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Abstract

NXT15906F6 (TamaFlex^®) is a food-derived herbal composition with synergistic anti-inflammatory properties and the potential to improve muscle–skeletal function in healthy populations. NXT15906F6 contains standardized ethanol/aqueous extracts of Tamarindus indica seeds and an ethanol extract of Curcuma longa rhizome in a 2:1 ratio. The present study was conducted to evaluate the broad-spectrum safety of NXT15906F6. All studies were conducted in compliance with good laboratory practice requirements and followed Organization for Economic Cooperation and Development Guidelines for Testing of Chemicals. The acute oral and acute dermal lethal dose of NXT15906F6 was greater than 2000 mg/kg body weight in Wistar rats. NXT15906F6 was classified as nonirritating to the skin and as a mild irritant to the eyes of New Zealand white rabbits. A repeated-dose 90-day subchronic study in Wistar rats was conducted. Groups of animals (n = 20, 10 male and 10 female) were orally supplemented with 0, 250, 500, and 1000 mg/kg/day of NXT15906F6 for 90 days. In parallel, two separate groups of animals (n = 10, five male and five female) were gavaged with 0 and 1000 mg/kg body weight of NXT15906F6 for 90 days and observed for another 28 days. No morbidity, mortality, or significant adverse events were observed during the study or the follow-up phase. The no observed adverse effect level for NXT15906F6 was 1000 mg/kg/day in both male and female rats. A bacterial reverse mutation test and an in vivo mouse bone marrow erythrocyte micronucleus test showed that the NXT15906F6 herbal blend is nonmutagenic and nongenotoxic. In summary, the safety studies conducted in various models demonstrate the broad-spectrum safety of NXT15906F6.

Keywords

NXT15906F6 acute oral acute dermal toxicity acute dermal irritation acute eye irritation repeated-dose 90-day subchronic toxicity study Ames bacterial reverse-mutation assay mammalian erythrocyte micronucleus test

Introduction

Although synthetic drugs reduce inflammation and pain, they work in a way that may be counterproductive to the purpose of treatment. Nonsteroidal anti-inflammatory drugs (NSAIDs) exert these effects by acting as cyclooxygenase (COX) inhibitors. However, inhibition of COX-1 in the gastrointestinal tract can result in serious adverse effects including ulcers, while inhibition of COX-2 in vascular cells can result in atherothrombosis and hypertension.¹

As consumers are increasingly interested in natural therapies that offer safe and effective inflammation and pain support, research suggests that botanical polyphenolic constituents are effective inhibitors of pro-inflammatory enzymes while preventing the formation of toxic cytokines generated in anti-inflammatory actions.² Unlike synthetic COX inhibitors, these botanicals generally have a century-long track record of safe use in food products.²

NXT15906F6 is a novel anti-inflammatory botanical formula that combines ethanol and aqueous extracts of Tamarindus indica fruit seeds (i.e. tamarind; Leguminosae family) and an ethanol extract of Curcuma longa rhizome (i.e. turmeric; Zingiberaceae family). These components are standardized for polyphenolic proanthocyanidins and curcuminoids, respectively.

Traditionally used in food applications, tamarind pericarp and seeds contain proanthocyanidins in various forms such as apigenin, catechin, procyanidin B2, epicatechin, procyanidin dimer, and procyanidin trimer, along with flavanones taxifolin, eriodictyol, and naringenin.³ Proanthocyanidins have anti-inflammatory effects; reduce lipid peroxidation and malondialdehyde production; inhibit N-acetyl-beta-d-glucosaminidase and nitric oxide (NO) synthase activity; and lower NO, interleukin-1beta (IL-1b), tumor necrosis factor (TNF)-alpha, and prostaglandin E₂ levels.^4

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Turmeric rhizomes are a source of polyphenolic curcumin and its derivatives demethoxycurcumin and bisdemethoxycurcumin, collectively known as curcuminoids. Curcuminoids have been used for centuries as a spice (curry), food preservative, and a coloring agent. They have been found to inhibit the arachidonic acid pro-inflammatory pathways (i.e. COX-1 and COX-2).¹ The results of a meta-analysis of clinical trials have shown that curcumin has a significant lowering effect on circulating levels of the pro-inflammatory cytokine IL-6.⁹ Furthermore, Daily et al.¹⁰ conducted a review and meta-analysis of randomized clinical trials involving the ability of turmeric extract and curcumin to alleviate symptoms of joint arthritis. These authors concluded that the data support the efficacy of turmeric extract (curcumin) in relieving pain and reducing the symptoms associated with arthritis.

The efficacy of NXT15906F6 has been evaluated in a double-blind, placebo-controlled clinical study of healthy men and women aged 35–70 years, for its potential beneficial effects on joint health. Researchers found the herbal blend benefitted joint health and comfort while improving physical function in healthy subjects in a variety of recognized health indices. The study participants did not report any major adverse events during the trial. The study findings are being published separately.

The present manuscript describes a systematic safety evaluation of NXT15906F6, including acute oral toxicity, acute dermal toxicity, and 90-day subchronic toxicity studies in Wistar rats; primary skin and eye irritation studies in New Zealand Albino rabbits; and a bacterial reverse-mutation assay and a mouse bone marrow erythrocyte micronucleus assay to evaluate safety.

Materials and methods

Test item

NXT15906F6 is an herbal blend containing the extracts of the T. indica seeds and C. longa rhizome. Dried seeds of T. indica were pulverized and extracted with ethanol. The leftover residue was extracted with water. The extracts were concentrated independently under vacuum and blended in a 9:1 ratio to obtain a novel T. indica seed extract (TSE). Each batch was assayed by high-performance thin-layer chromatography and standardized to contain not less than (NLT) 65% proanthocyanidins.

Dried C. longa rhizomes were pulverized, extracted with ethanol, and concentrated to a thick paste under vacuum. The C. longa rhizome extract (CLE) contains a minimum of 15% total curcuminoids as determined by high-performance liquid chromatography. The test item NXT15906F6 is a blend of TSE, cyclic guanosine monophosphate (CLE), and excipients in a 6:3:1 ratio. It was produced in a cGMP-compliant manufacturing facility and stored at room temperature. The test item contained 8% (w/w) microcrystalline cellulose powder and 2% (w/w) Syloid silica as excipients. NXT15906F6 was standardized to contain NLT 39% of proanthocyanidins and 4.5% of total curcuminoids.

Chemicals, reagents, and bacterial strains

The clinical chemistry and hematology reagents were obtained from I.LAB (Bergamo, Italy) and Siemens (Munich, Germany), respectively. Bacto Agar was purchased from Becton Dickinson (Sparks, Maryland, USA). Sodium carboxymethyl cellulose (CMC-Na), polyethylene glycol 400, dimethyl sulfoxide, nicotinamide adenine dinucleotide phosphate, glucose-6-phosphate, sodium azide, 2-nitrofluorene, 9-aminoacridine, 4-nitroquinoline-1-oxide, 2-aminoanthracene, fetal bovine serum, absolute methanol, and cyclophosphamide monohydrate (CPA) were obtained from Sigma Chemical Co. (St. Louis, Missouri, USA). Sodium thiopentone barbiturate, isoflurane, magnesium chloride, potassium chloride, and sodium phosphate salts were also purchased from Sigma Chemical Co. Salmonella typhimurium TA98, TA100, TA1535, and TA1537 strains were obtained from the National Collection of Type Cultures (London, England). Escherichia coli WP2 uvrA/pKM101 was obtained from National Collection of Industrial, Food and Marine Bacteria (Edinburgh, Scotland). Lyophilized rat liver S9 fraction was purchased from Celsis In Vitro Technologies (Baltimore, Maryland, USA).

Experimental animals and housing conditions

All studies were conducted at Liveon Biolabs Pvt. Ltd (Karnataka, India). Specific pathogen-free experimental animals were used in various studies to evaluate the safety of NXT15906F6. Male and female Wistar rats were obtained from Vivo BioTech Ltd (Hyderabad, India), Swiss albino mice were procured from Palamur Biosciences Private Limited (Hyderabad, India), and New Zealand white rabbits were acquired from Mahaveera Enterprises (Hyderabad, India). Animals were allowed free access to standard diet and provided reverse-osmosis filtered water ad libitum. The animals were acclimatized to laboratory conditions for 5–7 days before treatment began. Animal rooms were maintained at 22°C ± 3°C, 40–70% relative humidity, and a 12-h light/12-h dark cycle. The protocol was approved by the Institutional Animal Ethics Committee and licensed and monitored by the Committee for the Purpose of Control and Supervision of Experiments on Animals, India. All studies were conducted in compliance with good laboratory practices and the test procedures were conducted following the Organization for Economic Cooperation and Development (OECD) Guidelines for the testing of chemicals.¹¹

Acute oral toxicity study in female rats

A limit test of the study product was performed using five 9-week-old female Wistar rats at a dose of 2000 mg/kg body weight, according to the OECD Test Guideline 425.⁹ The NXT15906F6 herbal blend was suspended in 0.5% w/v CMC-Na in water and a total volume of 10 mL was made. The herbal blend in this solution had a final concentration of 200 mg/mL to allow a constant dosage volume of 10 mL/kg body weight. This suspension was administered through oral gavage at a dose of 2000 mg/kg body weight. All animals were observed for morbidity/mortality or clinical signs at pre-dose, 30 min, 1 h, 2 h, 3 h, and 4 h post-dosing on day 1. Thereafter, animals were observed once daily for 14 consecutive days. Body weights were measured on a weekly basis during the study period. Gross pathology was performed at the end of the experimental period (i.e. day 15).

