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Abstract

Introduction

Black pepper (Piper nigrum) is a rich source of natural and bioactive components such as N-trans-feruloyltyramine (NFT). In this paper, we discuss the results of the subchronic toxicity and mutagenicity studies conducted to understand the potential for adverse effects if any, of Black Pepper Extract Preparation (BPE).

Methods

To evaluate mutagenicity, an Ames test was conducted with BPE in the presence and absence of S9 metabolic activation. Long-term safety was inferred through a 90-day subchronic toxicology study using adult rats. Dose levels were selected with expected human intake levels of NFT (120 mg/kg/day), with an acceptable safety factor, for preclinical safety and tolerability. Sprague Dawley rats were fed diets targeting dietary intakes (doses) of 0, 125, 350, or 700 mg/kg/day of BPE for 90 days, an NFT dose level equivalent to 68, 190, and 380 mg/kg/day.

Results

In vitro Ames test up to 5000 µg/plate with and without S9 metabolic activation showed no BPE-related increases in revertant colony numbers and was non-mutagenic. There were no BPE-related changes in viability, clinical signs, body weight, food consumption, and organ weights. BPE dietary administration did not induce any treatment-related changes in hematology, clinical chemistry, other macroscopic or microscopic endpoints.

Discussion and conclusion

The highest dose tested with BPE (700 mg/kg/day) was the no-observed-adverse-effect level (NOAEL) that revealed no adverse effects. Based on toxicological endpoints evaluated, this NOAEL for BPE corresponded to a human equivalent NFT dose level of 380 mg/kg/day, dependent upon a (∼50%) concentration of NFT in BPE.

Keywords

Black pepper extract Ames’s test subchronic toxicity clinical investigation Black pepper extract-No observed adverse effect level

Introduction

Black pepper (Piper nigrum) is commonly used throughout the world as a spice and seasoning in foodstuffs. It is the fruit of a perennial woody climber plant that grows mainly in hot and humid climates, particularly in countries such as India, Vietnam, and Sri Lanka.^1,2 Now available in most countries, it is consumed safely by millions worldwide.

Black pepper has been safely used for a millennium in the areas of its production and worldwide since the beginning of its exportation³ and its consumption has been associated with many health benefits.⁴ Historical⁵ records exist of the properties of black pepper,³ and recent research has focused on elucidating the metabolic mechanisms of these health benefits.^6–8 Black pepper, P. nigrum L.⁸ is a rich source of natural and bioactive compounds (e.g., piperine, N-trans-feruloyltyramine), and it is from these compounds that its beneficial properties are derived. N-trans-feruloyltyramine (NFT) has been described as having antioxidant, antimicrobial, anti-melanogenesis, and anticancer activities.⁹ It is a phenolic amide originally isolated from the roots of Solanum melongena L.¹⁰ It has also been identified in other foods, such as aged garlic extract,¹¹ and in plants used for herbal medicines, such as Datura metel L.¹² and Pilosocereus gounellei.¹³

NFT present in black pepper is known to play an important role in modulating hepatic nuclear factor 4α (HNF4α). HNF4α is a nuclear receptor transcription factor that regulates downstream genes crucial for cellular metabolic processes and homeostasis.^14,15 One of these important functions is fat storage regulation in the liver. HNF4α regulates this by inducing lipophagy, which reduces the amount of fat stored in the liver and removing excess fat.¹⁶ However, fatty acids in the liver can sometimes bind to HNF4α′s ligand binding pocket, inhibiting its ability to initiate lipophagy, thus leading to excessive fat build-up in the liver (fatty liver disease).¹⁷ Researchers have shown that NFT can reverse this effect by serving as an agonist for HNF4α. Lee et al.¹⁶ demonstrated in vitro that inhibited HNF4α expression and fat clearance mechanisms can be recovered after treatment with NFT. This result is consistent with other data showing that HNF4α agonist treatments can prevent steatosis and even prevent weight gain.¹⁵ These results, indicate the potential health benefits of NFT, however, a comprehensive search of the literature did not identify any studies relevant to the safety of NFT.

Therefore, this study aimed to establish safety, by investigating potentially adverse effects of a black pepper extract preparation (BPE) enriched with ∼50% NFT. An Ames assay was performed to test for mutagenicity while long-term safety was inferred through a 90-day subchronic toxicology study using adult rats. Different doses of BPE were administered based on a 14-day toxicology study that was conducted in rats to determine the appropriate amount of BPE and corresponding levels of NFT. Range-finding dose levels were selected and derived from expected human intake levels of 120 mg/kg/day, with an acceptable safety factor, for preclinical safety and tolerability. The main objective of the 90-day toxicology study was to evaluate the potential oral subchronic toxicity in male and female rats continuously exposed to BPE in the diet.

Materials and methods

Test material preparation

The source for the BPE was dried black peppercorns that were fractionated by peppercorn fruit size and density and pre-screened for the bioactive (NFT) of interest. The peppercorns were optionally steam-treated to reduce bioburden. The peppercorns were then mechanically milled through a 40 to 80 mesh-sized screen, resulting in a powder. Prior to the extraction step, the moisture level of the ground pepper was adjusted by the addition of water, as needed, to compensate for the inherent variability in the starting raw material and to maintain NFT within specifications. Extraction of the ground black pepper using supercritical CO₂ as solvent (temperature of 40°C to 60°C, pressure 4000 to 10,000 psi) resulted in ∼50% NFT-enriched BPE as depicted in Figure 1.

Figure 1.

Chromatogram of 100 mg/mL dose formulation of BPE enriched with ∼50% N-trans-feruloyltyramine.

The nutrient composition of BPE is detailed in Supplemental Table S1. The quantitative analysis of the bioactive NFT in the BPE-enriched formulation was conducted as follows. Approximately 50 mg of the powdered formulation was resuspended in 20 mL of 50% acetonitrile in water. NFT was extracted by sonication and vortexing, followed by centrifugation. The resulting extract, containing NFT, was injected into an Agilent Infinity II HPLC and subjected to gradient separation using water and acetonitrile containing 0.1% formic acid to isolate NFT. Separation was achieved using a Thermo Scientific Hypersil Gold C18 column maintained at 60 °C with a constant flow rate of 1 mL/min. NFT identification was carried out using a DAD-UV detector set at 315 nm, and quantitation was performed using a calibration curve prepared with analytical standards in a linear range of 10 mg/L to 1000 mg/L.

Mutagenicity study

Ames test

The Ames test was performed according to Good Laboratory Practice (GLP) regulations in accordance with the Organization for Economic Co-operation and Development (OECD) 471 to assess BPE’s potential for mutagenicity in bacterial strains.¹⁸ Substances known to cause increases in revertant colony counts were used as the positive control. The positive control substances used in assays with or without the metabolic activation system were 2-nitrofluorene, sodium azide, 4-Nitroquinoline N-oxide, 9-aminoacridine, 2-aminoanthracene, and benzo(a)pyrene.

The Ames test was conducted with BPE at dose levels of 1.58, 5.0, 15.8, 50, 158, 500, 1580, and 5000 µg/plate. The highest dose tested was the standard limit for this test. The initial (main) test was conducted with Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and Escherichia coli strains WP2 uvrA using the plate incorporation method in both the presence and absence of the metabolic activation system (S9 mix) (Molecular Toxicology Inc.). The confirmatory test employed the pre-incubation modification of the plate incorporation test wherein either the test or control substances along with bacterial suspension and the S9 or substitution buffer were incubated under agitation for 30 min at 37°C before mixing with the overlay agar and pouring onto the minimal agar plates.

