A Safety Evaluation of a Nature-Identical l -Ergothioneine in Sprague Dawley Rats

Abstract

l-(+) Ergothioneine is a naturally occurring thiol amino acid with antioxidant properties and potential benefits as a dietary supplement. Despite its century-old identification and wide distribution in human food, little is known of its mechanism of action and safety. The nature-identical biomimetic of l-(+) ergothioneine, produced by Mironova Labs and supplied as Mironova (EGT+), has been investigated in the present studies for its mutagenic and toxicologic potential. In a plate incorporation and preincubation assay with Salmonella typhimurium strains TA98, 100, 1,535, and 1,537 and Escherichia coli WP2uvrA strain, at dose concentrations of 1.58, 5, 15.8, 50, 158, 500, 1,580, and 5,000 μg/plate with and without metabolic activation, no cytotoxicity or mutagenicity was observed. Following a preliminary 28-day study, a repeated dose 90-day gavage study at dose levels of 0, 400, 800, and 1,600 mg/kg body weight (bw)/d in Sprague Dawley rats, in which dose-proportional systemic absorption was confirmed by plasma analysis, no adverse clinical, body weight/gain, food consumption and efficiency, clinical pathology, or histopathological changes associated with the administration of the nature-identical ergothioneine were observed. In conclusion, EGT+ administered over 90 days was well tolerated with a no adverse effect level at 1,600 mg/kg bw/d, the highest dose tested for male and female rats. In addition, the nature-identical test substance, EGT+ was not mutagenic in a bacterial reverse mutation assay at plate concentrations of up to 5,000 μg/mL in the presence or absence of metabolic activation.

Keywords

ergothioneine safety toxicity dietary supplement antioxidant Mironova EGT+

Introduction

Oxidative stress resulting from the increased formation of reactive oxygen species or the perturbation of antioxidant defenses has been implicated in a number of disease states including cancer, arthritis and inflammatory processes, and cardiovascular, neurologic, and immune deficit, among others.^1

-4 First identified in the sclerotia of the ergot fungus, l-(+) ergothioneine is a naturally occurring, water-soluble sulfonated thiol amino acid derivative of betaine histidine synthesized by nonyeast-like fungi and certain types of bacteria of order Actinomycetales and Cyanotypes,^5,6 which has been increasingly reported for its antioxidant potential and possible role in disease mitigation.^7
-9 Since humans and other vertebrates are unable to synthesize ergothioneine, it must be acquired in the diet, assimilated from fungi or plant fixation in the soil.¹⁰ Enhanced by small intestine uptake and absorbed and distributed under the sole selective control of the mitochondrial cation membrane transporter protein (formerly OCTN1), expressed by the SLC22A4 gene, ergothioneine does not accumulate in cells that do not possess the transporter.¹¹ As such, susceptibility to oxidative stress may be considered the result of SLC22A4 as one purported genetic determinant and the variable presence, dependent upon species and individual, of the ergothioneine transporter, which has the capability to accumulate l-ergothioneine in high levels (millimolar concentrations) and serve as a biomarker for l-ergothioneine activity. Levels of ergothioneine are highest in erythrocytes, bone marrow, liver, kidney, the lens and cornea of the eyes, seminal fluid and skin, and tissues exposed to high levels of oxidative stress.^10,12 The ability to accumulate l-ergothioneine in these tissues has been reported to confer increased antioxidant potential, the reduction of which can lead to mitochondrial DNA damage, lipid peroxidation, and protein oxidation.^12

-16 Case–control and in vitro studies of inflammatory disease, such as Crohn’s, ulcerative colitis and diabetes, have reported an association with polymorphism in the SLC22A4 gene responsible for l-ergothioneine production and expression.^17

-21

Beneficial physiologic properties of l-ergothioneine include its rapid clearance from the circulation into retained tissues with minimal metabolism and high stability (low redox potential), long half-life (approximately 30 days), and its reduced tendency to auto-oxidize or generate free radicals from peroxide and Fe²⁺ at physiologic pH, apart from that of glutathione.^11,22,23 Generally considered an intracellular antioxidant, the potency of ergothioneine compared with glutathione has been disputed, while its antioxidant scavenging activities appear similar.^24
-26 Since no reports exist of deficiency-related disease, ergothioneine is not currently considered an essential dietary micronutrient, though its abundance in a wide variety of foods, including beans, mushrooms, grains, red meat, and organ liver and kidney meats, has implications for potential toxicity considering its high level of absorption, slow degradation, and slow turnover due to renal reabsorption and low urinary excretion (e.g., net result of glomerular filtration, active tubular secretion, and tubular reabsorption).^9,25,27 Nonetheless, despite the extensive historical evidence of the potential advantages of this amino acid, the only one with a known specific transport system,²⁸ little is known of its mechanism of action or of its safety as a dietary supplement.