Acute dermal toxicity study in rats

An acute dermal toxicity test was conducted in Wistar rats to determine the potential of NXT15906F6 to produce toxicity from a single dermal application. A limit test was performed using five male (weighing 245–270 g) and five female (weighing 205–235 g) Wistar rats, according to OECD Test Guideline 402.¹² One day before applying the test material, hair was removed by clipping the dorsal area and the trunk. NXT15906F6 at a dose of 2000 mg/kg body weight was calculated based on the body weight of the individual animals. The test item was evenly distributed on surgical gauze patch and moistened with a minimal amount of distilled water. The prepared surgical gauze was then applied to the intact skin (4 × 4 cm²) and held in place with nonirritating adhesive tape for 24 h. Thereafter, the residual test item was wiped off the skin using water-soaked cotton. Animals were observed for mortality/morbidity and clinical signs of toxicity at various time intervals, and body weights were recorded on a weekly basis during the study period. Gross pathology was performed on day 15.

Acute dermal irritation study in rabbits

The acute dermal irritation test was conducted on male New Zealand white rabbits to determine the irritation potential of NXT15906F6 after a single topical application per the OECD Test Guideline 404.¹³ Three healthy young adult rabbits (weighing 2.0–2.5 kg) were used for the study. On the day before the application, fur was removed from the dorsal and trunk area using a clipper. A single dermal dose of 500 mg of NXT15906F6 moistened with a small amount of distilled water was applied to a 6 × 6 cm² clipped area of skin. The application area was covered with two-ply gauze patch that was held in place with nonirritating tape and covered with porous tape for a semiocclusive dressing. The rabbits were exposed to the test item for 4 h after which it was removed. Test sites were evaluated by Draize score¹⁴ for signs of dermal irritation approximately 1, 24, 48, and 72 h after test item removal. Initially, the test was carried out using one animal and then the results were confirmed in two additional animals. After 72 h of observation, the animals were killed using an overdose of sodium thiopentone and subjected to necropsy and gross pathological examination.

Acute eye irritation study in rabbits

An acute eye irritation study was conducted in three healthy young adult male New Zealand white rabbits (weighing 2.0–2.8 kg) to determine the irritation potential of NXT15906F6 from a single instillation via the ocular route per the OECD Test Guideline 405.¹⁵ Both eyes of each animal were examined approximately 24 h before the application of NXT15906F6. The lower left eyelid was gently pulled away from eyeball and a single dose of 100 mg of the test item was instilled into the conjunctival sac of the left eye. The eyelids were then gently held together for a few seconds to prevent the loss of the test item. The right eye remained untreated and served as the control. The eyes of the test animals were washed with distilled water at 24 h following the application of the test item. Initially, the test was carried out using one animal. The results from the initial test were confirmed by repeating the experiment in two additional rabbits. The conjunctiva, iris, and cornea of both treated and control eyes were evaluated and scored according to the Draize method¹⁴ at 1, 24, 48, and 72 h following the application of the test item. All the animals were killed at the end of the observation period using an overdose of sodium thiopentone and subjected to necropsy and gross pathological examination.

Subchronic (90-day) repeated-dose oral toxicity study

A 90-day subchronic repeated-dose oral toxicity study evaluated the potential toxicity of NXT15906F6 according to the OECD Guideline 408.¹⁶ The herbal blend was administered through oral gavage to 7- to 8-week-old Wistar rats for 90 consecutive days. The recovery animals (vehicle control recovery [G1R] and 1000 mg/kg high-dose recovery [G4R] groups) were assessed further for any effects over the subsequent recovery period of 28 days. NXT15906F6 was weighed and mixed into the vehicle (0.5% w/v CMC-Na in water). The suspension was prepared fresh daily, immediately prior to dose administration, and continuously stirred throughout dosing administration. The suspension was administered by gavage to rats for 90 consecutive days at graduated dose levels of 250, 500, and 1000 mg/kg body weight per day for low-dose (G2), mid-dose (G3), high-dose (G4), and G4R groups, respectively. The rats in the vehicle control (G1) and G1R groups received the vehicle alone. The dose volume administered was 10 mL/kg body weight per day. The G1–G4 groups consisted of 20 rats (10 male and 10 female), while each of the recovery groups (G1R or G4R) consisted of 10 rats (five male and five female rats). The vehicle or test item dose formulations were not administered to the G1R or G4R groups during the 28 days of recovery period following 90 days of treatment.

Animals were observed twice daily for morbidity/mortality and clinical signs were observed once daily after administration of the test item. Observations included changes in skin and fur, eyes, mucous membranes, respiratory system, circulatory system, autonomic/central nervous systems, somatomotor activity, and behavior patterns. Attention was directed to observations of tremors, convulsions, salivation, diarrhea, lethargy, sleep, and coma. Detailed clinical observations were conducted on the day before test item administration and weekly during the experimental period. Body weight and feed consumptions were measured weekly. Functional observation battery (FOB) tests and the ophthalmological examinations were carried out during preinitiation of dosing and at the termination of respective test groups (G1–G4R).

The animals were fasted overnight (approximately 16–18 h) before blood collection. At day 91 for G1–G4 groups and at day 119 for recovery groups, blood samples were collected using a retro-orbital plexus puncture under mild isoflurane anesthesia with the help of a fine capillary tube containing potassium EDTA and heparin tubes for hematology and clinical chemistry analysis, respectively.

Terminal necropsy of all animals was carried out and the organ weights (kidneys, adrenals, spleen, heart, liver, thymus, brain, testes/ovaries, uterus/epididymides, seminal vesicles with coagulating glands, and thyroid/parathyroid) were measured. Absolute and relative organ weights were recorded. Subsequently, various organs and tissues were collected for histopathological investigation according to the OECD Test Guideline 408¹⁴ on day 91 for G1–G4 groups and on day 119 for the recovery groups. The organs and tissue were weighed and preserved in phosphate-buffered 10% formalin for histopathology examination. Tissues from control (G1) and high-dose (G4) groups were processed, embedded in paraffin wax, sectioned at 3–5 µm, and stained with hematoxylin and eosin. The sections were examined by light microscopy using a ZEISS AXIO Scope.A1 microscope (Carl Zeiss, Munich, Germany).

Statistical analyses

Statistical analysis was performed using GraphPad Prism software version 6 (GraphPad Software, La Jolla, California, USA). One-way analysis of variance (ANOVA) followed by Dunnett’s post hoc tests was used to analyze the differences between different dosage groups and the control group. Student’s t test was used for the recovery group. All values are reported as mean ± standard deviation. All comparisons were evaluated at the 95% level of confidence with statistical significance of p < 0.05.

Bacterial reverse-mutation assay

The Ames test was conducted according to the OECD Test Guideline 471¹⁷ to determine the potential of NXT15906F6 to induce point mutations at selected histidine loci of the tested strains of S. typhimurium (TA98, TA100, TA1535, and TA1537) and tryptophan loci of the WP2 uvrA/pKM101 strain of E. coli in the presence or absence of metabolic activation system.^18,19 Suspensions of bacterial cells were exposed to the test item in triplicate at concentrations of 100, 266, 707, 1880, and 5000 μg/plate. The suspensions were mixed with an overlay agar and plated immediately onto minimal medium. After 67 h of incubation at 37°C ± 1°C, revertant colonies in the test item–treated plates were counted manually and compared to the number of revertant (spontaneous) colonies on vehicle control plates. Appropriate strain-specific positive controls were included in this study (see Table 12).

Mouse bone marrow erythrocyte micronucleus assay

The mouse bone marrow erythrocyte micronucleus assay study was conducted following the OECD Test Guideline 474 (OECD 474).²⁰ Healthy 6- to 10-week-old mice were randomly placed into five groups (n = 10 per group; five male and five female). Control group animals received the vehicle (0.5% w/v CMC-Na). The herbal blend was administered orally twice at 500, 1000, and 2000 mg/kg body weight, at an interval of approximately 24 h. The positive control (CAP; 40 mg/kg body weight) was administered through intraperitoneal route only once on the second day of test item administration. The bone marrow sampling was performed after 22 h of the second day treatment. All animals were killed using carbon dioxide (CO₂) inhalation and both femurs were collected. A total of 4000 polychromatic (immature) erythrocytes (PCEs)/animal were counted for evaluation of micronucleus frequency. The toxicity of NXT15906F6 was determined by a ratio of immature PCEs and total erythrocytes (TE = immature + mature).