Specific criteria were used in determining the validity of the results obtained in the assay. The background lawn for vehicle control plates is expected to appear normal (i.e., slightly hazy with abundant microscopic non-revertant bacterial colonies). The colony numbers, the mean revertant colony counts for each strain treated with the vehicle are expected to be within the laboratory’s historical control range and/or published values.^19,20

The Mutation Factor (MF) was calculated for each test group (dose levels 1.58, 5.0, 15.8, 50, 158, 500, 1580, and 5000 µg/plate) by dividing the mean revertant colony count by the mean revertant colony count for the corresponding concurrent vehicle control group. The mutagenic activity of BPE was assessed by applying the following criteria. The results were considered positive (mutagenic) if the results for the BPE showed a substantial increase in revertant colony counts, i.e., response MF ≥2 for strains TA98, TA100, and WP2 uvrA or MF ≥3 for strains TA1535 and TA1537, with mean value(s) outside the laboratory historical control range. Otherwise, results were considered negative. Also, the increase must be dose related and/or reproducible (at least one strain, more than one dose level, more than one occasion or with different methodologies). If the second criterion was not met, the results were classified as equivocal. If BPE produced neither a concentration related increase in revertant colonies nor a reproducible significant increase in revertant colonies, the test group was considered non-mutagenic in this test system.

90-day toxicology study

Study design

This study was conducted in compliance with the U.S. FDA GLP: 21 CFR Part 58, 1987. Which are compatible with the OECD Principles of Good Laboratory Practice (as revised in 1997) published in ENV/MC/CHEM (98)17.²¹ The study design was conducted based on the OECD Guidelines for Testing of Chemicals and Food Ingredients, Section 4 (Test No. 408): Health Effects, Repeated Dose 90-Day Oral Toxicity Study in Rodents (OECD 2018). Also, adhered to the US FDA Toxicological Principles for the Safety Assessment of Food Ingredients, Redbook 2000, IV.C. 4. a.²²

Animals

All animal care and experimental protocols were under the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals.²³ The Sprague-Dawley® rat was the system of choice because, historically, it has been a preferred and commonly used species for dietary toxicity tests. Eighty healthy Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) (40 males; 40 females) were used in the test. Animals were selected for this study based on adequate body weight gain, free from clinical signs of disease or injury, and a body weight. Selected rats were distributed by randomization according to stratification by body weight so that there was no statistically significant difference among group body weight means within a sex. The animals weighed 229-333 g (males) and 183-232 g (females) and were approximately seven to 8 weeks of age at initial test substance administration. Ophthalmologic evaluations were conducted during the acclimation period, when the eyes of the rats were examined by focal illumination, indirect ophthalmoscopy and sometimes by slit-lamp microscopy. These procedures were repeated on all test animals prior to test termination on Day 92.

Treatment

The BPE was administered in the diet because human exposure may occur via this route. Test diets were prepared using Inotiv Teklad Global Rodent Diet® and were available ad libitum during the study. Filtered tap water was also available ad libitum from individual bottles attached to the cages or from an automatic watering access system. Animals were randomly assigned, stratified by body weight, to test groups. One group was the control group (Group 1). The dose levels were selected based on a 14-day palatability/toxicity study and the selected target intake (dose) levels were 125 (Group 2), 350 (Group 3), and 700 mg/kg/day (Group 4). To maintain target dose levels throughout the study, concentrations in the test diets were calculated based on the most recent group body weight and food consumption data. Alternatively, historical control values, relevant to the age and weight of the rats at corresponding intervals were used. Diets for males and females at each dose level were made separately each week.

The BPE and selected prepared diets were sampled in duplicate. Additional samples were collected and analyzed to ensure stability, accuracy, and homogeneity of the dietary concentrations over the course of the study. Samples were collected from representative animal diets of the initial, middle, and final diet preparations. Samples were analyzed to verify the concentration of the test diets.

Evaluations

Clinical observations

All animals were observed at least twice daily for viability. Cage-side observations of all animals were performed daily during the study. On Day 0, prior to the first treatment with the BPE, and weekly thereafter, a detailed observation was conducted while handling the animal, generally occurring on days that the animals were weighed, and food consumption measurements taken. Potential signs noted included changes in skin, fur, eyes, and mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Likewise, changes in gait, posture, and response to handling, and the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling), or bizarre behavior (e.g., self-mutilation, walking backwards) were also recorded.

Neurobehavioral assessment

Once towards the end of the exposure period on Day 82 for males and Day 81 for females, a Functional Observational Battery of assessments (FOB) were performed on all study animals. Each rat was assessed while handling, as well as in an open field for excitability, autonomic function, gait, and sensorimotor coordination (open field and manipulative evaluations), reactivity and sensitivity (elicited behavior), and other abnormal clinical signs, including convulsions, tremors unusual or bizarre behavior emaciation, dehydration, and general appearance. The rats were observed in random order and the observed was blind to the treatment group. Forelimb and hindlimb grip strength and foot splay measurements were also obtained and recorded.

Concurrently, motor activity was also evaluated. This assessment coincided with FOB assessments for the same animals. Activity was monitored using an automated Photobeam Activity System^®, San Diego Instruments, Inc. An equal number of animals assigned to Motor Activity assessment from all dose groups was evaluated in each session. Each animal was placed into a polycarbonate solid bottom cage and the evaluation phase began immediately for that animal. Each animal was evaluated for a single one-hour phase, with photobeam count accumulated over six 10-min intervals.

Body weight and food consumption

Individual body weights were recorded twice during acclimation. Test animals were weighed on Day 0 (prior to study start) and weekly thereafter (intervals of 7 days ±1). The animals were also weighed immediately prior to sacrifice to calculate organ to body weight ratios. Body weight gain was calculated for selected intervals. Individual food consumption was measured and recorded correlating with body weight measurements. Food efficiency and dietary intake of the BPE (mg/kg/day) was also calculated and reported.

Estrus cycle determination

The estrus cycle determination occurred during weeks 6-7, and again during weeks 12-13. Vaginal smears and estrus cycle stages were taken and recorded daily for a duration of 14 days on all study females for reproductive functionality. At terminal sacrifice (Day 95), vaginal smears were collected from all female rats on the day of terminal sacrifice to determine the stage of estrus.

Sperm analysis

For motility assessment, the left vas deferens was excised and prepared for sperm dispersal. Following this dispersal period, a sample of sperm was collected for motility analysis. For accuracy, motility results were calculated based on a minimum number of sperm (200) counted in all fields, total. Five fields per animal were selected and stored as digital images, until 200 sperm were counted, for a maximum of 20 fields. Twenty fields per animal were selected, reported, and adjusted for caudal epididymal weight. A 100 µL sample of the suspension was stained with a dye that uniquely stains sperm heads. Twenty fields/animal were selected, reported, and adjusted for testis weight. Two eosin-stained slides were prepared for each animal. The slides were evaluated, and where possible, a minimum of 200 sperm cells/animal were examined for morphological development using a light microscope.

Clinical pathology (hematology, clinical chemistry, coagulation, urinalysis, and thyroid hormones)

Clinical pathology was performed on all animals including hematology, clinical chemistry, coagulation, urinalysis, and thyroid hormone analysis. Animals were fasted overnight prior to blood collection. Blood samples were collected from the sublingual vein or vena cava/abdominal aorta (clinical chemistry, coagulation), under isoflurane anesthesia. The method of bleeding was dependent upon biological time constraints that would be required to generate a quality sample for accurate data assessment. Hematology and thyroid hormone assessment was collected in a relatively short period of time from all study animals to reduce variability between subjects (samples).