Although numerous reports detail the bioavailability of l-ergothioneine concentrations in various human tissues both in vivo, including in blood and glomerular filtration rate,^29

-32 and with evaluation of protection from oxidative stress in vitro, including that in dermal fibroblasts, erythrocytes, endothelial cells, and keratinocytes,^16,20,33
-35 limited data are available to support ergothioneine safety. Abbreviated pharmacological studies from Tainter³⁶ failed to show any deleterious effects of ergothioneine at physiologically relevant concentrations. The carcinogenic potential of the compound is considered low with several investigators reporting that ergothioneine actually protects against mutagen production by intercepting the mutagenic or carcinogenic potential in critical cells such as stem cells.^37
-39

Early in vivo studies in rats at 70 to 100 mg/kg confirm l-ergothioneine’s rapid urinary clearance (e.g., excretion into the urine by the kidneys) and its accumulation in the liver where, with the kidney, it is reported to modulate oxidative damage from polyunsaturated fatty acids.^40,41 Most recently, preclinical studies testing the genotoxicity of l-ergothioneine showed no mutagenic or clastogenic activity in in vitro Ames and chromosome aberration assays up to 5,000 µg/mL or in an in vivo mouse micronucleus test at concentrations of up to 150 mg/mL or 1,500 mg/kg.^42,43 No adverse effects were reported during a 2,000 mg/kg oral acute limit test, a 14-day toxicity test, or reproductive fertility or developmental toxicity test in rats at concentrations of 0.1%, 0.3%, or 0.9% in the diet.⁴⁴

To date, an acceptable daily intake or tolerable daily intake for l-ergothioneine has not been established in humans. However, based on consumption of the king bolete-type mushroom (Boletus edulis) for its high concentration, estimated average intakes of l-ergothioneine by adult consumers in Europe (70 kg) and the United States (80 kg) are calculated between 0.051 and 0.255 mg/kg bw/d.^5,45

In order to qualify safety of this purported antioxidant for its suitability as a dietary supplement, nature-identical ergothioneine, EGT+, manufactured from a proprietary process and derived as l-ergothioneine, was investigated for its safety profile in subchronic oral toxicity studies in rats and in an in vitro Ames genotoxicity study.

Materials and Methods

Test Substance

The test substance, a synthetic l-(+) ergothioneine (CAS 497-30-03), under the name of Mironova EGT+, derived from batch numbers ERG-F-0913-01 (28 day study) and ERG-F-0414-011 and ERG-F-0814-01 (90-day and genotoxic studies, respectively) purity > 99%, was manufactured by Mironova Labs Inc. (Fairfield, New Jersey) using sustainable, proprietary process under current Good Manufacturing Practices controls and designated as Mironova EGT+. The product is verified by optical rotation ([α]20D = +120° − +126.5°, c = 1, H20) and high-performance liquid chromatography (HPLC) with mass spectroscopy detection using a positive-mode electrospray ionization (criteria purity > 98%).

Dose levels for the bacterial reverse mutation (Ames) test and oral toxicity studies were chosen based on recommended guidelines and an expected human dose of 5 to 10 mg/d, respectively, derived from studies that quantified the average amount of l-ergothioneine in various mushroom species by eating a serving per day.^25,46
-48 The doses for the oral toxicity studies were selected based on safety factors for the extended, long-term daily intended use of the nature-identical dietary supplement use of 0.167 mg/kg bw/d l-ergothioneine for a 60-kg individual (wt/wt).

For the dose-analysis phase of the 90-day study, neat EGT+ samples were collected on days 1, 43, and 92 and dose mixture samples were collected on day 1 for homogeneity and on days 43, 71, and 92 for concentration verification. l-Ergothioneine concentration (%) was determined in each of the samples by an HPLC method of validation for linearity, system suitability (instrumentation precision), specificity, accuracy (spike recovery), and precision. All samples were received frozen and maintained frozen prior to extraction. Samples were considered stable from the point at which they were frozen.

Toxicity Studies—Methods

Mutagenicity and 90-day subchronic toxicity were conducted in conformance with Office of Economic Cooperative Development (OECD) Principles of Good Laboratory Practices⁴⁹ and US Food and Drug Administration Good Laboratory Practices.^50,51 The genotoxicity and repeated-dose subchronic studies (28- and 90-day studies) were conducted by Eurofins/Product Safety Labs (Dayton, New Jersey) under OECD Guidelines for the Testing of Chemicals, Section 4, No. 471, “Bacterial Reverse Mutation Test”, adopted July 21, 1997⁵²; Commission Regulation (EC) No. 440/2008 B. 13/14 B: “Mutagenicity-Reverse Mutation test using Bacteria, dated May 3, 2008⁵³; and EPA Health Effects Test Guidelines, OPPTS 870.5100, “Bacterial Reverse Mutation Assay” EPA 712-C-98-247, August 1998.⁵⁴ The in vivo toxicity study undertaken by the testing laboratory was in accordance with the most recent Guide for the Care and Use of Laboratory Animals,⁵⁵ according to the Association for Assessment and Accreditation of Laboratory Animal Care standards and accreditation and the OECD Guidelines for the Testing of Chemicals, Section 4, No. 407, “Repeated Dose 28-day Oral Toxicity Study in Rodents,” adopted October 16, 2008; and the OECD Guidelines for the Testing of Chemicals, Section 4, No. 408 “Repeated Dose 90-day Oral Toxicity Study in Rodents,” adopted September 21, 1998.