Results

Acute oral toxicity study in rats

At the limit dose of 2000 mg/kg body weight, NXT15906F6 did not cause mortality/morbidity and did not show any signs of toxicity in rats following dosing and during the following observation period of 15 days. The experimental rats gained body weight normally. Gross pathological examination did not reveal any abnormalities. Based on these findings, the median oral lethal dose (LD₅₀) of NXT15906F6 was found to be greater than 2000 mg/kg body weight in female Wistar rats.

Acute dermal toxicity in rats

All animals survived, gained normal body weight, and appeared active and healthy. No signs of gross toxicity, dermal irritation, adverse pharmacological effects, or abnormal behavior were observed. Based on the above findings, the median dermal LD₅₀ of NXT15906F6 was found to be greater than 2000 mg/kg body weight in male and female Wistar rats.

Acute dermal irritation study in rabbits

None of the animals treated with NXT15906F6 showed dermal reactions at 1, 24, 48, and 72 h after patch removal. All animals appeared active and healthy, with no signs of toxicity, adverse pharmacological effects, or abnormal behavior. Body weight gain of the individual animal was normal during the study period. Under the study conditions, the primary dermal irritation index for NXT15906F6 is 0.0, thus NXT15906F6 is classified as a nonirritant when applied to the skin of New Zealand white rabbits.

Primary eye irritation study in rabbits

One hour following the test item instillation, all treated eyes exhibited conjunctival redness. The severity increased at the 24-h observation and subsided after 48 h. The affected eyes appeared normal at day 7. No corneal opacity or iritis was observed in any treated eye during the study. Apart from the eye irritation noted, all animals appeared active and healthy. There were no other signs of gross toxicity, adverse pharmacological effects, or abnormal behavior. Thus, NXT15906F6 is classified per the Harmonized Integrated Classification System¹² as a “mild irritant” to rabbit eyes.

The 90-Day (subchronic) repeated-dose oral toxicity study

Mortality, clinical signs, and FOB

Oral administration of NXT15906F6 for 90 days did not cause any mortality or morbidity in any of the treatment groups throughout the experimental period. No treatment-related cage-side clinical signs were noticed during course of the study.

No significant treatment-related toxicological changes were observed in FOB at any of the doses tested during the experimental period. No test item–related abnormalities in the home-cage observations, observation during removal from the home cage and handling, open-field observation, sensory reactive measurements, or landing hind limbs foot splay were noted in any treatment group. No test item–related difference in the forelimb grip strength values was noted in any treatment group. No treatment-related abnormalities were observed in the mean locomotor activity (vertical and horizontal counts) in male and female treatment groups (G1–G4). A significant decrease in locomotor activity (only vertical counts) was observed in the G4R female animals (data not shown). However, this observation was not found in the female animals of G4. Hence, this change was considered sporadic or incidental.

Body weight and feed consumption

No significant changes in the body weights were observed in any of the treatment groups (G1–G4, G1R, and G4R) of either sex at any time point of the study when compared with the respective control groups. The development of animals in all groups during the experimental period corresponded to their species and age (Tables 1 and 2). No significant differences in feed consumption were seen between groups. A few intergroup differences attained statistical significance but lacked consistency that is unlikely to be of biological significance. The feed consumption of G2 males decreased during the first week (day 8) when compared with the respective control. In female rats, a decrease in feed consumption was observed in G2 females on day 22, whereas an increase in feed consumption was observed in G4 females at various time intervals (days 8, 15, 36, 43, 71, and 78) when compared with the respective control. These changes are not related to the treatment with any toxicological significance because they had a nonlinear response with respect to body weight gain. In the G4R group, a significant decrease in feed intake was observed during the experimental period at various occasions (days 15, 85, and 99 for males and days 22 and 36 for females). These changes were considered sporadic and unrelated to the treatment because no similar changes were observed in the same dose main-group (G4) animals and no related changes in the corresponding animal body weight gains (Tables 3 and 4).

Table 1.

Body weights (mean ± SD) of male Wistar rats given NXT15906F6 daily by gavage for 90 days.^a

		NXT15906F6
Days	Control (0 mg/kg)	250 mg/kg	500 mg/kg	1000 mg/kg	Control recovery	1000 mg/kg recovery
1	183.17 ± 10.91	183.00 ± 9.55	182.20 ± 8.05	187.87 ± 14.05	177.31 ± 11.70	177.70 ± 6.65
8	228.74 ± 13.45	222.32 ± 18.49	228.32 ± 9.05	232.39 ± 17.85	222.64 ± 16.83	217.77 ± 6.87
15	273.05 ± 14.81	261.37 ± 22.01	271.06 ± 10.42	276.38 ± 18.83	265.81 ± 21.59	257.18 ± 8.69
22	306.50 ± 16.79	296.08 ± 19.70	302.76 ± 14.29	310.64 ± 22.89	302.69 ± 26.72	294.10 ± 9.21
29	334.15 ± 20.00	323.43 ± 23.65	330.18 ± 18.96	339.92 ± 28.86	335.32 ± 29.90	319.53 ± 10.04
36	356.81 ± 22.38	342.94 ± 25.50	352.45 ± 24.03	362.35 ± 31.89	358.30 ± 34.57	345.32 ± 12.93
43	376.54 ± 25.24	361.47 ± 28.44	372.02 ± 27.75	380.99 ± 33.61	379.15 ± 37.98	366.67 ± 13.13
50	392.10 ± 28.58	370.86 ± 29.19	387.66 ± 30.21	394.76 ± 39.10	390.35 ± 33.00	381.21 ± 14.50
57	404.10 ± 30.25	386.25 ± 33.92	397.12 ± 29.87	406.07 ± 41.80	408.53 ± 40.71	394.74 ± 16.32
64	415.49 ± 31.33	397.94 ± 36.98	408.28 ± 31.17	418.57 ± 39.15	421.01 ± 43.48	404.46 ± 20.17
71	424.75 ± 34.94	405.45 ± 38.20	415.80 ± 34.86	425.73 ± 39.63	430.54 ± 48.21	412.64 ± 21.45
78	433.07 ± 37.01	413.14 ± 40.24	425.10 ± 37.02	430.05 ± 36.84	442.29 ± 50.47	422.65 ± 20.47
85	441.49 ± 36.76	419.13 ± 40.16	432.13 ± 38.34	433.61 ± 37.67	450.15 ± 54.09	430.19 ± 20.19
92					459.97 ± 55.22	434.17 ± 21.34
99					467.88 ± 59.81	437.89 ± 25.30
106					474.26 ± 60.42	444.94 ± 27.60
113					481.58 ± 65.00	449.97 ± 29.08
118					487.10 ± 68.07	453.82 ± 30.76

SD: standard deviation.

^a n = 10 in control, 250, 500, and 1000 mg/kg NXT15906F6 groups; n = 5 in recovery groups.

Table 2.

Body weights (mean ± SD) of female Wistar rats given NXT15906F6 daily by gavage for 90 days.^a

		NXT15906F6
Days	Control (0 mg/kg)	250 mg/kg	500 mg/kg	1000 mg/kg	Control recovery	1000 mg/kg recovery
1	147.07 ± 10.71	143.83 ± 5.24	147.15 ± 11.47	149.53 ± 7.12	151.35 ± 9.92	147.57 ± 5.21
8	167.64 ± 14.78	164.69 ± 7.08	166.53 ± 11.50	168.71 ± 7.79	167.59 ± 12.99	166.31 ± 10.70
15	184.83 ± 11.53	182.13 ± 9.45	183.50 ± 11.34	188.38 ± 8.30	187.37 ± 17.73	189.13 ± 6.10
22	199.65 ± 13.73	194.22 ± 11.98	196.78 ± 13.27	201.96 ± 7.47	202.88 ± 18.35	199.05 ± 8.51
29	209.60 ± 13.00	206.46 ± 10.77	205.76 ± 14.85	212.68 ± 10.12	213.79 ± 17.83	210.98 ± 7.48
36	215.34 ± 11.53	216.59 ± 16.36	214.78 ± 14.69	220.23 ± 10.20	223.81 ± 22.69	216.37 ± 7.14
43	225.29 ± 13.06	224.21 ± 14.82	222.35 ± 15.67	228.87 ± 10.94	230.18 ± 23.00	225.64 ± 8.19
50	229.37 ± 13.97	226.83 ± 12.59	228.40 ± 17.38	233.71 ± 12.05	238.94 ± 26.01	228.94 ± 9.02
57	232.89 ± 14.20	231.92 ± 12.67	231.45 ± 16.53	237.58 ± 13.32	247.78 ± 26.91	232.70 ± 10.84
64	235.18 ± 14.03	234.52 ± 11.30	234.82 ± 16.74	240.20 ± 13.11	244.96 ± 28.3	234.50 ± 7.98
71	238.30 ± 14.20	237.74 ± 11.52	237.86 ± 17.18	242.69 ± 13.06	249.22 ± 29.51	239.64 ± 10.83
78	240.73 ± 15.13	241.02 ± 11.89	241.86 ± 19.30	246.36 ± 12.25	253.43 ± 28.89	243.35 ± 11.65
85	242.47 ± 15.16	242.43 ± 11.25	242.69 ± 19.05	246.84 ± 13.92	253.89 ± 28.93	244.89 ± 9.51
92					259.55 ± 31.61	245.78 ± 9.22
99					256.78 ± 34.84	242.54 ± 7.93
106					257.71 ± 33.53	246.48 ± 10.58
113					258.48 ± 36.50	247.20 ± 12.08
118					261.22 ± 35.06	248.50 ± 12.24

SD: standard deviation.