All blood samples were evaluated for quality by visual examination. Approximately 500 µL of blood was collected in a pre-calibrated tube containing K2EDTA for hematology assessments. The following parameters were evaluated: hematocrit, platelet count, hemoglobin concentration, red blood cell count, mean corpuscular hemoglobin, red cell distribution width, mean corpuscular volume, reticulocytes count, white blood cell and differential leukocyte count, and mean corpuscular hemoglobin concentration. Coagulation was assessed by collecting approximately 1.8 mL of blood in a pre-calibrated tube containing 3.2% sodium citrate. The parameters evaluated were activated partial thromboplastin time and prothrombin time.

Clinical chemistry was assessed by collecting approximately 1000 µL of blood. The following parameters were evaluated: albumin, lipoprotein (high density), alkaline phosphatase, lipoprotein (low density), bilirubin (total), potassium, blood creatinine, serum alanine aminotransferase, calcium, serum aspartate aminotransferase, chloride, serum protein (total), cholesterol (total), sodium, fasting glucose, sorbitol dehydrogenase, globulin triglycerides, inorganic phosphorous, urea, and nitrogen.

Urinalysis was performed the day before collection of samples for the clinical pathology. The parameters evaluated were quality, pH, ketone, color, glucose, bilirubin, clarity, specific gravity, blood protein (total), urobilinogen, microscopic urine, and sediment volume.

Blood was collected for assessment of effects on the pituitary-thyroid axis from all animals. Samples were analyzed for Triiodothyronine (T3), Thyroxine (T4), and Thyroxine Stimulating Hormone (TSH). Determination of the levels of TSH, T3 and T4 were performed using an ELISA method.

Histopathology

All animals in the study were subjected to a gross necropsy, which included examination of the external surface of the body, all orifices, musculoskeletal system, and the cranial, thoracic, abdominal, and pelvic cavities, with their associated organs and tissue. The following tissue were weighed wet as soon as possible after dissection to avoid drying: adrenals (combined), kidneys, testes (combined), brain, liver, thymus, epididymides (combined), ovaries with oviducts (combined), uterus, heart, and spleen.

Thyroid/parathyroid, pituitary, prostate, and seminal vesicles with coagulating gland (combined) were weighed at least 24 h after preservation in 10% neutral buffered formalin. The following organs and tissues from all surviving animals were preserved in 10% neutral buffered formalin for possible future histopathological examination: accessory genital organs (prostate and seminal vesicles), ileum with Peyer’s patches, rectum, jejunum, salivary glands (sublingual, submandibular, and parotid), adrenals, kidneys, all gross lesions, larynx, aorta, liver, skeletal muscle, bone (femur), lungs, skin, bone marrow (from femur and sternum), lymph node mandibular, lymph node mesenteric, spinal cord (cervical, mid-thoracic, and lumbar), brain (sections including medulla/pons, cerebellar and cerebral cortex), mammary gland, nasal turbinates, spleen, cecum, nose, sternum, cervix, ovaries, stomach, colon, oviducts, thymus, thyroid, duodenum, pancreas (with islets), trachea, parathyroid, esophagus, peripheral nerve (sciatic), urinary bladder, Harderian gland, pharynx, uterus, heart, pituitary gland, and vagina.

Histological examination was performed on the preserved organs and tissues of the animals from both the control and high dose groups (Groups 1 and 4, respectively). At scheduled necropsy, all male rats were evaluated for reproductive capacity by evaluation of sperm samples collected at termination for motility, epididymal sperm count, homogenization- resistant spermatid (HRS; from the testis) count, and morphology. On the day of necropsy, all males were prepared for left testis and epididymis sperm evaluation.

Statistical analysis

Statistical analysis was performed on all data collected during the in-life phase of the study, as well as clinical pathology results and organ weight data. Statistical analysis was conducted by using one or more of the following software applications: Provantis^™ version 10, Tables and Statistics, Instem LSS, Staffordshire, UK and Prism Biostatistics, GraphPad Software, San Diego, CA. Significance was judged at a probability value of p < 0.05. Mean and standard deviation were calculated for all quantitative data. Male and female rats were evaluated separately.

For all in-life endpoints that were identified as multiple measurements of continuous data over time (e.g., body weight parameters, food consumption, food efficiency, and FOB endpoints such as foot splay and grip strength, when applicable), treatment and control groups were compared using a repeated measures analysis of variance (RMANOVA), testing the effects of both time and treatment.²⁴ Significant interactions observed between treatment and time as well as main effects were further analyzed by a post hoc multiple comparisons test^25,26 of the individual treated groups to control.

Results

Mutagenicity test

Revertant colony counts for each strain both for the plate incorporation and pre-incubation methods are presented in Tables 1 and 2, respectively.

Table 1.

Results from plate incorporation method (AMES).

Test substance	Dose level µg/plate)	S9 mix	Revertant colony counts (mean)^a
Test substance	Dose level µg/plate)	S9 mix	TA 98	Mut. Factor^d	TA 100	Mut. Factor^d	TA1535	Mut. Factor^d	TA 1537	Mut. Factor^d	WP2 uvra	Mut. Factor^d
Black pepper extract preparation	0^b	̅	24	1.00	118	1.00	15	1.00	16	1.00	42	1.00
	1.58		23	0.96	118	1.00	14	0.93	19	1.19	40	0.95
	5		22	0.92	115	0.97	18	1.20	18	1.13	39	0.93
	15.8		24	1.00	112	0.95	15	1.00	14	0.88	41	0.98
	50		25	1.04	119	1.01	15	1.00	13	0.81	37	0.88
	158		22	0.92	112	0.95	18	1.20	17	1.06	40	0.95
	500		22	0.92	110	0.93	14	0.93	16	1.00	39	0.93
	1580		21	0.88	120	1.02	12	0.80	19	1.19	40	0.95
	5000		19	0.79	118	1.00	15	1.00	16	1.00	39	0.93
NaN₃c	0.5				574	4.86	521	34.73
9ACc	50								380	23.75
2NFc	1		162	6.75
NQOc	0.5										427	10.17
Black pepper extract preparation	0^b	+	29	1.00	119	1.00	12	1.00	16	1.00	42	1.00
	1.58		29	1.00	125	1.05	14	1.17	15	0.94	51	1.21
	5		27	0.93	118	0.99	12	1.00	15	0.94	51	1.21
	15.8		29	1.00	122	1.03	14	1.17	15	0.94	44	1.05
	50		27	0.93	112	0.94	13	1.08	14	0.88	47	1.12
	158		29	1.00	115	0.97	10	0.83	15	0.94	52	1.24
	500		29	1.00	114	0.96	12	1.00	17	1.06	51	1.21
	1580		22	0.76	123	1.03	13	1.08	18	1.13	49	1.17
	5000		20	0.69	111	0.93	12	1.00	17	1.06	37	0.88
2AA^c	2						400	33.33	211	13.19
2AA^c	10										186	4.43
BAP^c	5		190	6.55	648	5.45

^amean of replicate (3) plates.

^bNegative: DMSO.

^cPositive control agents: NaN₃ = sodium azide; 9AC = 9-aminoacridine; 2NF = 2-Nitrofluorene; NO = 4-Nitroquinoline N-oxide; 2AA = 2-aminoanthracine; BAP = Benzo[a]pyrene.

^dMutation Factor = mean revertants (BPE)/mean revertants (Negative Control).

Table 2.

Results from pre-incubation method (AMES).