Mutagenicity Studies

Nature-identical ergothioneine EGT+ was tested for potential cytotoxicity and mutagenicity in a bacterial reverse mutation assay according to the plate incorporation test (experiment 1) and the preincubation test (experiment 2) at concentrations of 0, 1.58, 5.0, 15.8, 50, 158, 500, 1,580, and 5,000 μg/plate solubilized in distilled water of Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of S9 liver microsomal fraction prepared from phenobarbital/benzoflavone-induced rats. Both the plate incorporation method and the preincubation method were performed.^56
-58 For each method, 2 independent experiments were run in triplicate for each test concentration. Negative (solvent and untreated) and positive controls were performed simultaneously. Positive controls for cultures without S9 were ICR 191 acridine (10 µg/mL) and daunomycin (60 µg/mL), respectively (TA98 and TA1537), sodium azide (15 µg/mL; TA1535 and TA100), and methyl methane sulfonate (25 µL/mL; WP2 uvrA). For cultures with S9, 2-aminoanthracene (100 µg/mL) was used for all strains. For the plate incorporation method at each concentration and bacterial strain, 100 µL test solution, negative control, or positive control was mixed in a test tube with 500 µL of S9 or S9 substitution buffer (plates without metabolic activation), 100 µL bacterial suspension, and 2,000 µL overlay agar. The mixture was poured over the surface of overlay agar Moltox (Molecular Toxicology, Boone, NC) minimal glucose agar plates with 2% glucose and allowed to solidify. For the preincubation assay, the tester strains (100 µL) were preincubated with 100 µL of test substance preparation and 500 µL of S9 or sterile buffer (plates without metabolic activation) at 37°C. After 60 minutes, 2,000 µL overlay agar was added and the mixture was poured onto Moltox agar plates with 2% glucose and allowed to solidify. In both methods, once the plates were solidified, bacteria were incubated in the dark at 37°C for at least 48 hours after which colonies were counted. A confirmatory test used the preincubation modification of the plate incorporation test. The test or control substances, bacteria suspension, and S9/substitution buffer were incubated under agitation for approximately 30 minutes at 37°C ± 2°C prior to mixing with the overlay agar and pouring onto the minimal agar plates before proceeding as described for the initial test. The study design for the confirmatory test, including strains, dose levels, and so on, was as described as above for the plate incorporation test.

Twenty-Eight–Day Oral Toxicity Study

In a preliminary 28-day range-finding study for potential toxicity indicated by any changes in clinical signs, body weight, body weight gain, food consumption, food efficiency, or macroscopic findings at necropsy, groups of 5 CD IGS Sprague Dawley (Crl: SD, Charles River Laboratories, Inc, Raleigh, North Carolina) rats/sex/dose were orally intubated daily with the nature-identical ergothioneine test substance dissolved in water at dose levels of 0 (distilled water vehicle control), 50, 200, or 500 mg/kg bw/d at a dose volume of 5 mL/kg bw/d adjusted weekly for body weight. Prior to treatment, rats were acclimated for 6 days. Rats were approximately 8 weeks of age and males weighed 241 ± 6.05 g and females weighed 189 ± 7.18 g at the start of treatment. Rats were individually housed in suspended stainless steel cages with mesh floors. Litter paper was placed beneath the cages and was changed at least 3 times per week. The room temperature ranged from 19°C to 22°C with a relative humidity of 48% to 78% and a 12-hour light/dark cycle. Humidity was above the targeted upper limit for 1 day during the study. A portable dehumidifier was used to lower the humidity levels during this time. Filtered tap water was available ad libitum from an automatic watering supply system. The dose preparations were prepared daily based on the most recent daily body weights. Neat test substance and dosing preparations were sampled and preserved. At terminal sacrifice, all animals were euthanized by carbon dioxide asphyxiation. 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 tissues. Animals were not fasted prior to necropsy. Tissues were not preserved or evaluated further unless indicated for possible toxicological interest.