^a n = 10 in control, 250, 500, and 1000 mg/kg NXT15906F6 groups; n = 5 in recovery groups.

Table 3.

Feed consumption (mean ± SD) by male Wistar rats given NXT15906F6 daily by gavage for 90 days.^a

		NXT15906F6
Days	Control (0 mg/kg)	250 mg/kg	500 mg/kg	1000 mg/kg	Control recovery	1000 mg/kg recovery
8	136.79 ± 5.11	127.86 ± 8.75^b	134.38 ± 1.13	140.49 ± 5.29	139.02 ± 8.83	138.52 ± 4.31
15	149.62 ± 10.50	144.26 ± 14.53	146.55 ± 5.54	156.49 ± 5.84	149.95 ± 4.66	134.59 ± 5.80^b
22	154.10 ± 7.62	149.66 ± 6.35	148.86 ± 7.77	159.70 ± 5.37	159.91 ± 9.15	150.69 ± 6.37
29	154.19 ± 11.85	150.48 ± 7.00	147.97 ± 6.29	158.03 ± 8.18	163.42 ± 11.93	152.56 ± 7.63
36	154.99 ± 13.41	150.70 ± 6.34	150.62 ± 8.09	157.78 ± 9.73	157.78 ± 10.55	154.07 ± 4.02
43	155.60 ± 14.78	157.62 ± 13.42	150.12 ± 8.54	154.73 ± 8.15	154.98 ± 12.30	152.41 ± 8.51
50	150.83 ± 11.89	146.71 ± 8.61	150.59 ± 10.09	150.40 ± 12.99	150.08 ± 2.73	145.61 ± 6.74
57	149.16 ± 9.91	144.68 ± 7.88	143.68 ± 5.29	149.15 ± 8.85	149.16 ± 5.49	149.01 ± 8.50
64	144.15 ± 11.97	142.64 ± 9.48	143.14 ± 5.33	143.93 ± 10.38	145.76 ± 5.65	141.09 ± 6.16
71	139.28 ± 12.21	137.47 ± 8.00	138.55 ± 11.18	136.92 ± 5.71	140.71 ± 3.18	137.79 ± 3.20
78	138.59 ± 15.52	136.11 ± 9.05	139.67 ± 9.68	129.78 ± 4.46	141.49 ± 5.47	142.56 ± 8.93
85	132.06 ± 12.38	126.61 ± 3.98	132.13 ± 9.67	129.86 ± 5.03	136.99 ± 8.49	126.25 ± 0.58^c
92	94.24 ± 8.16	89.31 ± 5.07	93.79 ± 4.72	91.60 ± 6.50	127.85 ± 6.56	122.47 ± 3.66
99					133.72 ± 9.28	116.62 ± 10.00^c
106					135.52 ± 8.22	128.07 ± 18.48
113					135.33 ± 6.00	126.04 ± 10.98
118					101.91 ± 3.71	94.69 ± 6.96

SD: standard deviation.

^a n = 10 in control, 250, 500, and 1000 mg/kg NXT15906F6 groups; n = 5 in recovery groups. ^b p < 0.05. ^c p < 0.01 versus control group.

Table 4.

Feed consumption (mean ± SD) by female Wistar rats given NXT15906F6 daily by gavage for 90 days.^a

		NXT15906F6
Days	Control (0 mg/kg)	250 mg/kg	500 mg/kg	1000 mg/kg	Control recovery	1000 mg/kg recovery
8	93.14 ± 4.78	93.34 ± 4.49	94.87 ± 2.96	98.95 ± 4.5^b	92.83 ± 2.91	94.48 ± 5.53
15	96.08 ± 5.50	97.72 ± 3.57	97.57 ± 3.41	102.35 ± 3.16^c	98.54 ± 2.98	100.81 ± 1.43
22	100.72 ± 5.60	98.53 ± 3.39	95.94 ± 2.18^b	102.31 ± 2.14	100.07 ± 3.03	96.66 ± 0.46^b
29	98.39 ± 5.83	97.47 ± 4.41	94.16 ± 3.91	97.67 ± 3.18	96.90 ± 5.83	94.02 ± 1.64
36	88.00 ± 15.26	94.67 ± 5.76	95.48 ± 2.50	98.44 ± 2.84^b	99.09 ± 0.21	94.32 ± 3.91^b
43	96.90 ± 5.82	96.20 ± 4.19	94.18 ± 2.29	101.91 ± 3.01^b	101.42 ± 10.09	98.19 ± 5.74
50	96.79 ± 7.18	95.38 ± 4.09	94.71 ± 1.95	100.41 ± 5.70	95.01 ± 7.26	96.35 ± 6.95
57	93.55 ± 6.19	93.81 ± 6.98	93.07 ± 3.00	97.70 ± 0.72	93.65 ± 5.21	97.88 ± 6.96
64	91.34 ± 7.56	94.44 ± 6.32	92.08 ± 3.09	96.36 ± 3.31	93.12 ± 3.96	93.56 ± 6.47
71	88.19 ± 7.59	90.52 ± 5.00	89.78 ± 2.39	95.20 ± 1.34^c	90.67 ± 3.15	91.68 ± 8.46
78	89.15 ± 6.48	89.54 ± 2.23	89.68 ± 4.39	94.83 ± 3.86^b	90.81 ± 5.91	95.12 ± 5.08
85	86.63 ± 4.81	84.99 ± 3.50	83.35 ± 3.63	88.27 ± 1.46	85.74 ± 6.32	90.11 ± 1.62
92	56.26 ± 5.70	55.72 ± 2.15	56.16 ± 1.45	57.98 ± 3.89	84.10 ± 0.63	81.40 ± 4.74
99					78.13 ± 6.79	76.12 ± 0.58
106					78.61 ± 5.30	84.09 ± 3.30
113					81.56 ± 5.46	85.84 ± 9.05
118					62.72 ± 2.80	60.40 ± 3.92

SD: standard deviation.

^a n = 10 in control, 250, 500, and 1000 mg/kg NXT15906F6 groups; n = 5 in recovery groups.

^b p < 0.05.

^c p < 0.01 versus control group.

Ophthalmological observation

No ocular changes were observed in any aspect of the eyes either at the start or at the end of the study at any of the doses of NXT15906F6 tested. Thus, NXT15906F6 is not an ocular toxicant.

Hematology and clinical chemistry

The effects of 90 days (subchronic) administration of NXT15906F6 on hematological parameters are presented in Tables 5 and 6. After 90 days of supplementation, no treatment-related changes were observed in hematological parameters in comparison with the control group, with some exceptions of marginal variations in certain parameters. In the G2 and G3 groups (female), there was a slight but significant reduction in mean corpuscular volume, and there was also a slight but significant reduction in basophil count in the G2 female group (Table 6). However, these variations are within the historical control range for Wistar rats in the test facility.

Table 5.