Test substance	Dose level µg/plate)	S9 mix	Revertant colony counts (mean)^a
Test substance	Dose level µg/plate)	S9 mix	TA 98	Mut. Factor^d	TA 100	Mut. Factor^d	TA1535	Mut. Factor^d	TA 1537	Mut. Factor^d	WP2 uvra	Mut. Factor^d
Black pepper extract preparation	0^b	̅	22	1.00	116	1.00	14	1.00	10	1.00	44	1.00
	1.58		25	1.14	123	1.06	14	1.00	11	1.10	40	0.91
	5		22	1.00	116	1.00	14	1.00	9	0.90	43	0.98
	15.8		26	1.18	115	0.99	16	1.14	9	0.90	43	0.98
	50		21	0.95	107	0.92	17	1.21	12	1.20	47	1.07
	158		21	0.95	121	1.04	11	0.79	10	1.00	44	1.00
	500		24	1.09	118	1.02	9	0.64	11	1.10	49	1.11
	1580		23	1.05	123	1.06	17	1.21	14	1.40	39	0.89
	5000		29	1.32	124	1.07	18	1.29	13	1.30	34	0.77
NaN₃^c	0.5				510	4.40	494	35.29
9AC^c	50								410	41.00
2NF^c	1		189	13.9
NQO^c	0.5										661	15.02
Black pepper extract preparation	0^b	+	26	1.00	127	1.00	15	1.00	9	1.00	50	1.00
	1.58		27	1.04	121	0.95	15	1.00	9	1.00	56	1.12
	5		27	1.04	124	0.98	11	0.73	10	1.11	51	1.02
	15.8		29	1.12	124	0.98	7	0.47	11	1.22	49	0.98
	50		29	1.12	120	0.94	12	0.80	11	1.22	56	1.12
	158		30	1.15	116	0.91	18	1.20	14	1.56	46	0.92
	500		25	0.96	119	0.94	18	1.20	14	1.56	53	1.06
	1580		26	1.00	119	0.94	10	0.67	10	1.11	53	1.06
	5000		22	0.85	118	0.93	13	0.87	13	1.44	39	0.78
2AA^c	2						395	26.33	231	25.67
2AA^c	10										132	2.64
BAP^c	5		157	6.04	508	4.00

^amean of replicate (3) plates.

^bNegative: DMSO.

^cPositive control agents: NaN₃ = sodium azide; 9AC = 9-aminoacridine; 2NF = 2-Nitrofluorene; NO = 4-Nitroquinoline N-oxide; 2AA = 2-aminoanthracine; BAP = Benzo[a]pyrene.

^dMutation Factor = mean revertants (BPE)/mean revertants (Negative Control).

The mean revertant colony counts for each strain treated with the vehicle were close to or within the expected range, and within the historical control range and/or published values.^19,20 The positive control substances caused the expected substantial increases in revertant colony counts in both the absence and presence of S9 in each phase of the test, confirming the sensitivity of the test and the activity of the S9 mix. No indicators of precipitation nor toxicity were observed in any strains used at all dose levels.

For all strains, at least eight non-toxic dose levels were evaluated; therefore, bacterial mutagenicity was adequately assessed. There were no concentration-related or substantial BPE- related increases in the number of revertant colonies observed with strains TA98, TA100, TA1535, TA1537, or E. coli WP2 uvrA in both the absence and presence of S9 using either the plate incorporation or the pre-incubation method (Tables 1 and 2). Even at the highest BPE dose level (5000 µg/plate), there were no bacterial revertant colonies. Therefore, the results of the study demonstrated that BPE was non-mutagenic.

Subchronic toxicology study

Survival, body weight, food consumption, food efficiency, and dietary intake

There were no changes in body weight parameters for male and female rats due to the dietary administration of the BPE (Figure 2). Mean weekly body weights and mean daily body weight gain for male and female treated rats with 125 mg/kg/day BPE (Group 2 male and female), 350 mg/kg/day BPE (Group 3 male and female) and 700 mg/kg/day BPE (Group 4 male and female) were comparable to the control (Group 1) animals throughout the study. There were also no BPE-related or other mortalities during this study.

Figure 2.

Body weight measurements during subchronic toxicity study.

Based on target weekly nominal concentrations, dietary intakes of 122.9, 344.6, and 688.2 mg/kg/day in male rats, and 124.6, 347.5, and 697.0 mg/kg/day in female rats were calculated for Groups 2-4, respectively. Food consumption (Figure 3) and food efficiency in male or female rats did not change based on BPE administration. There were no clinical observations considered attributable to the dietary administration of BPE. Mean daily food consumption and mean food efficiency for male and female treated rats in Groups 2-4 were comparable to the control Group 1 animals throughout the study. Body weight and food consumption measurements collected throughout the study were used to calculate the mean overall daily intake of BPE, based on the expected achievement of weekly diet concentrations, targeting intake (dose) levels 125, 350, and 700 mg/kg/day (Groups 2-4, respectively).

Figure 3.

Mean daily food consumption during subchronic toxicity study.

Clinical observations and neurobehavioral assessments

Clinical observations in males included: noticeable lacrimation in 1/10 Group 2 and 1/10 Group 4 animals and hair loss in 1/10 Group 2 animals. Mean forelimb/hindlimb grip strength and mean foot splay measurements were comparable to control values for animals observed on Day 82. Clinical observations in females included: hair loss in 1/10 control Group 1, 1/10 Group 2, and 2/10 Group 4 animals; eschar in 1/10 Group 2 animals; no auditory response in 1/10 control Group 1 and 1/10 Group 2 animals; and no tail pinch response in 1/10 Group 2 animals.

There were no BPE-related changes in any of the parameters evaluated in the functional observational battery.

BPE administration caused no changes in motor activity. Mean total movements observed over the course of the study were considered comparable to that of controls.

Ophthalmologic tests

Both eyes of all animals on study were examined by focal illumination, slit lamp biomicroscopy, and indirect ophthalmoscopy prior to study initiation and near termination of the study on Day 92. All animals included in the study were normal upon ophthalmic exam, thus BPE was not considered an ocular irritant.

Estrus cycle determination

Vaginal smears were collected daily from all females during weeks 6-7 and 12-13. The mean cycle length and number of cycles for the animals over the course of each time period did not generally differ from control for the BPE treated groups. Therefore, the administration of BPE did not influence estrus cyclicity in females. Measuring estrus phase to estrus phase, the mean cycle length during Days 42-55 (weeks 6-7) was 3.85, 4.10, 4.40, and 4.25 days, with the mean number of cycles over that time observed as 2.2, 1.9, 2.0, and 2.0 for Groups 1-4, respectively. Similarly, the mean cycle length during Days 77-95 (weeks 12-13) was 4.50, 4.73, 5.02, and 4.73 days, with the number of cycles over that time observed as 2.6, 2.6, 2.4, and 2.5 for Groups 1-4, respectively.

Clinical pathology

Hematology, urinalysis, coagulation, clinical chemistry, and hormone analysis

There were no BPE-related changes in hematology parameters (Table 3). Increased reticulocyte count in Group 4 males (700 mg/kg/day) was considered toxicologically insignificant as it was not associated with any change in bone marrow (Table 3). Increased white blood cell count in Group 3 females (350 mg/kg/day) was considered toxicologically insignificant as there was no dose progression. All other changes in hematology parameters were considered unrelated to BPE administration, There were no BPE -related changes in coagulation parameters. Increased activated partial thromboplastin time (APTT) in Group 3 females was considered toxicologically insignificant as there was no dose progression (Supplemental Table 2). There were no BPE-related changes in clinical chemistry parameters (Table 4). There were no BPE-related changes in thyroid hormones at any of the dose levels tested. Increased TSH levels in Group 3 males and females was considered toxicologically insignificant as there was no dose progression (Supplemental Tables 3 and 4).

Table 3.

Hematology parameters on BPE treated rats.

