1). Histopathological changes in the testes were also observed at 5 mg piperine/kg bw/day, but to a lesser extent than in the high-dose group. Within this low-dose group, the other parameters mentioned above showed changes in the same direction as seen in the high-dose group, but these changes were also of smaller magnitude and, in the majority of cases, no longer statistically significant. The reduced testicular weight was attributed to disturbed spermatogenesis and the reduced total lipid content of the testes caused by piperine administration [58,59].


**Table 1.** Paternal reproductive effects of piperine observed in animal studies.


**Table1.***Cont.*



169


**Table1.***Cont.* significantlystatisticallysignificantly;*p*reproductiveorganweightsstudy,publicationstestes weights and calculated indexes or a coefficient based on testes and body weights [62]. For this reason, no further detailed information on the weights of the male reproductive organs is provided here. Onlypotentiallyreprotoxicologicallyrelevanteffectsreported.othereffectsobservedthisstudy,(2)

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 see

 in Section 4.2.2.

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In another study [60], D'Cruz and co-workers administered 0, 1, 10 and 100 mg piperine/kg bw/day dissolved in a vehicle (10% DMSO in ethanol and groundnut oil at the ratio of 1:1) to young adult male rats via a micropipette for 30 days and observed in the 10 mg/kg bw group statistically significant (*p* < 0.05) reductions in the cauda epididymal sperm count and sperm motility, statistically significantly reduced absolute weight of the testes and caput, corpus and cauda epididymides, and reduced activity of the antioxidant enzymes in the testes and corpus and cauda epididymides, accompanied by increased hydrogen peroxide generation and lipid peroxidation. In this study, the observed effects in the 10 mg/kg bw group on epididymal sperm count and testicular and epididymal weight were markedly smaller than in the study by Malini and co-workers. In the highdose group (100 mg/kg bw), the effects were more pronounced and additionally included statistically significantly reduced sperm viability and significantly reduced relative testes weight (relative to body weight) (for details, see Table 1). Further immunofluorescence studies by the same group [61] revealed a dose-dependent increase in caspase 3 and FAS protein in testicular germ cells that was related to piperine administration and was accompanied by dose-dependent changes in the testicular antioxidant system (reduced activity of antioxidant enzymes, increases in hydrogen peroxide generation and in lipid peroxidation). In the low-dose group (1 mg/kg bw), no relevant reproductive toxicological effects were observed in either of the studies by D'Cruz et al. [60,61]. These studies used small animal groups with only four animals each, which reduces the scientific significance of the study findings.

In young adult male rats that orally received 10 mg piperine/kg bw/day (suspended in normal saline containing 0.5% carboxymethyl cellulose) for 60 days, Chinta and coworkers observed a statistically significantly (*p* < 0.05) reduced epididymal sperm count, sperm motility and sperm viability compared to the control group (the latter receiving the vehicle only), accompanied by histopathological changes in the testes and epididymides; statistically significantly increased serum gonadotropins (FSH, LH), with a reduced intratesticular testosterone concentration; and statistically significantly reduced activity of anti-oxidant enzymes or other enzymes in the testes and epididymides (for details, see Table 1) [16,62]. Data on reproductive organ weights were also recorded in this study, but in one of the two publications dealing with this study, inconsistencies were noticed between the testes weights and calculated indexes or a coefficient based on testes and body weights [62]. For this reason, no further detailed information on the weights of the reproductive organs is provided here. After a recovery period of 60 days without piperine administration, the observed changes were reversible.