Hematological findings (mean ± SD) in male rats after 90 days oral administration of NXT15906F6.^a

		NXT15906F6
Parameters	Control (0 mg/kg)	250 mg/kg	500 mg/kg	1000 mg/kg	Control recovery	1000 mg/kg recovery
RBC (10⁶ cells/µL)	9.50 ± 0.39	9.59 ± 0.50	9.65 ± 0.36	9.70 ± 0.32	9.65 ± 0.66	9.66 ± 0.32
HB (g/dL)	16.03 ± 0.24	16.09 ± 0.44	16.40 ± 0.75	16.23 ± 0.50	16.76 ± 0.34	16.6 ± 0.29
Hct (%)	50.16 ± 1.52	51.23 ± 1.84	51.73 ± 2.23	52.40 ± 3.65	51.80 ± 1.91	51.64 ± 1.45
MCV (fL)	52.83 ± 1.77	53.45 ± 1.46	53.62 ± 1.45	54.06 ± 3.65	53.82 ± 2.97	53.50 ± 0.75
MCH (pg)	16.88 ± 0.58	16.81 ± 0.80	17.02 ± 0.68	16.75 ± 0.77	17.44 ± 1.38	17.20 ± 0.48
MCHC (g/dL)	31.98 ± 0.64	31.43 ± 0.82	31.71 ± 0.57	31.05 ± 1.69	32.34 ± 0.92	32.14 ± 0.54
Plt (10³ cells/µL)	843.70 ± 94.59	792.80 ± 91.21	891.20 ± 108.11	864.90 ± 127.13	758.00 ± 29.72	778.00 ± 76.69
WBC (10³ cells/µL)	8.38 ± 2.26	7.93 ± 1.96	7.72 ± 1.77	7.77 ± 1.95	8.08 ± 1.28	8.39 ± 1.19
Neu (%)	17.78 ± 5.57	15.85 ± 4.51	17.82 ± 4.97	19.31 ± 5.71	18.96 ± 6.60	19.48 ± 6.65
Lym (%)	76.24 ± 6.24	78.64 ± 4.82	75.68 ± 5.79	74.02 ± 6.50	72.94 ± 8.21	72.66 ± 7.72
Mono (%)	3.02 ± 0.86	2.54 ± 0.58	3.15 ± 0.63	3.11 ± 0.79	4.30 ± 1.00	4.14 ± 0.80
Eoso (%)	1.43 ± 0.47	1.42 ± 0.73	1.86 ± 1.15	1.91 ± 1.09	2.38 ± 1.01	1.96 ± 0.78
Baso (%)	0.30 ± 0.11	0.45 ± 0.28	0.36 ± 0.08	0.62 ± 0.49	0.44 ± 0.11	0.44 ± 0.15
LUC (%)	1.23 ± 0.33	1.08 ± 0.32	1.16 ± 0.51	0.99 ± 0.41	1.02 ± 0.29	1.34 ± 0.38
Rec (%)	1.97 ± 0.47	1.74 ± 0.60	1.96 ± 0.56	1.84 ± 0.50	1.62 ± 0.54	1.46 ± 0.13
CT (s)	154.70 ± 15.54	147.40 ± 25.70	151.80 ± 20.05	150.10 ± 23.53	149.40 ± 9.81	157.40 ± 16.23

SD: standard deviation; RBC: red blood cells; HB: hemoglobin; Hct: hematocrit; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; Plt: platelets; WBC: white blood cell; Neu: neutrophils; Lym: lymphocyte; Mono: monocyte; Eoso: eosinophil; Baso: basophil; LUC: large unstained cell; Rec: reticulocyte; CT: clotting time.

^a n = 10 in control, 250, 500, and 1000 mg/kg NXT15906F6 groups; n = 5 in recovery groups.

Table 6.

Hematological findings (mean ± SD) in female rats after 90 days oral administration of NXT15906F6.^a

		NXT15906F6
Parameters	Control (0 mg/kg)	250 mg/kg	500 mg/kg	1000 mg/kg	Control recovery	1000 mg/kg recovery
RBC (10⁶ cells/µL)	8.63 ± 0.34	8.83 ± 0.25	8.89 ± 0.39	8.87 ± 0.29	8.67 ± 0.28	8.70 ± 0.48
Hb (g/dL)	15.59 ± 0.41	15.45 ± 0.55	15.70 ± 0.27	15.63 ± 0.36	15.46 ± 0.38	15.90 ± 0.23
Hct (%)	48.79 ± 1.65	48.00 ± 1.25	48.70 ± 0.99	49.28 ± 1.46	47.94 ± 1.27	56.26 ± 17.19
MCV (fL)	56.62 ± 1.90	54.35 ± 0.79^b	54.87 ± 1.79^b	55.62 ± 2.07	55.30 ± 1.01	65.34 ± 22.55
MCH (pg)	18.09 ± 0.67	17.50 ± 0.48	17.70 ± 0.92	17.63 ± 0.43	17.84 ± 0.63	18.32 ± 0.96
MCHC (g/dL)	31.96 ± 1.03	32.19 ± 0.52	32.22 ± 0.67	31.74 ± 0.57	32.22 ± 0.88	29.78 ± 6.31
Plt (10³ cells/µL)	895.40 ± 93.92	977.00 ± 77.65	977.22 ± 77.56	917.00 ± 111.34	905.80 ± 73.64	872.80 ± 92.49
WBC (10³ cells/µL)	5.34 ± 1.47	10.14 ± 0.35	5.22 ± 0.96	4.16 ± 0.72	4.59 ± 0.89	4.26 ± 1.16
Neu (%)	10.33 ± 4.66	11.47 ± 4.41	11.41 ± 3.96	13.12 ± 5.44	16.70 ± 5.33	14.68 ± 4.03
Lym (%)	84.82 ± 4.85	83.94 ± 4.58	83.69 ± 4.06	81.22 ± 6.17	77.10 ± 6.31	79.60 ± 3.66
Mono (%)	2.02 ± 0.80	2.01 ± 0.67	1.77 ± 0.40	2.53 ± 1.07	2.90 ± 0.59	2.72 ± 0.88
Eoso (%)	1.43 ± 0.50	1.42 ± 0.39	1.84 ± 0.42	1.56 ± 0.46	1.96 ± 1.18	1.90 ± 0.32
Baso (%)	0.41 ± 0.12	0.27 ± 0.09^b	0.34 ± 0.10	0.42 ± 0.13	0.36 ± 0.09	0.28 ± 0.08
LUC (%)	1.02 ± 0.29	0.89 ± 0.32	0.94 ± 0.28	1.14 ± 0.43	1.06 ± 0.38	0.80 ± 0.35
Rec (%)	1.97 ± 0.52	1.56 ± 0.36	1.58 ± 0.55	1.75 ± 0.42	1.38 ± 0.57	1.50 ± 0.23
CT (s)	162.40 ± 12.51	151.60 ± 10.72	154.00 ± 17.26	151.10 ± 14.81	152.00 ± 17.31	164.60 ± 12.05

^a n = 10 in control, 250, 500, and 1000 mg/kg NXT15906F6 groups; n = 5 in recovery groups.

^b p < 0.01 versus control.

Clinical chemistry parameters (Tables 7 and 8) revealed values within the normal healthy range except a few sporadic changes at various time points. In the male group, a significant decrease in glucose levels was observed in all the treatment groups (G2, G3, and G4; Table 7), but these groups did not show any significant differences in body weight gain and feed intake when compared with the control group (G1). In female animals, creatinine levels significantly decreased in the G2, G3, and G4 groups, compared to the vehicle control (Table 8). Other sporadic and statistically significant changes observed in clinical chemistry include a decrease in serum creatinine in the G4 group and an increase in serum potassium levels in the male cohort (Table 7). A decrease in triglycerides and an increase in serum potassium levels were observed in the G4 reversal group of female cohort (Table 8). A decrease in serum alanine aminotransferase (ALT) and chloride levels was observed, respectively, in G4 and G2 groups of female cohort. The above changes observed in serum creatinine, triglycerides, potassium, ALT, and chloride levels were of low magnitude and were within the physiological limits and therefore were not considered to be of toxicological significance.

Table 7.

Clinical chemistry data (mean ± SD) of male rats after oral administration of NXT15906F6 for 90 days.^a

		NXT15906F6
Parameters	Control (0 mg/kg)	250 mg/kg	500 mg/kg	1000 mg/kg	Control recovery	1000 mg/kg recovery
Glu (mg/dL)	150.40 ± 17.73	127.20 ± 13.16^b	122.00 ± 14.51^c	125.80 ± 10.93^b	132.00 ± 7.58	122.20 ± 6.30
BUN (mg/dL)	13.50 ± 4.25	14.10 ± 2.51	15.50 ± 3.27	15.70 ± 2.11	13.80 ± 2.28	17.00 ± 2.92
Ur (mg/dL)	28.80 ± 8.83	30.10 ± 5.67	33.40 ± 7.01	33.60 ± 4.60	29.80 ± 4.97	36.20 ± 6.10
Crea (mg/dL)	0.64 ± 0.08	0.610 ± 0.07	0.57 ± 0.03	0.55 ± 0.08^d	0.61 ± 0.03	0.64 ± 0.06
T.Chol (mg/dL)	74.90 ± 16.48	75.90 ± 7.46	79.00 ± 15.73	72.50 ± 9.40	74.60 ± 15.98	72.00 ± 11.25
Trig (mg/dL)	151.30 ± 76.55	136.00 ± 66.40	147.60 ± 54.90	153.70 ± 55.89	193.40 ± 91.81	159.40 ± 37.79
T.Bil (mg/dL)	0.11 ± 0.03	0.12 ± 0.03	0.09 ± 0.02	0.10 ± 0.04	0.13 ± 0.03	0.09 ± 0.02
AST (U/L)	82.2 ± 10.72	82.80 ± 20.62	87.70 ± 12.09	90.10 ± 23.32	90.60 ± 12.74	85.00 ± 9.14
ALT (U/L)	43.00 ± 11.22	40.40 ± 11.94	45.30 ± 10.33	43.00 ± 16.46	48.20 ± 10.55	47.60 ± 8.44
ALP (U/L)	72.10 ± 16.79	74.40 ± 19.44	79.60 ± 17.24	75.60 ± 16.49	70.60 ± 15.34	67.40 ± 8.68
TP (g/dL)	6.86 ± 0.45	7.00 ± 0.36	6.94 ± 0.42	6.85 ± 0.43	7.08 ± 0.28	6.98 ± 0.19
ALB (g/dL)	3.82 ± 0.13	3.92 ± 0.14	3.92 ± 0.13	3.87 ± 0.19	3.94 ± 0.11	3.92 ± 0.08
Ca (mg/dL)	9.93 ± 0.42	10.12 ± 0.51	10.24 ± 0.34	10.04 ± 0.40	10.58 ± 0.4	10.50 ± 0.12
Phos (mg/dL)	5.29 ± 0.59	5.26 ± 0.61	5.40 ± 0.42	5.16 ± 0.43	4.88 ± 0.24	4.78 ± 0.49
Na (mmoL/L)	141.67 ± 0.72	142.02 ± 1.12	141.80 ± 0.48	141.74 ± 0.68	143.94 ± 0.71	142.94 ± 0.73
Pot (mmoL/L)	3.50 ± 0.16	3.54 ± 0.16	3.68 ± 0.14^d	3.73 ± 0.14^b	3.53 ± 0.17	3.77 ± 0.10
Cl (mmoL/L)	104.76 ± 1.33	104.85 ± 1.07	105.04 ± 0.85	104.75 ± 1.05	106.54 ± 0.75	106.66 ± 0.97