Parameter (unit)	Test substance dose levels (mg/kg/day)
	0		125		350		700
	Males	Females	Males	Females	Males	Females	Males	Females
RBC (x10⁶/µL)	8.950 ± 0.374	8.259 ± 0.138	9.047 ± 0.448	8.075 ± 0.360	8.948 ± 0.510	8.101 ± 0.220	8.914 ± 0.499	8.102 ± 0.263
HGB (g/dL)	15.41 ± 0.41	14.91 ± 0.38	15.72 ± 0.79	15.01 ± 0.60	15.49 ± 0.70	15.10 ± 0.51	15.29 ± 0.79	14.94 ± 0.31
HCT (%)	47.96 ± 1.78	44.36 ± 1.38	48.44 ± 2.37	44.64 ± 1.82	47.61 ± 2.41	45.58 ± 1.64	47.74 ± 2.32	45.08 ± 1.28
MCV (fL)	53.60 ± 1.38	53.69 ± 1.19	53.56 ±0.95	55.34 ± 2.21	53.31 ± 1.68	56.27 ± 1.49^S1	53.58 ± 1.35	55.68 ± 2.13^S1
MCH (pg)	17.25 ± 0.60	18.08 ± 0.32	17.36 ± 0.46	18.62 ± 0.82	17.33 ± 0.57	18.62 ± 0.44	17.18 ± 0.60	18.46 ± 0.63
MCHC (g/dL)	32.16 ± 0.61	33.69 ± 0.50	32.42 ± 0.43	33.66 ± 0.36	32.56 ± 0.33	33.07 ± 0.51^S1	32.06 ± 0.39	33.15 ± 0.55^S1
RDW (%)	13.10 ± 0.80	11.70 ± 0.40	13.60 ± 1.77	11.73 ± 0.43	14.19 ± 1.12	12.12 ± 0.51	14.59 ± 1.74	12.22 ± 0.71
PLT (×10³/µL)	1062.80 ± 123.37	1080.70 ± 163.61	1006.70 ± 245.15	1182.20 ± 113.25	1173.80 ± 152.31	1168.10 ± 211.76	1093.70 ± 78.44
WBC (×10³/µL)	10.330 ± 3.350	4.935 ± 1.968	9.972 ± 2.522	5.007 ± 1.510	10.320 ± 2.453	6.570 ± 1.217^S1	11.346 ± 2.547	6.157 ± 1.936
ANEU 0.475	1.565 ± 0.631	1.214 ± 0.348	1.618 ± 0.489	1.172 ± 0.410
ALYM (×10³/µL)	8.031 ± 3.001	3.646 ± 1.727	7.370 ± 1.479	3.589 ± 1.270	8.073 ± 1.881	4.887 ± 1.055	8.965 ± 2.080	4.590 ± 1.586
AMON (×10³/µL)	0.323 ± 0.076	0.164 ± 0.083	0.306 ± 0.137	0.139 ± 0.048	0.307 ± 0.134	0.221 ± 0.105	0.384 ± 0.075	0.188 ± 0.082
AEOS (×10³/µL)	0.143 ± 0.033	0.094 ± 0.051	0.148 ± 0.090	0.104 ± 0.056	0.155 ± 0.055	0.115 ± 0.051	0.145 ± 0.059	0.097 ± 0.040
ABAS (×10³/µL)	0.147 ± 0.077	0.071 ± 0.030	0.164 ± 0.074	0.062 ± 0.029	0.140± 0.043	0.081 ± 0.032	0.137 ± 0.068	0.066 ± 0.030
ALUC (×10³/µL)	0.105 ± 0.058	0.043 ± 0.033	0.071 ± 0.014	0.027 ± 0.011	0.082 ± 0.044	0.052 ± 0.018	0.094 ± 0.041	0.043 ± 0.019
ARET (×10³/µL)	197.710 ± 31.600	160.370 ± 38.041	204.350 ± 70.364	149.700 ± 30.243	238.710 ± 30.010	186.530 ± 42.301	276.600 ± 79.692^S1	193.200 ± 31.383

^S1 – p < 0.05.

WBC - white blood cell count.

ANEU - absolute neutrophil (all forms).

ALYM - absolute lymphocyte.

AMON - absolute monocyte.

AEOS - absolute eosinophil.

ABAS - absolute basophil.

ALUC - absolute large unstained cell.

ARET - absolute reticulocyte.

RBC - red blood cell count.

HGB - hemoglobin.

HCT - hematocrit.

MCV - mean corpuscular (cell) volume.

MCH - mean corpuscular (cell) hemoglobin.

MCHC - mean corpuscular (cell) hemoglobin concentration.

RDW - red cell distribution width.

PLT - platelet count.

Table 4.

Clinical chemistry values for BPE treated rats.

Parameter (unit)	Test substance dose levels (mg/kg/day)
	0		125		350		700
	Males	Females	Males	Females	Males	Females	Males	Females
AST (U/L)	79.5 ± 15.3	104.2 ± 28.0	74.7 ± 8.7	115.2 ± 73.2	91.2 ± 34.4	134.1 ± 125.6	80.9 ± 20.3	110.5 ± 36.6
ALT (U/L)	28.7 ± 4.4	39.2 ± 15.0	27.1 ± 4.3	47.8 ± 42.9	40.0 ± 26.4	43.5 ± 34.4	27.1 ± 6.9	44.2 ± 18.5
SDH (U/L)	11.75 ± 1.82	18.37 ± 5.41	12.82 ± 4.40	21.69 ± 13.38	16.84 ± 11.34	28.80 ± 34.96	13.39 ± 4.14	26.34 ± 16.32
ALKP (U/L)	64.6 ± 10.6	29.0 ± 7.3	69.8 ± 10.3	31.5 ± 13.1	55.7 ± 9.2	31.3 ± 12.4	60.8 ± 9.7	27.9 ± 6.5
BILI (mg/dL)	0.069 ± 0.025	0.082 ± 0.012	0.083 ± 0.016	0.078 ± 0.019	0.079 ± 0.020	0.099 ± 0.045	0.071 ± 0.018	0.068 ± 0.021
BUN (mg/dL)	15.9 ± 1.2	20.5 ± 2.6	16.4 ± 2.8	22.2 ± 5.1	15.1 ± 1.4	20.2 ± 4.0	15.8 ± 2.6	20.9 ± 4.5
CREA (mg/dL)	0.226 ± 0.047	0.322 ± 0.039	0.244 ± 0.057	0.322 ± 0.042	0.214 ± 0.037	0.333 ± 0.035	0.220 ± 0.034	0.336 ± 0.059
CHOL (mg/dL)	64.4 ± 16.0	91.8 ± 23.7	73.4 ± 12.6	81.8 ± 21.0	73.1 ± 18.3	83.4 ± 20.9	84.0 ± 15.8	87.4 ± 19.2
TRIG (mg/dL)	71.0 ± 24.9	56.0 ± 42.4	71.3 ± 25.0	54.2 ± 27.3	61.4 ± 15.3	45.6 ± 17.2	68.5 ± 32.8	46.3 ± 17.5
GLUC (mg/dL)	205.4 ± 25.9	213.2 ± 64.9	196.3 ± 17.8	197.9 ± 41.2	182.5 ± 31.3	197.1 ± 26.7	191.2 ± 33.6	188.0 ± 48.2
TP (g/dL)	5.84 ± 0.15	6.77 ± 0.73	5.92 ± 0.32	6.66 ± 0.63	5.94 ± 0.31	6.71 ± 0.51	5.90 ± 0.23	6.59 ± 0.47
ALB (g/dL)	3.84 ± 0.10	5.08 ± 0.52	3.89 ± 0.15	4.95 ± 0.59	3.94 ± 0.23	4.96 ± 0.49	3.92 ± 0.13	4.86 ± 0.47
GLOB (g/dL)	2.00 ± 0.15	1.69 ± 0.33	2.03 ± 0.27	1.71 ± 0.14	2.00 ± 0.16	1.75 ± 0.16	1.98 ± 0.17	1.73 ± 0.18
CALC (mg/dL)	11.26 ± 0.66	12.56 ± 1.05	11.52 ± 0.71	12.68 ± 1.20	11.25 ± 0.53	12.74 ± 0.94	11.19 ± 0.47	12.23 ± 0.95
IPHS (mg/dL)	8.10 ± 0.53	9.08 ± 1.13	8.58 ± 0.99	9.83 ± 1.73	8.43 ± 0.75	10.49 ± 1.98	8.14 ± 1.02	9.71 ± 1.50
NA (mmol/L)	140.70 ± 1.64	142.40 ± 2.12	140.60 ± 2.12	142.00 ± 1.70	140.90 ± 1.66	141.80 ± 2.49	140.80 ± 1.32	140.50 ± 3.57
K(mmol/L)	6.815 ± 0.927	7.950 ± 2.041	6.989 ± 0.888	8.456 ± 1.970	6.982 ± 1.062	8.618 ± 2.691	6.668 ± 1.078	9.777 ± 3.192
CL (mmol/L)	104.10 ± 1.15	105.76 ± 1.62	104.32 ± 1.64	106.42 ± 1.50	104.26 ± 1.39	105.38 ± 1.80	103.78 ± 1.43	105.72 ± 0.98
LDL (mmol/L)	0.300 ± 0.133	0.218 ± 0.069	0.380 ± 0.103	0.194 ± 0.076	0.380 ± 0.140	0.211 ± 0.074	0.470 ± 0.183	0.220 ± 0.059
HDL (mmol/L)	1.030 ± 0.250	1.840 ± 0.445	1.130 ± 0.231	1.646 ± 0.410	1.170 ± 0.320	1.657 ± 0.380	1.300 ± 0.294	1.748 ± 0.362