Animals receiving the piperine dose (10 mg/kg bw) every 4th day in the same study displayed some of the abovementioned adverse effects that were also observed with the daily piperine administration, e.g., reduced sperm viability and mobility, which were less pronounced but in some cases still statistically significant (for details, see Table 1). With piperine administration (10 mg/kg bw) every 7th day, the individual parameters that were mentioned in relation to the daily piperine administration showed changes in the same direction but the changes were considerably milder and no longer statistically significant (e.g., sperm count, testicular testosterone). Chinta and co-workers concluded from their data that piperine might be a good lead molecule for the development of a reversible oral male contraceptive [16,62]. Animals receiving piperine every day displayed decreased body weight of about 10% after 60 days of piperine administration, which was not statistically significant. All animal groups receiving piperine (n = 6 per study group) showed statistically significantly reduced absolute liver weights (approximately 35–39% reduction compared to the control group) at the end of the piperine administration period. The authors did not comment on those data. These reductions were still existent in the group that had received piperine every day (about −9%) and the group that had received piperine every 4th day (about −41%) after the recovery period, without reaching statistical significance (*p* < 0.05). Due to the administration of piperine suspended with carboxymethyl cellulose, it is assumed that piperine was given as a bolus once per day.

In a fourth study with male rats (juvenile animals, 35 days old at baseline; n = 6 per group) that involved administration of 0, 5 or 10 mg piperine/kg bw/day for 30 days by gavage, stimulation of pubertal Leydig cell development (increased Leydig cell number and promoted maturation) and an inhibited spermatogenesis were observed. Regarding the latter effect, the authors only cite histological findings of the testes and epididymides and indicated that already the dose of 5 mg piperine/kg bw/day reduced the epididymal sperm count, but no concrete figures on sperm counts were provided. This lack of data presentation reduces the scientific weight of the evidence provided in this study. Serum testosterone levels were elevated and the FSH levels were lowered in both piperine groups [63,64]. In this regard, the findings in juvenile rats differ from those observed in older rats [59,62].

Overall, the findings from these four studies [16,58–64] largely point in the same direction, with some differences of observed adverse reproductive effects between young adult and juvenile male rats. Concordantly, in young adult male rats, reproductive toxicological effects, i.e., disturbed spermatogenesis (and accompanying effects on testes, epididymides and accessory male reproductive organs of different nature and degrees) were observed with intakes of 10 mg/kg bw/day [16,58–62]. The less pronounced effects, which were only partly statistically significant, were observed in adult male rats already at 5 mg/kg bw/day. From these studies, a LOAEL of 5 mg piperine/kg bw/day [58,59] and a NOAEL of 1 mg/kg bw/day [60,61] can be identified for the endpoint male reproductive toxicity (disturbed spermatogenesis). However, the study of D'Cruz and co-workers used a wide spacing between the tested piperine doses (factor of 10) [60,61]. In one study, adverse paternal reproductive effects observed with repeated daily piperine (bolus) doses of 10 mg/kg bw were reversible after piperine discontinuation for several weeks [16,62]. Based on information of the four studies on piperine administration, it can be assumed that piperine administration in these studies was carried out via bolus administration. In three of these four studies, piperine administration was via gavage, micropipette or gastric catheter, suggesting bolus administration. The piperine dosage form of the fourth study, i.e., piperine in carboxymethyl cellulose, suggests bolus application as well.

It is noted that these studies are afflicted with certain limitations (statistical analysis of organ weights mainly comprising data on the absolute organ weights and the data on relative organ weights not being available in most cases; small animal group sizes in some studies; in the study of Chen et al., only histological findings were cited but no concrete data on sperm counts were provided; and reduced absolute liver weights not having been reported in other animal studies at this daily dose and contradictory information on the parameters related to testes weights in the study of Chinta and co-workers). However, taken as a whole, the aggregated study findings all point in the same direction and the paternal toxicological reproductive effects, i.e., disturbed spermatogenesis, are corroborated by findings at different levels, such as histopathology, sperm parameters, hormonal changes and changes at the level of enzyme activities, as well as changes in absolute organ weights. The limited scientific significance of absolute organ weights is acknowledged; however, a statistically significant change in the relative testes weights was seen at least in one study [58] with daily doses of 10 mg/kg bw. The observed differences in hormone levels between the studies of Malini et al. and Chinta et al. on the one hand compared to Chen et al. on the other hand may be related to the different life stages of the investigated male animals (juvenile versus young adult rats) [16,58,59,62–64].