Ninety-Day Oral Toxicity Study

Nature-identical ergothioneine EGT+ was tested in a 90-day oral toxicity study for potential toxicity in CD IGS Sprague Dawley (Crl: SD) rats following regulatory guidelines. Based on the negative results of the 28-day study and the expected human intake, dose levels of 0 (control), 400, 800, and 1,600 mg/kg bw/d (increased from the 28-day study and 2,400-9,600 times the expected exposure for a 60-kg human) were chosen and orally intubated to 10 rats/sex/group at a uniform volume of 10 mL/kg bw, adjusted weekly for body weight, for at least 90 days daily. The control group received distilled water vehicle only at the same dose volume as the test animals. Prior to treatment, rats were acclimated for 5 days. At the start of treatment (day 0), rats were approximately 7 to 8 weeks of age and males weighed 191 to 243 g and females weighed 139 to 160 g. Rats were individually housed in suspended stainless steel cages with mesh floors. Litter paper was placed beneath the cages and was changed at least 3 times per week. The room temperature ranged between 18°C and 23°C with a relative humidity of 38% to 60% and a 12-hour light/dark cycle. Feed 2016CM Harlan Teklad Global Rodent Diet (Harlan Teklad, Inc, Indianapolis, Indiana) and filtered tap water from an automatic watering system were provided ad libitum except when animals were fasted overnight prior to blood sampling. Test animals housed in the same room for a sentinel health monitoring program evaluated for the absence of viruses near the end of the in-life portion of the study showed no viral contaminants. The nature-identical ergothioneine at the appropriate concentrations was prepared and thoroughly stirred just prior to use daily. Samples of the test substance both neat and in the dosing solution were collected at the beginning, middle, and end of the in-life phase of the study and analyzed by validated HPLC methodology against an ergothioneine standard to evaluate stability and to verify dose concentration and homogeneity. Ophthalmologic evaluations were conducted prior to the commencement of the study and on day 89 by focal illumination and indirect ophthalmoscopy after pharmacologic mydriasis with 1% tropicamide solution. During week 12, blood samples were collected 2 hours postdosing via sublingual bleeding of fasted rats while under isoflurane anesthesia for hematology (erythrocyte count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width, absolute reticulocyte count, platelet count, total white blood cell and differential leukocyte count such as lymphocyte, neutrophil, eosinophil, monocyte, and basophil, and large unstained cell), clinical chemistry (serum aspartate aminotransferase, serum alanine aminotransferase, sorbitol dehydrogenase, alkaline phosphatase, total bilirubin, urea nitrogen, blood creatinine, total cholesterol, triglycerides, fasting glucose, total serum protein, albumin, globulin, calcium, inorganic phosphorus, sodium, potassium, chloride), and plasma analysis for l-ergothioneine determination. Blood samples taken for coagulation (prothrombin time, activated partial thromboplastin time) were collected via the inferior vena cava under isoflurane anesthesia at termination. Prior to the scheduled blood collection for the clinical pathology evaluation during week 12, rats were fasted for at least 15 hours and placed in metabolism cages to collect urine (quality, pH, ketone, color, glucose, bilirubin, clarity, specific gravity, blood, volume, protein, urobilinogen, microscopic urine sediment examination). At the end of the study, animals were euthanized by exsanguination from the abdominal aorta under isoflurane anesthesia and subjected to full necropsy (ie, examination of external surface of the body, all orifices, and the thoracic and abdominal cavities and their contents; weighing of selected organs; preservation of selected organs and tissues in 10% neutral-buffered formalin or modified Davidson fixative). Histopathological examinations of preserved organs and tissues from control and high-dose groups and any gross lesions of potential toxicological significance of the low- and mid-dose groups were performed under light microscopy by a board-certified veterinary pathologist following processing and staining with hematoxylin and eosin. Plasma analysis was performed on the blood of all surviving animals.

Statistical Analysis

Bacterial reverse mutation assay

The mutation factor (MF) is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values).

A statistical evaluation is not regarded as necessary. Toxic effects of the nature-identical ergothioneine are indicated by the partial or complete absence of a background lawn of nonrevertant bacteria (colony counts, if any, should not be reported), a substantial dose-related reduction in revertant colony counts compared with lower dose levels and concurrent vehicle control taking into account the laboratory historical control range or biologically relevant increases in revertant colony numbers. The results were considered positive if the results for the test substance showed a substantial increase in revertant colony counts, that is, 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; and the above increase is dose related and/or reproducible, that is, increases were obtained at more than one experimental point (at least one strain, more than one dose level, more than one occasion, or with different methodologies). When the results indicated neither a concentration-related increase in the number of revertant colonies nor a reproducible substantial increase in revertant colonies, the test substance was considered to be nonmutagenic in this test system.

Repeated dose oral toxicology

Group means and standard deviations were calculated for body weight, daily body weight gain, daily feed consumption, daily feed efficiency, organ weight, and organ-to-body/brain weight ratio. Data within groups were evaluated for homogeneity of variances and normality by Bartlett test. Where Bartlett test indicated homogeneous variances, treated and control groups were compared using analysis of variance, followed by comparison of the treated groups to control by Dunnett t test for multiple comparisons. Where variances were considered significantly different by Bartlett test, groups were compared using a nonparametric method (Kruskal-Wallis nonparametric analysis of variance followed by Dunn test). For clinical pathology, means and standard deviations were calculated. Data within groups were initially analyzed using Levene test for variance homogeneity and Shapiro-Wilk test for normality. If variances were considered to be not significantly different, groups were compared using analysis of variance followed by Dunnett t test for multiple comparisons. If the Shapiro-Wilk test was not significant but the Levene test was significant, a robust version of Dunnett test was used. Where variances were considered significantly different by Levene test, groups were compared using a nonparametric method (Kruskal-Wallis nonparametric analysis of variance followed by Dunn test). Differences among groups were judged to be statistically significant at a probability value of P < .05. Male and female rats were evaluated separately.

Results

Mutagenicity Reverse Mutation Assay

An Ames test using S typhimurium tester strains TA98, TA100, TA1535, and TA1537 and E coli tester strain WP2 uvrA in the plate incorporation and preincubation tests with and without metabolic activation verified that EGT+ did not cause gene mutation activity by base pair changes or frame shifts in the genome of the tester strains used (Tables 1 and 2). Specifically, there was no precipitation, no toxic effects, or biologically relevant increases in revertant colony numbers in any of the test strains of dose group with or without metabolic activation. The reference mutagens induced a distinct increase in revertant colonies indicative of the validity of the experiments.

Table 1.