TP – total serum protein.

ALB – albumin.

GLOB – globulin.

CALC – calcium.

IPHS – inorganic phosphorus.

NA – sodium.

K – potassium.

CL – chloride.

LDL – Low Density Lipoprotein.

HDL – High Density Lipoprotein.

^S1 – p < 0.05.

AST – serum aspartate aminotransferase.

ALT – serum alanine aminotransferase.

SDH – sorbitol dehydrogenase.

ALKP – alkaline phosphatase.

BILI – total bilirubin.

BUN – urea nitrogen.

CREA – blood creatinine.

CHOL – total cholesterol.

TRIG – triglycerides.

GLUC – fasting glucose.

For urinalysis, pH, specific gravity, urobilinogen, and protein levels of Groups 2-4 for males and females were comparable to controls (Group 1) (Table 5).

Table 5.

Mean absolute organ weight values for BPE treated rats.

Parameter	Test substance dose levels (mg/kg/day)
	0		125		350		700
	Males	Female	Males	Female	Males	Female	Males	Female
Terminal BW	539.5 ± 64.0	274.1 ± 23.3	522.2 ± 50.9	278.2 ± 27.8	535.0 ± 39.9	287.6 ± 19.4	508.7 ± 57.4	276.0 ± 19.7
Adrenal	0.0629 ± 0.0099	0.0800 ± 0.0121	0.0697 ± 0.0128	0.0772 ± 0.0123	0.0689 ± 0.0082	0.0801 ± 0.0137	0.0576 ± 0.0088	0.0889 ± 0.0118
Brain	2.276 ± 0.124	2.033 ± 0.129	2.225 ± 0.122	1.989 ± 0.078	2.208 ± 0.086	2.005 ± 0.126	2.268 ± 0.122	2.002 ± 0.116
Heart	1.581 ± 0.188	0.956 ± 0.084	1.512 ± 0.139	0.992 ± 0.094	1.548 ± 0.127	0.985 ± 0.046	1.517 ± 0.251	1.003 ± 0.110
Kidneys	3.196 ± 0.472	2.097 ± 0.231	3.037 ± 0.259	2.138 ± 0.199	3.272 ± 0.265	2.080 ± 0.203	3.203 ± 0.473	2.077 ± 0.164
Liver	12.667 ± 1.585	7.914 ± 1.392	12.090 ± 1.650	8.578 ± 1.020	12.815 ± 1.083	8.764 ± 0.774	12.867 ± 1.815	8.815 ± 0.653
Pituitary gland	0.0158 ± 0.0034	0.0213 ± 0.0044	0.0154 ± 0.0031	0.0215 ± 0.0039	0.0147 ± 0.0032	0.0204 ± 0.0032	0.0150 ± 0.0033	0.0195 ± 0.0034
Spleen	0.825 ± 0.099	0.474 ± 0.053	0.829 ± 0.167	0.502 ± 0.067	0.851 ± 0.116	0.534 ± 0.088	0.853 ± 0.099	0.526 ± 0.094
Thymus	0.2709 ± 0.0895	0.2331 ± 0.0457	0.3064 ± 0.0908	0.2237 ± 0.0678	0.3318 ± 0.1175	0.2324 ± 0.0529	0.2494 ± 0.1014	0.2257 ± 0.0624
Thyroid /para-thyroid	0.0271 ± 0.0049	0.0230 ± 0.0065	0.0298 ± 0.0081	0.0278 ± 0.0050	0.0312 ± 0.0060	0.0301 ± 0.0049	0.0267 ± 0.0038	0.0253 ± 0.0048
Uterus		0.717 ± 0.172		0.706 ± 0.234		0.627 ± 0.080		0.970 ± 0.347
Ovaries-oviduct		0.1282 ± 0.0196		0.1162 ± 0.0262		0.1158 ± 0.0176		0.1329 ± 0.0209
Epididymides	1.5377 ± 0.2630		1.6315 ± 0.1396		1.6734 ± 0.1044		1.5394 ± 0.1621
Pro, SV, CG	3.618 ± 0.533		3.701 ± 0.496		3.396 ± 0.406		3.197 ± 0.446
Testes	3.642 ± 0.251		3.542 ± 0.308		3.558 ± 0.243		3.168 ± 0.585

Organ weights and histopathological examinations

Mean absolute and relative organ weights for male rats in Groups 2-4 were comparable to control Group values (Table 6). Mean absolute and relative organ weights for female rats in Groups 2-4 were comparable to control Group 1 values with the following exceptions: liver-to-body weight ratios increased (p < 0.05) in Group 4 females (700 mg/kg/day BPE); thyroid/parathyroid terminal organ weights increased (p < 0.05) in Group 3 females (350 mg/kg/day BPE); thyroid/parathyroid-to-brain weight ratios increased (p < 0.05) in Group 3 females (350 mg/kg/day BPE).

Table 6.

Mean urinalysis values for BPE treated rats.