SD: standard deviation; Glu: glucose; BUN: blood urea nitrogen; Ur: urea; Crea: creatinine; T.Chol: total cholesterol; Trig: triglyceride; T.Bil: total bilirubin; AST: aspartate aminotransferase; ALT: alanine aminotransferase; ALP: alkaline phosphatase; TP: total protein; ALB: albumin; Ca: calcium; Phos: phosphorus; Na: sodium; Pot: potassium; Cl: chloride.

^a n = 10 in control, 250, 500, and 1000 mg/kg NXT15906F6 groups; n = 5 in recovery groups.

^b p < 0.01.

^c p < 0.001 versus control group.

^d p < 0.05.

Table 8.

Clinical chemistry data (mean ± SD) of female rats after oral administration of NXT15906F6 for 90 days.^a

		NXT15906F6
Parameters	Control (0 mg/kg)	250 mg/kg	500 mg/kg	1000 mg/kg	Control recovery	1000 mg/kg recovery
Glu (mg/dL)	116.20 ± 15.12	118.00 ± 11.84	127.80 ± 19.02	119.30 ± 11.12	110.00 ± 6.16	113.80 ± 5.63
BUN (mg/dL)	18.70 ± 2.83	19.20 ± 3.08	21.00 ± 2.83	17.80 ± 2.70	18.00 ± 2.55	19.80 ± 4.44
Ur (mg/dL)	39.90 ± 5.88	40.70 ± 6.57	44.60 ± 5.76	38.30 ± 5.42	38.20 ± 5.26	42.00 ± 9.25
Crea (mg/dL)	0.70 ± 0.08	0.64 ± 0.03^b	0.64 ± 0.03^b	0.61 ± 0.04^c	0.63 ± 0.15	0.64 ± 0.03
Total Chol. (mg/dL)	55.50 ± 9.00	67.10 ± 19.74	64.40 ± 15.74	54.30 ± 14.54	56.00 ± 11.77	60.20 ± 13.55
Trig (mg/dL)	75.00 ± 23.70	93.90 ± 45.18	77.00 ± 23.31	77.00 ± 26.57	105.20 ± 21.11	55.40 ± 11.28^c
T.Bil (mg/dL)	0.14 ± 0.05	0.11 ± 0.03	0.11 ± 0.03	0.10 ± 0.04	0.16 ± 0.06	0.11 ± 0.02
AST (U/L)	78.60 ± 13.11	77.10 ± 10.80	74.90 ± 14.57	72.80 ± 7.55	84.20 ± 27.52	70.80 ± 8.14
ALT (U/L)	38.30 ± 11.33	34.30 ± 4.19	31.50 ± 5.06	30.20 ± 5.92^b	35.80 ± 6.22	32.40 ± 4.77
ALP (U/L)	24.50 ± 7.17	29.10 ± 7.81	26.60 ± 4.88	28.10 ± 5.43	26.00 ± 3.16	27.20 ± 7.53
TP (g/dL)	6.99 ± 0.28	7.23 ± 0.45	7.02 ± 0.23	7.14 ± 0.36	7.32 ± 0.18	7.36 ± 0.30
ALB (g/dL)	4.26 ± 0.21	4.40 ± 0.23	4.33 ± 0.16	4.42 ± 0.21	4.52 ± 0.16	4.48 ± 0.13
Ca (mg/dL)	10.25 ± 0.49	10.10 ± 0.46	10.07 ± 0.55	10.08 ± 0.49	10.76 ± 0.09	10.78 ± 0.31
Phos (mg/dL)	4.42 ± 0.70	4.02 ± 0.65	4.07 ± 1.26	3.82 ± 0.69	3.50 ± 0.69	3.86 ± 0.79
Na (mmoL/L)	141.44 ± 1.47	141.20 ± 1.07	141.51 ± 1.09	142.17 ± 0.67	143.38 ± 0.89	142.84 ± 0.47
K (mmoL/L)	3.47 ± 0.20	3.38 ± 0.21	3.31 ± 0.15	3.35 ± 0.34	3.41 ± 0.22	3.82 ± 0.16^c
Cl (mmoL/L)	105.78 ± 1.03	104.09 ± 0.95^c	105.60 ± 1.46	106.09 ± 1.22	108.08 ± 0.78	107.94 ± 0.68

^a n = 10 in control, 250, 500, and 1000 mg/kg NXT15906F6 groups; n = 5 in recovery groups.

^b p < 0.05.

^c p < 0.01 versus control group.

Organ weights

There was no treatment-related variations in the absolute and relative organ weights in any of the treatment groups of either sex when compared to respective control groups (Tables 9 and 10). Some of the incidental variations that attained statistical significance as compared with the respective control included an increase in the relative organ weight of the liver in the G2-treated male and female groups and an increase in the absolute organ weight of the thymus in the G3 female group. In addition, an increase in the relative organ weight of the thymus was observed in G4 female animals (Table 11) and an increase in the relative organ weight of the heart in G4R-treated male group (Table 12). These changes were considered unrelated to the treatment because no dose dependency was exhibited when taking the main groups into consideration.

Table 9.

Absolute organ weights (g; mean ± SD) in male rats after 90 days oral administration of NXT15906F6.^a

		NXT15906F6
Organs	Control (0 mg/kg)	250 mg/kg	500 mg/kg	1000 mg/kg	Control recovery	1000 mg/kg recovery
Liver	11.7774 ± 1.3443	12.4743 ± 1.4868	12.6643 ± 1.7574	12.6366 ± 1.3665	12.8036 ± 1.5659	12.1572 ± 0.9167
Kidney	2.5789 ± 0.2864	2.4639 ± 0.2070	2.7768 ± 0.3364	2.6592 ± 0.3032	2.6354 ± 0.3232	2.5664 ± 0.1985
Adrenals	0.0554 ± 0.0095	0.0578 ± 0.0090	0.0622 ± 0.0044	0.0603 ± 0.0072	0.0511 ± 0.0085	0.0620 ± 0.0138
Heart	1.1160 ± 0.1167	1.0932 ± 0.0930	1.1594 ± 0.0956	1.1388 ± 0.0672	1.1346 ± 0.0673	1.1917 ± 0.0423
Brain	2.1567 ± 0.1326	2.1284 ± 0.0866	2.1517 ± 0.1577	2.1806 ± 0.1297	2.1323 ± 0.1279	2.1693 ± 0.1278
Spleen	0.6667 ± 0.1068	0.6883 ± 0.1027	0.7621 ± 0.1351	0.7515 ± 0.1234	0.6616 ± 0.0764	0.7229 ± 0.1272
Thymus	0.3832 ± 0.0604	0.3627 ± 0.0612	0.3639 ± 0.0526	0.3798 ± 0.0535	0.4486 ± 0.0707	0.3644 ± 0.0464
Testes	3.5410 ± 0.3456	3.6177 ± 0.2770	3.7012 ± 0.2649	3.8097 ± 0.2030	3.6436 ± 0.2077	3.2987 ± 0.8128
Epididymides	1.1670 ± 0.0863	1.1705 ± 0.1019	1.1820 ± 0.0974	1.1688 ± 0.0621	1.1423 ± 0.0693	1.1570 ± 0.1893
SV-CG and prostate gland	2.4386 ± 0.2255	2.3875 ± 0.2098	2.3075 ± 0.2517	2.3323 ± 0.1361	3.3052 ± 0.0837	3.1119 ± 0.2608

SD: standard deviation; SV-CG: seminal vesicles with coagulating glands.