Results of a Plate Incorporation Test (Experiment 1) of EGT+ on Salmonella typhimurium/Escherichia coli in the Presence (+) and Absence (−) of S9 Mixture.^a

Test substance	Dose level (µg/plate)	S9 mixture	Revertant colony counts (mean)^b
Test substance	Dose level (µg/plate)	S9 mixture	TA 98	Mutation factor	TA 100	Mutation factor	TA 1535	Mutation factor	TA 1537	Mutation factor	WP2 uvrA	Mutation factor
EGT+	0^c	−	33	1.00	126	1.00	14	1.00	10	1.00	65	1.00
	1.58		32	0.97	123	0.98	12	0.86	11	1.10	65	1.00
	5.00		30	0.91	114	0.90	13	0.93	10	1.00	66	1.02
	15.8		33	1.00	131	1.04	14	1.00	12	1.20	64	0.98
	50.0		28	0.85	132	1.05	15	1.07	12	1.20	57	0.88
	158		28	0.85	117	0.93	15	1.07	11	1.10	60	0.92
	500		28	0.85	125	0.99	14	1.00	9	0.90	54	0.83
	1,580		30	0.91	123	0.98	14	1.00	11	1.10	59	0.91
	5,000		28	0.85	110	0.87	14	1.00	9	0.90	58	0.89
NaN₃ ^d	1.5		NA	NA	510	4.05	539	38.5	NA	NA	NA	NA
MMS	2.5 µL		NA	NA	NA	NA	NA	NA	NA	NA	542	8.34
ICR 191^d	1		NA	NA	NA	NA	NA	NA	677	67.70	NA	NA
Daunomycin	6		578	17.52	NA	NA	NA	NA	NA	NA	NA	NA
EGT+	0^c	+	33	1.00	138	1.00	10	1.00	16	1.00	65	1.00
	1.58		38	1.15	133	0.96	13	1.30	13	0.81	64	0.98
	5.00		34	1.03	110	0.80	13	1.30	14	0.88	68	1.05
	15.8		32	0.97	133	0.96	9	0.90	14	0.88	60	0.92
	50.0		35	0.06	119	0.86	13	1.30	14	0.88	64	0.98
	158		33	1.00	128	0.93	14	1.40	13	0.81	64	0.98
	500		35	1.06	133	0.96	12	1.20	14	0.88	61	0.94
	1,580		31	0.94	124	0.90	16	1.60	11	0.69	61	0.94
	5,000		34	1.03	120	0.87	12	1.20	11	0.69	61	0.94
2-AA^d	10.0		1,364	41.33	1,426	10.33	278	27.80	353	22.06	158	2.43

Abbreviations: 2-AA, 2-aminoanthracene; ICR 191, acridine; MMS, methyl methane sulfonate; NaN₃, sodium azide.

^a $Mutation factor = \frac{mean revertants (ET)}{mean revertants (solvent control)} .$

^bMean of replicate (3) plates.

^cNegative (solvent) control: distilled water.

^dPositive control agents.

Table 2.

Results of a Preincubation Test (Experiment 2) of EGT+ on Salmonella typhimurium/Escherichia coli in the Presence (+) and Absence (−) of S9 Mixture.^a

Test substance	Dose level (µg/plate)	S9 mixture	Revertant Colony Counts (Mean)^b
Test substance	Dose level (µg/plate)	S9 mixture	TA 98	Mutation factor	TA 100	Mutation factor	TA 1,535	Mutation factor	TA 1,537	Mutation factor	WP2 uvrA	Mutation factor
EGT+	0^c	−	22	1.00	141	1.00	11	1.00	12	1.00	73	1.00
	1.58		23	1.05	139	0.99	12	1.09	12	1.00	73	1.00
	5.00		25	1.14	124	0.88	10	0.91	11	0.92	63	0.86
	15.8		28	1.27	154	1.09	15	1.36	12	1.00	58	0.79
	50.0		24	1.09	143	1.01	12	1.09	9	0.75	66	0.90
	158		24	1.09	128	0.91	9	0.82	16	1.33	62	0.85
	500		22	1.00	139	0.99	10	0.91	15	1.25	64	0.88
	1,580		19	0.86	129	0.91	15	1.36	12	1.00	67	0.92
	5,000		18	0.82	133	0.94	11	1.00	11	0.92	64	0.88
NaN₃ ^d	1.5		NA	NA	607	4.30	561	51.00	NA	NA	NA	NA
MMS	2.5 µL		NA	NA	NA	NA	NA	NA	NA	NA	457	6.26
ICR^d	1		NA	NA	NA	NA	NA	NA	1,363	113.58	NA	NA
Daunomycin	6.0		567	25.77	NA	NA	NA	NA	NA	NA	NA	NA
EGT+	0^c	+	33	1.00	183	1.00	10	1.00	19	1.00	72	1.00
	1.58		31	0.94	191	1.04	11	1.10	14	0.74	66	0.92
	5.00		30	0.91	158	0.86	12	1.20	16	0.84	71	0.99
	15.8		32	0.97	152	0.83	12	1.20	12	0.63	67	0.93
	50.0		34	1.03	160	0.87	11	1.10	16	0.84	72	1.00
	158		25	0.76	174	0.95	12	1.20	14	0.74	75	1.04
	500		30	0.91	157	0.86	9	0.90	13	0.68	63	0.88
	1,580		30	0.91	145	0.79	9	0.90	14	0.74	68	0.94
	5,000		37	1.12	135	0.74	12	1.20	16	0.84	65	0.90
2-AA^d	10.0		1,395	42.27	1,228	6.71	249	24.90	332	17.47	173	2.40

Abbreviations: 2-AA, 2-aminoanthracene; ICR 191, acridine; MMS, methyl methane sulfonate; NaN₃, sodium azide.