Parameter (unit)	Test substance dose levels (mg/kg/day)
	0		125		350		700
	Males	Females	Males	Females	Males	Females	Males	Females
UVOL (mL)	3.25 ± 2.24	1.25 ± 1.25	4.80 ± 3.56	3.17 ± 3.49	5.25 ± 3.62	1.30 ± 1.21	2.95 ± 1.42	4.25 ± 5.12
pH	6.15 ± 0.34	6.15 ± 0.34	6.00 ± 0.41	6.06 ± 0.17	6.10 ± 0.46	6.25 ± 0.49	5.85 ± 0.47	5.75 ± 0.68
SG	1.0295 ± 0.0016	1.0295 ± 0.0016	1.0295 ± 0.0016	1.0278 ± 0.0036	1.0285 ± 0.0034	1.0290 ± 0.0032	1.0300 ± 0.0000	1.0270 ± 0.0048
URO (EU/dL)	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00
UMTP (mg/dL)	42.5 ± 30.7	27.0 ± 6.3	66.5 ± 88.2	21.7 ± 13.2	46.5 ± 37.5	34.0 ± 24.0	88.0 ± 114.3	50.5 ± 43.4
Urine Ketone (mmol/L)	12.0 ± 11.4	0.5 ± 1.6	12.5 ± 11.6	0.6 ± 1.7	9.0 ± 12.9	2.0 ± 4.8	6.0 ± 5.2	0.0 ± 0.0
Urine Glucose (mg/dL)	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0

UVOL - volume.

pH - the logarithm of the reciprocal of the hydrogen ion concentration.

SG - specific gravity.

URO - urobilinogen.

UMTP - protein.

There were no BPE-related microscopic changes of collected tissues. Several changes were identified but were not considered BPE-related due to sporadic occurrences, the lack of supporting changes to suggest a pattern or biological mechanism, or the fact that they were common background findings in laboratory rats.

Sperm analysis

There were no changes in percent motile sperm, epididymal sperm count, homogenization- resistant sperm count, or percent abnormal sperm (Table 7). Morphological abnormalities noted were restricted to amorphous heads, small heads, coiled tails and bent tails. None of these were attributed to administration of BPE. There were no unscheduled deaths during this study to affect this phase. Group 4 male sperm motility was significantly decreased (p < 0.05) from control Group 1 values. Group 2 and 3 male sperm motility was comparable to control Group 1. There were no statistically significant changes that were BPE-related in epididymal sperm count, in homogenization-resistant (HRS) spermatid count, or percent abnormal sperm that were noted for Group 2 or Group 3. The percent of abnormal cells was statistically increased (p < 0.05) in Group 4 as compared to control Group 1 values.

Table 7.

SPERM analysis values for BPE treated rats.

Parameter	0		125		350		700
Parameter	Males	N	Males	N	Males	N	Males	N
% motility	86.8 ± 3.71	10	81.1 ± 8.16	10	85.6 ± 7.18	10	77.7 ± 7.48	9
Caudal epididymal (million sperm/gram)	1119.16 ± 355.98	9	1054.03 ± 615.45	10	1319.07 ± 465.32	10	989.06 ± 528.23	9
Testicular (HRS) (million sperm/gram)	710.07 ± 238.98	10	621.27 ± 219.64	10	644.37 ± 266.27	10	506.9 ± 227.64	9
% abnormal	0.5 ± 0.71	10	0.6 ± 0.52	10	1.1 ± 1.11	10	1.3 ± 0.79	9

Discussion

The source material of BPE, black pepper, has been used as foodstuff for centuries. The use of black pepper, its essential oil, oleoresin and natural extractives in food for human consumption is considered Generally Recognized As Safe (GRAS) by the FDA for use as a flavoring agent in foods. The safety of black pepper and piperine has been previously evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 2006, and more recently, by the European Food Safety Authority (EFSA) in 2015.^27,28 However, safety data on NFT is lacking even though it is another major bioactive component of black pepper besides piperine.

Therefore, the present study is the first toxicology study that aimed to investigate the safety of approximately 50% enriched NFT in BPE (Figure 1) and understand the adverse effects, if any. An Ames test was performed to assess mutagenicity of BPE with results that demonstrated no substantial increase in revertant colony counts with or without metabolic activation and within accepted ranges based on historical controls (Tables 1 and 2). Hence it was concluded that BPE was non-mutagenic. These results are consistent with the mutagenic potential of piperine that was assessed using Salmonella typhimurium strains and shown to be negative for mutagenicity.^29,30

To further test for safety, a 90-day toxicology study was performed with BPE with several evaluations, including: mortalities, clinical or functional observations, motor activity, changes in hematology, clinical chemistry, coagulation, urinalysis, thyroid hormone parameters, and general pathology. Qualitative assessment and statistical analysis of the results showed that there were no observed nor significant changes in all evaluated parameters caused by dietary BPE administration. There were no BPE-related mortalities or functional deprivations.

Changes were found in relative organ weights but they had no histologic correlation and were considered unrelated to BPE (Table 6). Sperm analysis was also performed as part of the 90-day toxicology study which showed some sperm abnormalities, however, these were also not attributed to BPE administration. Hence, the results were considered toxicologically insignificant, since there were no other corresponding observations. Further results of the sperm analysis showed no statistically significant changes in percent motile sperm, epididymal sperm count, homogenization- resistant sperm count, or percent abnormal sperm, demonstrating again that other changes were unrelated to BPE (Table 7).

Therefore, as with a few parameters discussed in the subchronic toxicity study, statistically significant changes found were considered unrelated to BPE. The values of all the significant changes were without an absolute dose-dependent pattern or there were no correlations with necropsy and histopathology; hence these changes were considered as biological variation instead of a treatment effect.

Overall, the subchronic toxicity study with BPE was consistent with the findings of a study that was carried out to evaluate acute and subchronic toxicities of the water extract from the dried fruits of Piper nigrum L.³¹ The subchronic toxicity was determined by oral feeding both male and female rats daily at the doses of 300, 600 and 1200 mg/kg body weight continuously for 90 days. The results indicated that the water extract from the dried fruits of P. nigrum L. does not cause subchronic toxicities in either male or female rats.³¹ The highest dose tested with BPE (700 mg/kg/day) was the NOAEL compared to 1200 mg/kg body weight of the tested water extract from the dried fruits of P. nigrum L. In summary the subchronic study with BPE revealed no adverse effects. The safety of a daily human intake of 120 mg/day NFT could be supported by the extrapolation of an acceptable daily intake (∼6.3 mg/kg/day) for a 60 kg individual, from the NOAEL (380 mg/kg bw/day NFT) established from this subchronic toxicity study in rats.

Although the mechanism of action and associated health benefits of plant-derived NFT have been investigated using in vitro and in vivo studies,^9,16 this was the first investigation of NFT in BPE demonstrating its safety for a duration of 90-days in vivo at equivalent dose levels of 68, 190, and 380 mg/kg/day.

In conclusion, the results of this study showed no adverse effects of BPE in administered diets to rats at levels 125, 350, and 700 mg/kg/day. The no-observed-adverse-effect level (NOAEL) for BPE was determined to be 700 mg/kg/day, the highest dose tested, equivalent to NFT dose level of 380 mg/kg/day. The findings from present investigations also revealed that BPE is unlikely to cause any mutagenic effects.

Supplemental Material

Supplemental Material - Assessment of safety through mutagenicity and subchronic toxicity studies with black pepper extract preparation

Supplemental Material for Assessment of safety through mutagenicity and subchronic toxicity studies with black pepper extract preparation by Lee Chae, Sungwon Lee, Swetha Mahadevan Mark R Bauter, Colleen Wojenski, Kristin Robertson, Brian Premkumar and Brinda Mahadevan in Human & Experimental Toxicology.

Footnotes

Author contributions

All the authors contributed to the conceptualization, methodology, investigation and review of the manuscript, while the majority of the writing, review and editing was performed by LC, MRB, BP and BM.