^a n = 10 in control, 250, 500, and 1000 mg/kg NXT15906F6 groups; n = 5 in recovery groups.

Table 10.

Relative organ weights (% of fasting body weight; mean ± SD) in female rats after 90 says oral administration of NXT15906F6.^a

		NXT15906F6
Organs	Control (0 mg/kg)	250 mg/kg	500 mg/kg	1000 mg/kg	Control recovery	1000 mg/kg recovery
Liver	2.7461 ± 0.1450	3.0575 ± 0.2054^b	2.9886 ± 0.2636	2.9784 ± 0.2910	2.8189 ± 0.3253	2.9145 ± 0.2447
Kidney	0.6934 ± 0.0827	0.6968 ± 0.0360	0.7061 ± 0.0445	0.6896 ± 0.0742	0.6776 ± 0.0607	0.7528 ± 0.0496
Adrenals	0.0283 ± 0.0032	0.0265 ± 0.0037	0.0276 ± 0.0022	0.0315 ± 0.0041	0.0271 ± 0.0061	0.0299 ± 0.0015
Heart	0.3061 ± 0.0209	0.3278 ± 0.0346	0.3287 ± 0.0357	0.3288 ± 0.0245	0.3308 ± 0.0446	0.3117 ± 0.0231
Brain	0.8470 ± 0.0636	0.8471 ± 0.0386	0.8674 ± 0.0670	0.8559 ± 0.0495	0.8423 ± 0.0930	0.8545 ± 0.0411
Spleen	0.2093 ± 0.0232	0.2151 ± 0.0179	0.2250 ± 0.0284	0.2226 ± 0.0273	0.2124 ± 0.0412	0.2242 ± 0.0409
Thymus	0.1384 ± 0.0189	0.1335 ± 0.0244	0.1699 ± 0.0351	0.1509 ± 0.0280^b	0.1184 ± 0.0267	0.1263 ± 0.0052
Uterus	0.2933 ± 0.0971	0.3165 ± 0.0651	0.2848 ± 0.0541	0.3091 ± 0.0906	0.3885 ± 0.1684	0.2496 ± 0.0390
Ovaries	0.0498 ± 0.0085	0.0484 ± 0.0051	0.0539 ± 0.0100	0.0528 ± 0.0090	0.0472 ± 0.0050	0.0482 ± 0.0019

SD: standard deviation.

^a n = 10 in control, 250, 500, and 1000 mg/kg NXT15906F6 groups; n = 5 in recovery groups.

^b p < 0.05 versus control group.

Table 11.

Absolute organ weights (g; mean ± SD) in female rats after 90 days oral administration of NXT15906F6.^a

		NXT15906F6
Organs	Control (0 mg/kg)	250 mg/kg	500 mg/kg	1000 mg/kg	Control recovery	1000 mg/kg recovery
Liver	6.412 ± 0.6376	7.1165 ± 0.4460	6.9750 ± 0.6396	7.0410 ± 0.8313	6.9637 ± 0.2408	6.9715 ± 0.5966
Kidneys	1.6152 ± 0.1980	1.6235 ± 0.1109	1.6501 ± 0.1420	1.6305 ± 0.2084	1.6824 ± 0.1589	1.7998 ± 0.1034
Adrenal glands	0.0658 ± 0.0069	0.0617 ± 0.0090	0.0646 ± 0.0065	0.0745 ± 0.0109	0.0676 ± 0.0156	0.0716 ± 0.0055
Heart	0.7134 ± 0.0649	0.7625 ± 0.0727	0.7671 ± 0.0847	0.7769 ± 0.0680	0.8165 ± 0.0503	0.7451 ± 0.0493
Brain	1.9675 ± 0.0789	1.9712 ± 0.0602	2.0207 ± 0.1001	2.0191 ± 0.0954	2.0821 ± 0.0861	2.0425 ± 0.0597
Spleen	0.4884 ± 0.0655	0.5013 ± 0.0482	0.5237 ± 0.0541	0.5255 ± 0.0666	0.5236 ± 0.0825	0.5383 ± 0.1074
Thymus	0.3243 ± 0.0588	0.3104 ± 0.0561	0.3944 ± 0.0710^b	0.3555 ± 0.0639	0.2893 ± 0.0306	0.3025 ± 0.0206
Uterus	0.6867 ± 0.2411	0.7358 ± 0.1445	0.6629 ± 0.1150	0.7336 ± 0.2272	0.9614 ± 0.4128	0.5995 ± 0.1120
Ovaries	0.1159 ± 0.0202	0.1127 ± 0.0120	0.1253 ± 0.0200	0.1250 ± 0.0236	0.1168 ± 0.0082	0.1152 ± 0.0051

SD: standard deviation.

^a n = 10 in control, 250, 500, and 1000 mg/kg NXT15906F6 groups; n = 5 in recovery groups. ^b p < 0.05 versus control group.

Table 12.

Evaluation of the genotoxicity of NXT15906F6 in reverse-mutation assay.

	Number of revertants/plate (mean ± SD)
Treatment (µg/plate)	TA98 (+S9)	TA98 (−S9)	TA100 (+S9)	TA100 (−S9)	TA1535 (+S9)	TA1535 (−S9)	TA1537 (+S9)	TA1537 (−S9)	WP2uvrA/pKM101 (+S9)	WP2uvrA/pK101 (−S9)
Vehicle control (0)	25.67 ± 4	24.67 ± 2	97.33 ± 9	92.67 ± 6	16.33 ± 2	15.00 ± 3	9.67 ± 2	9.33 ± 2	129.67 ± 5	132.67 ± 6
100	24.33 ± 3	22.67 ± 2	91.00 ± 7	95.67 ± 5	14.67 ± 2	14.67 ± 2	9.00 ± 1	9.00 ± 1	130.67 ± 8	127.00 ± 6
266	24.00 ± 2	22.67 ± 2	98.67 ± 4	91.33 ± 3	15.00 ± 3	13.67 ± 1	10.00 ± 1	7.33 ± 2	128.67 ± 3	131.33 ± 5
707	24.00 ± 2	22.00 ± 1	97.67 ± 3	95.33 ± 8	15.00 ± 2	16.33 ± 2	9.00 ± 1	9.67 ± 2	131.67 ± 9	132.00 ± 7
1880	23.33 ± 2	26.00 ± 2	97.67 ± 8	95.33 ± 6	14.00 ± 2	15.33 ± 2	9.00 ± 1	9.67 ± 2	128.00 ± 4	131.67 ± 6
5000	21.33 ± 1	22.33 ± 2	92.33 ± 4	88.67 ± 6	13.67 ± 1	14.33 ± 2	7.33 ± 1	7.33 ± 2	121.67 ± 2	124.00 ± 2
Positive control	571.67 ± 18^a	282.33 ± 16^c	889.00 ± 7^a	575.33 ± 11^d	165.67 ± 9^a	157.00 ± 9^d	149.00 ± 7^a	157.67 ± 9^e	583.33 ± 13^b	575.33 ± 10^f

SD: standard deviation.

^a2-Aminoanthraence (4 µg/plate).

^b2-Aminoanthraence (30 µg/plate).

^c2-Nitrofluorence (2 µg/plate).

^dSodium azide (1 µg/plate).

^e9-Aminoacridine (50 µg/plate).

^f4-Nitroquinoline (4 µg/plate).

Gross pathology and histopathology

The animals of G1–G4 on day 90 and the animals of G1R and G4R on day 118 were fasted overnight, weighed, and subjected to necropsy. Animals were killed by CO₂ asphyxiation followed by exsanguination. Gross pathological changes were recorded for each rat.

External and internal examination of all terminally killed animals of either sex across all groups (G1–G4, G1R, and G4R) did not reveal any abnormalities of pathological significance. Red discoloration of the thymus in three animals (two males in G1 and one female in G4) and excessive abdominal fat in one animal (one male in G1R) were noted. These findings observed in thymus and abdominal fat were incidental in nature and were considered unrelated to the test item administration.

No test item–related histopathological findings were recorded in male and female rats from the G4 groups treated with NXT15906F6 at 1000 mg/kg body weight per day. Various microscopic findings were recorded in different organs/tissues of both control (G1) and G4 group animals of either sex at a low occurrence rate with no indication of a treatment effect. These findings were minimal to mild in nature, comparable, inconsistent, and nonspecific.

Bacterial reverse-mutation assay

No significant increases in the mean number of revertant colonies in any of the five test strains were observed following treatment with NXT15906F6 at any concentration level (i.e. 100, 266, 707, 1880, and 5000 µg/plate) either in the presence or absence of metabolic activation (Table 13). These results indicated that NXT15906F6 is nonmutagenic in the reverse mutation test at 5000 µg/plate.

Table 13.