The question of whether the mode of piperine administration in the study by D'Cruz and co-workers (piperine dissolved in 10% DMSO, ethanol and groundnut oil) [60,61] or in the study by Chinta and co-workers (together with carboxymethyl cellulose) [16,62] could possibly affect the bioavailability of piperine, leading to increased adverse effects, remains elusive. Adequate data to compare the influence of these modes of administration with the influences of the currently available piperine-containing dietary supplements and the food additives or galenic technics used in their manufacturing on the bioavailability of piperine, are currently not available.

In contrast to the largely consistent findings from the four studies cited above, different results are available from the 90-day toxicity study with rats used by EFSA (2015) for the evaluation of piperine as a flavouring agent, and which has already been described in (2) in Section 4.2.2 [29,32]. In this study, intakes of 0, 5, 15 or 50 mg piperine/kg bw/day were administered via feed. In male animals of the 5 and 50 mg/kg bw-groups, statistically significantly reduced relative epididymis weights (relative to brain weight) were observed. EFSA attributed only limited toxicological relevance to these findings due to the small changes and the non-existent dose dependence, and these changes were without histopathological findings (see also (2) in Section 4.2.2 and Table 1). This study did not include any specific examinations of sperm parameters or LH and FSH blood levels, as these types of investigations are not common in 90-day toxicity studies.

In its assessment, EFSA (2015) did not address the findings of Malini et al. [58,59] and D'Cruz and co-workers [60,61], which were available at that time (since a review of the available scientific literature was not foreseen at this time as part of this assessment procedure).

A major difference between the four animal studies cited first [16,58–64] and the 90 day toxicity study used by EFSA seems to be that in the 90-day toxicity study, piperine was administered via feed, resulting in multiple intakes of small quantities spread throughout the day, whereas in the four first-cited animals studies, it can be assumed that piperine was administered as a bolus dose, possibly resulting in higher maximum blood or tissue levels or otherwise increased bioavailability. The bolus administration of piperine in the first four animal studies more closely resembles the usual human use of food supplements, which often bear recommendations relating to 1–3 doses per day.

In this context, it is noted that Daware et al. observed no increased numbers of abnormal sperm cells in a sperm shape abnormality test performed with male mice receiving daily doses of 35–75 mg piperine/kg bw for 5 days [54]. However, this test is primarily performed regarding a genotoxicity assessment.

In in vitro studies, reduced viability and motility of goat sperm cells were seen with high doses of piperine (40–100 μmol/L) added to the sperm culture media [65], as well as impaired fertilization ability of hamster sperms directly exposed to high piperine doses (180–1005 μmol/L) in the capacitation medium [66]. However, the scientific relevance of these in vitro findings remains elusive due to the high piperine concentrations used and the direct exposure of the sperm cells to piperine via culture media, which differs from the exposure of sperm cells resulting from oral piperine intakes.

The mode of action of the bolus doses of piperine on spermatogenesis and the accompanying effects on male reproductive organs remains elusive. With young adult male rats, it has been hypothesized that induced oxidative stress due to depletion of antioxidant enzymes and increased generation of reactive oxygen species (ROS) in epididymis and testis, and activation of the Fas-mediated pathway in testicular germ cells, may contribute to the observed antifertility effects. However, inhibition of the cytochrome P-450 enzymes or other enzymes involved in the synthesis of testicular steroid hormones, interaction of piperine with the active site of the androgen binding protein, induction of hormonal imbalances (effects on serum levels of FSH, LH, sex hormone-binding globulin and testicular testosterone) or other effects on the functional integrity of the testis and the male reproductive organs are also being discussed, and appear possible [16,61,62].