Ethical Approval

All animal experiments described in this study were conducted at Product Safety Labs (PSL), an AAALAC-accredited facility, in compliance with the U.S. Animal Welfare Act Regulations (9 CFR) and the Guide for the Care and Use of Laboratory Animals (8th edition, National Research Council, 2011). Study protocols were reviewed and approved by the PSL Institutional Animal Care and Use Committee (IACUC) prior to study initiation P700.01 BRI (14-Day Dietary Toxicity/Palatability Study in Rats) and P703.01 BRI (90-Day Dietary Study in Rats) (IACUC Protocols Approved November 6, 2019). Humane care and use of laboratory animals were ensured throughout the experimental phases of this research.

Declaration of conflicting interests

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Brightseed sponsored the study. LC, SL are employees of Brightseed. SM is a former employee of Brightseed. BM has worked as a consultant with Brightseed Inc., under a cost-reimbursable contract for this project.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Lee Chae

Mark R Bauter

Supplemental Material

Supplemental material for this article is available online.

References

Bui

Piao

Song

, et al. Piper nigrum extract ameliorated allergic inflammation through inhibiting Th2/Th17 responses and mast cells activation. Cell Immunol 2017; 322: 64–73.

Takooree

Aumeeruddy

Rengasamy

, et al. A systematic review on black pepper (Piper nigrum L.): from folk uses to pharmacological applications. Crit Rev Food Sci Nutr 2019; 59(sup1): S210–S243.

Ravindran

Divakaran

Pillai

. Handbook of herbs and spice. Oxford, UK: Woodhead Publisher, 2012, vol 1, pp. 86–115.

Butt

Pasha

Sultan

, et al. Black pepper and health claims: a comprehensive treatise. Crit Rev Food Sci Nutr 2013; 53(9): 875–886.

Selvendiran

Prince Vijeya Singh

Sakthisekaran

. In vivo effect of piperine on serum and tissue glycoprotein levels in benzo (a) pyrene induced lung carcinogenesis in Swiss albino mice. Pulm Pharmacol Ther 2006; 19(2): 107–117.

Vellaichamy

Balakrishnan

Panjamurthy

, et al. Chemopreventive potential of piperine in 7, 12-dimethylbenz [a] anthracene-induced skin carcinogenesis in Swiss albino mice. Environ Toxicol Pharmacol 2009; 28(1): 11–18.

Kapoor

Singh

, et al. Chemistry and in vitro antioxidant activity of volatile oil and oleoresins of black pepper (Piper nigrum). J Agric Food Chem 2009; 57(12): 5358–5364.

Nakatani

Inatani

Fuwa

. Structures and syntheses of two phenolic amides from Piper nigrum L. Agric Biol Chem 1980; 44(12): 2831–2836.

Jiang

Wang

. N-trans-feruloyltyramine inhibits LPS-induced NO and PGE2 production in RAW 264.7 macrophages: involvement of AP-1 and MAP kinase signalling pathways. Chem Biol Interact 2015; 235: 56–62.

10.

Yoshihara

Takamatsu

Sakamura

. Three new phenolic amides from the roots of eggplant (Solanum melongena L). Agric Biol Chem 1978; 42(3): 623–627.

11.

Matsutomo

Stark

Hofmann

. Targeted screening and quantitative analyses of antioxidant compounds in aged-garlic extract. Eur Food Res Tech 2018; 244: 1803–1804.

12.

Liu

Xiang

, et al. Metabolites identification of bioactive compounds daturataturin A, daturametelin I, n-trans-feruloyltyramine, and cannabisin F from the seeds of Datura metel in rats. Front Pharmacol 2018; 9: 731.

13.

Maciel

Chaves

Brito Filho

, et al. New alcamide and anti-oxidant activity of Pilosocereus gounellei A. Weber ex K. Schum. Bly. ex Rowl. (Cactaceae). Molecules 2015; 21(1): 11.

14.

Yeh

Bosch

Daoud

. Role of hepatocyte nuclear factor 4-alpha in gastrointestinal and liver diseases. World J Gastroenterol 2019; 25(30): 4074–4091.

15.

Veeriah

Lee

Levine

. Long-term oral administration of an HNF4α agonist prevents weight gain and hepatic steatosis by promoting increased mitochondrial mass and function. Cell Death Dis 2022; 13(1): 89.

16.

Lee

Veeriah

Levine

. Liver fat storage is controlled by HNF4α through induction of lipophagy and is reversed by a potent HNF4α agonist. Cell Death Dis 2021; 12(6): 603.

17.

Kiselyuk

Lee

Farber-Katz

, et al. HNF4α antagonists discovered by a high-throughput screen for modulators of the human insulin promoter. Chem Biol 2012; 19(7): 806–818.

18.

OECD . Test No. 408: Repeated Dose 90-Day Oral Toxicity Study in Rodents, OECD guidelines for the testing of chemicals, Section 4. Paris: OECD Publishing, 2018. https://doi.org/10.1787/9789264070707-en

19.

Mortelmans

Zeiger

. The Ames Salmonella/microsome mutagenicity assay. Mutat Res 2000; 455(1-2): 29–60.

20.

Gatehouse

. Bacterial mutagenicity assays: test methods. Methods Mol Biol 2012; 817: 21–34.

21.

OECD . OECD Principles on Good Laboratory Practice, OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring, No. 1. OECD Publishing, Paris. 1998; 98(17). https://doi.org/10.1787/9789264078536-en

22.

FDA . US FDA toxicological Principles for the safety assessment of food Ingredients. Redbook 2000 2007. https://www.fda.gov/files/food/published/Toxicological-Principles-for-the-Safety-Assessment-of-Food-Ingredients.pdf. IV.C.4.a.

23.

National Research Council . Guide for the care and use of laboratory animals. 8th edition Washington, DC: The National Academies Press, 2011.

24.

JECFA . Aliphatic and aromatic amines and amides. In: Safety Evaluation of certain food additives: sixty-fifth meeting of the joint FAO/WHO expert committee on food additives (JECFA). (WHO food additives series no. 56). Rome, Italy: Food and Agriculture Organization of the United Nations (FAO) / Geneva, Switzerland: World Health Organization (WHO), International Programme on Chemical Safety (IPCS), 2006, pp. 327–403.

25.

EFSA Panel on Food Contact Materials Enzymes Flavourings and Processing Aids CEF . Scientific opinion on flavouring group evaluation 86, revision 2 (FGE.86Rev2): consideration of aliphatic and arylalkyl amines and amides evaluated by JECFA (65th meeting): flavouring group evaluation 86 revision 2. EFSA J 2015; 13(1): 3998.

26.

Karekar

Mujumdar

Joshi

, et al. Assessment of genotoxic effect of piperine using Salmonella typhimurium and somatic and somatic and germ cells of Swiss albino mice. Arzneimittelforschung 1996; 46(10): 972–975.

27.

Thiel

Buskens

Woehrle

, et al. Black pepper constituent piperine: genotoxicity studies in vitro and in vivo. Food Chem Toxicol 2014; 66: 350–357.

28.

Chunlaratthanaphorn

Lertprasertsuke

Ngamjariyawat

, et al. Acute and subchronic toxicity study of the water extract from dried fruits of Piper nigrum L. in rats. Health 2007; 29: 109–124.

29.

Karekar

Mujumdar

Joshi

, et al. Assessment of genotoxic effect of piperine using Salmonella typhimurium and somatic and somatic and germ cells of Swiss albino mice. Arzneimittel-forschung. 1996; 46(10): 972–975.

30.

Thiel

Buskens

Woehrle

Etheve

, et al. Black pepper constituent piperine: Genotoxicity studies in vitro and in vivo. Food Chem Toxicol. 2014; 66: 350–357.

31.

Chunlaratthanaphorn

Lertprasertsuke

Ngamjariyawat

, et al. Acute and subchronic toxicity study of the water extract from dried fruits of Piper nigrum L. in rats. Health. 2007; 29: 109–124.

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