^a $Mutation factor = \frac{mean revertants (ET)}{mean revertants (solvent control)} .$

^bMean of replicate (3) plates.

^cNegative (solvent) control: distilled water.

^dPositive control agents.

Twenty-Eight–Day Oral Toxicity Study

Nature-identical ergothioneine was considered stable under the conditions of storage over the course of this study. Similarly, the test substance was considered to be both homogeneously distributed in the preparations and at the targeted concentration throughout the study. There was no mortality in any of the test groups and no treatment-related abnormal clinical findings. There were no daily or detailed clinical observations noted for male or female rats in all dose groups throughout the study attributable to the test administration. Clinical changes of alopecia found intermittently in males and females of all groups, and ocular and nasal discharge in 1 male in the high-dose group was attributed to a malocclusion of the upper incisors at the end of the study. There were no body weight, body weight gain, food consumption, food efficiency, or macroscopic observations at necropsy attributable to test substance administration. Significant, increases in overall mean weekly body weight (10%, P < 0.05) and body weight gain (25%, P < 0.01) in low-dose males were consistent, associated with increased food consumption at the end of the study (day 29), were without dose dependency, and considered unassociated with nature-identical ergothioneine test substance administration. There were no macroscopic observations at necropsy except for 1 female in each of the control, low-, and mid-dose groups with fluid-filled uterus attributable to normal variation in the estrus cycle in individual animals. These tissues were not evaluated microscopically. Based on this study, animals were expected to tolerate equal to or greater than 500 mg/kg bw/d in the 90-day study, and dose levels were selected accordingly.

90-Day Oral Toxicity Study

Nature-identical ergothioneine, as measured by ergothioneine recovery by HPLC, was found to be stable, homogeneously mixed, and met the target concentrations in the dosing solution for all dose levels under the storage conditions over the course of the study. The percentage change in stability over the course of the 13-week study was +1.9 (101.9% of initial) for a measured active ingredient percentage of 98.8% overall for the course of the study. The test substance as a prepared solution was reported to be stable and homogenously dispersed (94.4%-101.4%; relative standard deviation 0.6%-5.7%) at measured targeted concentrations (94.4%-104.2%) under the conditions of the study. Ergothioneine, analytically determined in rat plasma from samples collected at terminal sacrifice, resulted in significant dose-proportional increases from control for all test group males and females. The significant increases in mean results of ergothioneine found in terminal rat plasma from treated low-, mid-, and high-dose males of 6,090, 8,869, and 18,256 ng/mL and in females of 7,997, 12,973, and 19,676 ng/mL, respectively, were significantly higher than endogenous levels of l-ergothioneine observed in control (1,602 and 2,484 ng/mL for males and females, respectively). The presence of l-ergothioneine in the blood plasma of treated rats in proportion to dosage confirmed that the test substance was systemically absorbed (Table 3).

Table 3.

Mean Plasma Analysis of l-Ergothioneine (Mean ± Standard Deviation, ng/mL) Following EGT+ Gavage for 90 Days.

Group	Number, (M/F)	Dose, mg/kg/d	Male (M)	Female (F)
1 (Control)	10/10	0	1,602 ± 314	2,484 ± 623
2 (Low dose)	10/10	400	6,090 ± 3,075^a	7,997 ± 3,698^a
3 (Mid dose)	9/9	800	8,869 ± 5,750^b	12,973 ± 9,899^c
4 (High dose)	10/10	1,600	18,256 ± 11,595^c	19,676 ± 21,050^c

^aStatistically significant from controls, P < 0.05.

^bStatistically significant from controls, P < 0.01.

^cStatistically significant from controls, P < 0.001.

Mortality and clinical observations

There were no mortalities related to the administration of nature-identical ergothioneine. One male in the mid-dose group was found dead on day 66. Microscopic laryngeal lesions associated with inflammatory infiltrate and multifocal, moderate muscle degeneration were consistent with death likely related to gavage administration procedure and unassociated with a test substance effect. One female in the same test group was found dead on day 85 with multifocal red discoloration of the distal aspect of the tail corresponding to locally extensive, moderate epidermal necrosis. There were no other clinical observations. Although the cause of mortality could not be determined for this animal, the lack of a dose-response effect in incidence and the absence of significant macroscopic or microscopic findings suggest that the death of this animal was not related to the nature-identical ergothioneine administration. Clinical observations of surviving animals attributed to oral administration of the nature-identical ergothioneine were restricted to males and consisted of soft feces in 6 of 10 high-dose animals on study days 85 to 95, as well as diarrhea (day 95) observed in 2 other high-dose males. These findings were considered nonadverse. Incidental clinical findings in surviving males included a lesion on the nose/snout (1 of 10 low dose); red ocular discharge in the right eye (1 of 10 high dose); brown staining on the face (1 of 10 mid dose and 1/10 high dose); slight to moderate alopecia on the right/left forelimb/forepaw, abdomen, or anogenital area (1 of 10 low dose and 1 of 10 high dose); enophthalmos (1 of 10 low dose); eschar on the face (1 of 10 mid dose); white oral discharge (1 of 10 mid dose and 1 of 10 high dose); moist rales (1/10 mid dose); a malocclusion of the upper incisors (1 of 10 high dose); a broken tooth of the upper incisors (1 of 10 mid dose); and a broken toenail (1 of 10 mid dose). There were no clinical signs in control males. Clinical signs in females included slight to moderate alopecia on left/right forelimb/hind limb, flank, chest, back, abdomen, head, or anogenital area (3 of 10 control, 4 of 10 low dose, 1 of 10 mid dose, and 4 of 10 high dose); eschar on the back, head, and neck (1 of 10 mid dose); and a broken tooth of the upper left incisor (1 of 10 mid dose). Ophthalmoscopical examinations showed eyes to be normal.