Effect of NXT15906F6 on micronucleus frequency in mouse bone marrow erythrocytes.^a

Test item	Dose (mg/kg body weight)	Mean PCE–TE	% Reduction	% MNPCE ± SD
Vehicle control	0	0.590	NA	0.10 ± 0.02
NXT15906F6	500	0.585	0.9	0.02 ± 0.04
NXT15906F6	1000	0.586	0.7	0.14 ± 0.16
NXT15906F6	2000	0.603	−2.3	0.15 ± 0.10
CAP	40	0.555	5.8	1.7 ± 0.18^b

SD: standard deviation; ANOVA: analysis of variance; PCE: polychromatic erythrocytes; TE: total erythrocyte; MNPCE: micronucleated polychromatic erythrocyte; CAP: chloramphenicol.

^aValues are represented as mean ± SD; n = 5.

^b p < 0.0001, comparison between treatment and control group in one-way ANOVA.

Mouse bone marrow erythrocytes in micronucleus assay

NXT15906F6 did not show any toxicity in the form of reduction in immature PCEs-TE ratio when compared with that of vehicle-treated animals. The data on PCE-TE are presented in Table 14. The frequency of micronucleated PCEs in the NXT15906F6-treated groups was compared with that of the vehicle-treated group, and there were no statistically significant increases up to 2000 mg/kg body weight. The micronucleus frequency in the animals treated with positive control (CPA, 40 mg/kg body weight) was increased significantly. The one-way ANOVA statistical method was used to compare the data.

Table 14.

Relative organ weights (% of fasting body weight; mean ± SD) in male rats after 90 days oral administration of NXT15906F6.^a.

		NXT15906F6
Organs	Control (0 mg/kg)	250 mg/kg	500 mg/kg	1000 mg/kg	Control recovery	1000 mg/kg recovery
Liver	2.7260 ± 0.1739	3.0456 ± 0.3012^b	3.0088 ± 0.3537	2.9823 ± 0.2980	2.7184 ± 0.1042	2.7678 ± 0.2283
Kidneys	0.5994 ± 0.0715	0.6019 ± 0.0362	0.6622 ± 0.0928	0.6278 ± 0.0671	0.5596 ± 0.0259	0.5836 ± 0.0368
Adrenal glands	0.0128 ± 0.0019	0.0141 ± 0.0022	0.0148 ± 0.0010	0.0143 ± 0.0021	0.0109 ± 0.0014	0.0141 ± 0.0028
Heart	0.2587 ± 0.0202	0.2669 ± 0.0145	0.2765 ± 0.0288	0.2696 ± 0.0250	0.2425 ± 0.0209	0.2713 ± 0.0143^b
Brain	0.5014 ± 0.0361	0.5228 ± 0.0541	0.5122 ± 0.0366	0.5151 ± 0.0323	0.4565 ± 0.0492	0.4952 ± 0.0538
Spleen	0.1543 ± 0.0205	0.1680 ± 0.0223	0.1803 ± 0.0237	0.1784 ± 0.0348	0.1421 ± 0.0244	0.1639 ± 0.0235
Thymus	0.0891 ± 0.0140	0.0895 ± 0.0191	0.0865 ± 0.0121	0.0900 ± 0.0151	0.0966 ± 0.0207	0.0828 ± 0.0092
Testes	0.8232 ± 0.0855	0.8851 ± 0.0695	0.8817 ± 0.0700	0.9011 ± 0.0701	0.7802 ± 0.0835	0.758 ± 0.2086
Epididymides	0.2713 ± 0.0218	0.2861 ± 0.0208	0.2812 ± 0.0206	0.2771 ± 0.0298	0.2442 ± 0.0213	0.2656 ± 0.0577
SV-CG and prostate	0.5682 ± 0.0710	0.5868 ± 0.0778	0.5490 ± 0.0556	0.5510 ± 0.0367	0.7101 ± 0.0898	0.7091 ± 0.0742

SD: standard deviation; SV-CG: seminal vesicles with coagulating glands.

^a n = 10 in control, 250, 500, and 1000 mg/kg NXT15906F6 groups; n = 5 in recovery groups.

^b p < 0.05 versus control group.

Discussion

NXT15906F6 (TamaFlex^®) is an herbal blend containing aqueous and ethanol extracts of T. indica fruit seeds and an ethanol extract of C. longa rhizomes. It represents a new category of food-derived anti-inflammatory composition. NXT15906F6 is a distinct food supplement category from other popular anti-inflammatory botanical compounds because it gradually addresses chronic inflammation and associated pain without disturbing the physiology of the aging organism. This new category is distinct from the botanical NSAID products derived from the nonfood botanicals such as Boswellia serrata tree gum resin and derived Boswellic acids which have been successfully used in the management of clinically diagnosed osteoarthritis and other forms of clinically overt arthritis and degenerative chronic conditions.^21,22

The two components within NXT15906F6 are popular and widely used in Asian culinary tradition. Several safety and toxicological studies conducted on T. indica seeds and C. longa-derived products indicate that these ingredients are safe for human consumption.^23

–27 However, a broad-spectrum safety study of a composition derived from these individual ingredients is needed to confirm whether this final formulation consisting of extracts of T. indica seeds and C. longa-rhizomes is safe for human consumption. Broad-spectrum safety studies in animal models are commonly considered to evaluate potential health hazards in humans caused by the intrinsic toxicity of a substance.²⁸ Therefore, a series of safety and toxicological evaluations of NXT15906F6 was conducted on various animal models using the appropriate OECD test guidelines.

The acute oral toxicity study did not reveal any significant changes in experimental rats when treated with NXT15906F6. Based on these results and under the conditions of this study, the oral median LD₅₀ of NXT15906F6 was considered to be more than 2000 mg/kg body weight in female rats. In the acute dermal toxicity study, there were no signs of dermal irritation, adverse pharmacological effects, or abnormal behavior. Based on these results, the acute dermal LD₅₀ of NXT15906F6 was determined to be more than 2000 mg/kg body weight in male and female rats. Observations from this study show that the herbal blend is a nonirritant to rabbit skin. However, NXT15906F6 was found to be mild irritant to eyes. However, no corneal opacity or iritis was observed in the study.

The results from the 90-day subchronic oral toxicity study with a 28-day recovery period did not reveal any dose-dependent adverse effects in the tested animals. No changes in body weight, food consumption, eye examination, FOB examination, hematology, clinical chemistry, or organ weights (both absolute and relative) were noted up to 1000 mg/kg/day of NXT15906F6. Although we observed significant modulations in some parameters in the study, those changes were inconsistent, without having any dose response. Importantly, those variations are within the physiological range. Hence, these changes were considered not related to the treatment. No gross pathological changes were noted in any organ or tissue. In addition, no abnormalities were found in microscopic observations on the histopathology of liver, kidney, lung, testes, ovary, and other vital organs of NXT15906F6-supplemented animals. Together, these observations indicate that no observed adverse effect level of NXT15906F6 for male and female Wistar rats is at least 1000 mg/kg/day.

In addition, observations from the reverse mutation assay on bacterial strains suggest that NXT15906F6 is nonmutagenic (Table 12). In parallel, NXT15906F6 is also classified as a nonclastogenic or nonaneugenic based on the mammalian erythrocyte micronucleus test (Table 13).

In conclusion, a series of safety and toxicological studies indicate that the novel herbal blend NXT15906F6 with synergistic anti-inflammatory activities is safe, tolerable, and nongenotoxic. Further, these observations also suggest that NXT15906F6 is safe and tolerable for human consumption. NXT15906F6 (TamaFlex) provides a new category of a standardized, synergistic dietary ingredient that may help maintain a healthy muscle–skeletal system in aging individuals and benefit those with an active lifestyle.

Footnotes

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: HV, VB, HV, FG and SJS are consultants without financial interest in the company funding the study.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was conducted at Liveon Biolabs PVT. Ltd, (Karnataka, India) under a grant from NXT CAP, Ltd, and Cyprus which also provided the study product.

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Safety and toxicological evaluation of NXT15906F6 (TamaFlex ® )

Abstract

Keywords

Introduction

Materials and methods

Test item

Chemicals, reagents, and bacterial strains

Experimental animals and housing conditions

Acute oral toxicity study in female rats

Acute dermal toxicity study in rats

Acute dermal irritation study in rabbits

Acute eye irritation study in rabbits

Subchronic (90-day) repeated-dose oral toxicity study

Statistical analyses

Bacterial reverse-mutation assay

Mouse bone marrow erythrocyte micronucleus assay

Results

Acute oral toxicity study in rats

Acute dermal toxicity in rats

Acute dermal irritation study in rabbits

Primary eye irritation study in rabbits

The 90-Day (subchronic) repeated-dose oral toxicity study

Mortality, clinical signs, and FOB

Body weight and feed consumption

Ophthalmological observation

Hematology and clinical chemistry

Organ weights

Gross pathology and histopathology

Bacterial reverse-mutation assay

Mouse bone marrow erythrocytes in micronucleus assay

Discussion

Footnotes

Declaration of conflicting interests

Funding

References