It is noted that even with high bolus doses of fine *Piper nigrum* fruit powder (25 or 100 mg/kg bw/day) administered for 20 or 90 days to male mice, negative effects on the sperm count in the cauda epididymis, sperm motility, viability and number of morphologically abnormal spermatozoa were observed (viability not affected with 25 mg/kg bw dose administered for 20 days), which increased with escalating daily dose and duration of application from 20 to 90 days. After 90 days of pepper powder administration, statistically significantly reduced relative weights (relative to body weight) of the testis, epididymis and seminal vesicle were observed in both dosage groups. No male animal receiving 100 mg/kg bw/day for 90 days (other animals were not examined) was fertile in mating trials with untreated female mice 24 h and 14 days after the termination of pepper administration,

respectively. The fertility of the treated male animals improved after an 8-week recovery period, but was still (statistically not significantly) reduced at this time point [67]. The fine fruit powder (suspended in water containing milk powder) was administered by a feeding needle; therefore, it can be assumed that the administration occurred as a bolus.

(4) Maternal Reproductive Toxicological and Embryotoxic Effects

Depending on the time point of piperine administration before or during pregnancy, different reproductive toxicological or embryotoxic effects were observed in female animals.

In the study by Daware et al. (2000), young female mice (n = 6 per group) receiving 0, 10 or 20 mg piperine/kg bw/day for 14 days until the day of mating with untreated male animals displayed a statistically significantly reduced mating rate in the high-dose group (mating performance: 50% versus 83% in control group) and in both piperine dose groups a statistically significantly reduced fertility index (fewer mated animals became pregnant; fertility index: 60% and 66%, respectively). With this piperine administration protocol, the litter size of the pregnant animals and the growth of the pups were not affected. When the piperine doses (10 or 20 mg/kg bw/day) were administered to female mice from Day 1 through to Day 5 of gestation, significantly reduced implantation rates were observed in both dose groups, with implantations in 1 out of 6 mated animals each in the low- or high-dose group, respectively, versus 6 out of 6 mated animals in the control group. The post-implantation survival was not affected. In this study, piperine was given suspended in a formulation containing 1% carboyxmethyl cellulose, which was most likely done by bolus administration [54].

Significant implantation-inhibiting effects were also observed in another study by Piyachaturawat et al. (1982) with mice receiving oral bolus doses of 2 × 12.5 or 2 × 50 mg piperine/kg bw/day (dissolved in equal volume of DMSO and 95% ethanol; n = 19–21) from Day 2 through to Day 5 of gestation, with the implantation rates reduced by 71% and 90%, respectively, compared to the control group receiving the vehicle only (other oral doses not investigated). In addition, significant abortive effects were observed in mice given the same piperine bolus doses (2 × 12.5 or 2 × 50 mg/kg bw/day; n = 17–21) from Day 8 through to Day 12 of gestation (interrupted pregnancies in 58.8% and 71.4% of pregnant animals, respectively; other oral doses not investigated). Bolus doses of 25 mg piperine/kg bw/day given from gestation Day 15 onwards (n = 8) resulted in delayed labour and significantly increased number of dead foetuses (6.1 dead foetuses/litter vs. 0.3 dead foetuses/litter in the control group; other oral doses not investigated) [68].

In a study with female rats (n = 6), a reduced implantation rate (33% reduction) was observed with an oral piperine dose of 100 mg piperine/kg bw/day administered from gestation Day 1 through to Day 7 (other doses not investigated) [69].

In female hamsters, which received intra-gastric daily doses of 50 or 100 mg piperine/kg bw/day from Day 1 through to Day 4 of the oestrus cycle, followed by hormonally induced superovulation and artificial insemination with spermatozoa of untreated male animals, increased fertilization of eggs in the early phase of fertilization 9 or 24 h after insemination were observed compared to the control animals [70]. However, due to the applied methodology (hormonally induced superovulation which might interfere with piperine effects on female reproduction, artificial insemination, no information on pregnancy outcome), the scientific significance of these findings remains elusive regarding the effects of piperine on maternal reproduction or embryonic development.

In conclusion, from these studies, a LOAEL of 10 mg/kg bw/day can be identified with regard to adverse maternal reproductive and embryotoxic effects in mice [54]. This was the lowest daily dose investigated in these studies.

(5) Interactions with Drugs

Animal studies on the interactions of piperine with drugs are discussed together with the corresponding human studies in Section 4.2.4.