Body weight, body weight gain, food consumption, and food efficiency

There were no significant changes in body weight, body weight gain, food consumption, and food efficiency changes in treated male and female rats attributable to the administration of the nature-identical ergothioneine. Changes in male or female body weight gain were transient and without correlated changes in body weight, while decreases observed in male food consumption were transient and without concurrent or dose-dependent body weight, body weight gain, or food efficiency changes. Overall (days 0-92) and weekly mean body weight and mean daily body weight gain (Figure 1) of all treated rats were comparable with controls with the following exceptions: males showed a significant decrease in daily body weight gain during days 8 to 15 (high-dose group); females showed a significant decrease in body weight on days 64 and 71 (high-dose group); and females showed a significant decrease in daily body weight gain during days 36 to 43 (high-dose group). Overall and weekly feed consumption and mean daily feed efficiency of all treated rats were generally comparable to controls with the following exceptions: males showed decreases in food consumption during days 1 to 15 and 22 to 29 (high-dose group); females showed a significant decrease in feed consumption during days 1 to 15 and 64 to 71 (high-dose group) and overall (days 0-92); and females showed a significant decrease in feed efficiency on days 36 to 43.

Figure 1.

Mean body weight and body weight gain (day ± standard deviation [SD]) of male and female Sprague Dawley rats administered EGT+ gavage for 90 days for groups 1 (control), 2 (400 mg/kg/d), 3 (800 mg/kg/d), and 4 (1,200 mg/kg/d); mean of 9 of 10 animals/sex/group.

Clinical pathology

Hematology, coagulation, and clinical chemistry parameters revealed no adverse changes and no morphologic changes. Statistically significant hematology changes (Table 4) reported included decreased monocyte concentration (mid-dose males), decreased hemoglobin concentration (low-dose females), decreased hematocrit (low- and mid-dose females), decreased mean corpuscular volume (low-, mid-, and high-dose females), increased red cell distribution width and reticulocyte concentration (high-dose females), and decreased absolute eosinophil count (high-dose females). Prothrombin time was minimally decreased in mid-dose males and females.

Table 4.

Mean Hematology Values, Day 86 (Mean ± Standard Deviation) of Sprague Dawley Rats Following EGT+ Gavage for 90 Days.^a

Group	1	2	3	4	1	2	3	4
	Concentration (mg/kg/d)
	0	400	800^b	1,600	0^b	400	800^b	1,600^b
	Male				Female
RBC (×10⁶/µL)	8.71 ± 0.37	8.79 ± 0.34	8.73 ± 0.45	8.79 ± 0.54	8.42 ± 0.26	8.22 ± 0.31	8.14 ± 0.32	8.35 ± 0.29
HGB (g/dL)	15.7 ± 0.8	15.6 ± 0.5	15.7 ± 0.8	15.9 ± 1.0	15.9 ± 0.6	15.2 ± 0.5^c (14-17.1)	15.3 ± 0.6	15.5 ± 0.7
HCT (%)	46.5 ± 2.5	46.3 ± 1.3	46.3 ± 2.0	47.0 ± 2.6	46.3 ± 1.3	44.0 ± 1.3^c (40.6-49.4)	43.6 ± 1.5^c	44.7 ± 2.0
MCV (fL)	53.4 ± 1.5	52.7 ± 1.5	53.1 ± 1.4	53.5 ± 2.1	55.0 ± 1.4	53.6 ± 0.9^d (50.2-58.0)	53.6 ± 1.0^d	53.5 ± 0.07^d
MCH (pg)	18.0 ± 0.5	17.8 ± 0.6	18.0 ± 0.5	18.2 ± 0.9	18.9 ± 0.6	18.5 ± 0.3	18.7 ± 0.5	18.6 ± 0.4
MCHC (g/dL)	33.7 ± 0.4	33.8 ± 0.4	34.0 ± 0.6	33.9 ± 0.6	34.4 ± 0.5	34.6 ± 0.6	35.0 ± 0.5	34.7 ± 0.6
RDW (%)	14.4 ± 1.4	13.7 ± 1.0	13.3 ± 1.4	13.6 ± 1.0	11.5 ± 0.4	11.5 ± 0.4	11.9 ± 0.3	12.1 ± 0.9^d (10.8-13.1)
PLT (×10³/µL)	1,104 ± 104	1,117 ± 114	1,179 ± 161	1,107 ± 143	1,014 ± 132	1,060 ± 86	1,092 ± 83	1,177 ± 198
WBC (×10³/µL)	15.92 ± 4.15	14.12 ± 2.53	14.18 ± 5.46	11.84 ± 2.41	8.77 ± 2.95	7.56 ± 1.93	8.27 ± 3.08	8.40 ± 3.43
ANEU (×10³/µL)	2.90 ± 0.87	2.35 ± 0.81	2.88 ± 1.93	1.87 ± 0.78	1.10 ± 0.68	0.99 ± 0.32	1.07 ± 0.41	1.53 ± 1.24
ALYM (×10³/µL)	12.04 ± 3.79	11.10 ± 2.51	10.45 ± 4.57	9.22 ± 1.79	7.23 ± 2.18	6.13 ± 1.69	6.73 ± 2.78	6.46 ± 2.53
AMON (×10³/µL)	0.55 ± 0.23	0.32 ± 0.07^d (0.14-0.78)	0.48 ± 0.22	0.44 ± 0.20	0.21 ± 0.09	0.24 ± 0.10	0.24 ± 0.09	0.29 ± 0.16
AEOS (×10³/µL)	0.22 ± 0.07	0.16 ± 0.05	0.19 ± 0.08	0.15 ± 0.04	0.14 ± 0.05	0.14 ± 0.05	0.15 ± 0.10	0.04 ± 0.06^c (0.05-0.35)
ABAS (×10³/µL)	0.08 ± 0.05	0.08 ± 0.04	0.07 ± 0.04	0.06 ± 0.02	0.02 ± 0.02	0.02 ± 0.01	0.02 ± 0.01	0.02 ± 0.02
ALUC (×10³/µL)	0.12 ± 0.07	0.10 ± 0.03	0.11 ± 0.08	0.10 ± 0.06	0.05 ± 0.04	0.05 ± 0.02	0.06 ± 0.03	0.06 ± 0.04
ARET (×10³/µL)	250.0 ± 49.4	226.9 ± 70.5	218.1 ± 60.0	214.8 ± 32.0	165.5 ± 29.4	164.8 ± 35.9	192.1 ± 28.9	224.0 ± 58.2^d (91.3-247.1)
Day 95/96 PT (seconds)	10.8 ± 0.2	10.8 ± 0.3	10.5 ± 0.2^d (9.5-11.5)	10.6 ± 0.2	10.2 ± 0.2	10.2 ± 0.3	10.0 ± 0.1^d (9.2-10.5)	10.0 ± 0.2
Day 95/96 APTT (seconds)	16.0 ± 1.2	16.0 ± 1.2	15.6 ± 1.1	15.6 ± 0.8	15.1 ± 1.1	14.4 ± 0.9	14.6 ± 0.9	15.6 ± 1.6

Abbreviations: ABAS, absolute basophil count; AEOS, absolute eosinophil count; ALUC, absolute large unstained cell; ALYM, absolute lymphocyte count; AMON, absolute monocyte count; ANEU, absolute neutrophil count; APTT, activated partial thromboplastin time; ARET, absolute reticulocyte count; HCT, hematocrit; HGB, hemoglobin concentration; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; PLT, platelet count; PPT, prothrombin time; RBC, erythrocyte count; RDW, red cell distribution width; WBC, total white blood cell.

^aLaboratory historical control values for this strain, age, and sex are given in parenthesis.

^bn = 9 for all group 1 females, group 3 males and females; all others n = 10.

^cTest: Dunnett nonparametric 2-sided P < 0.05.

^dTest: Dunnett 2-sided P < 0.05.

Clinical chemistry changes (Table 5) included increased alkaline phosphatase concentration (high-dose males and females), decreased glucose concentration (high-dose males), decreased sodium concentration (mid-dose males), decreased triglyceride concentration (high-dose males), decreased chloride concentration (mid- and high-dose males), decreased creatinine, protein, and albumin concentration (high-dose females), and increased triglyceride concentration (high-dose females). These changes were considered nonadverse and not related to exposure because the magnitude of the changes was considered not clinically significant, fell within historical control range of the laboratory, and were not accompanied by any other corresponding clinical or pathological change. There were no test substance-related changes in blood cell morphology. The only statistically significant changes reported in urinalysis was increased urine volume in mid-dose males, decreased urinary protein in low- and mid-dose males, and decreased urinary pH in high-dose females. These changes, without supporting clinical, macroscopic, or microscopic changes, were not adverse and, as such, considered unrelated to the administration of the nature-identical ergothioneine.

Table 5.

Mean Clinical Chemistry Values, Day 86 (Mean ± Standard Deviation) of Sprague Dawley Rats Following EGT+ Gavage for 90 Days.^a

Group	1	2	3	4	1	2	3	4
				Concentration (mg/kg/d)
	0	400	800^b	1,600	0^b	400	800^b	1,600
	Male				Female
AST (U/L)	95 ± 19	99 ± 19	103 ± 17	98 ± 37	79 ± 18	76 ± 19	69 ± 14	66 ± 9
ALT (U/L)	39 ± 5	35 ± 6	40 ± 6	45 ± 24	32 ± 7	37 ± 18	31 ± 10	36 ± 9
SDH (U/L)	7.8 ± 3.0	6.0 ± 2.7	5.4 ± 2.5	5.4 ± 2.1	6.4 ± 2.9	7.7 ± 2.5	6.5 ± 1.6	5.5 ± 1.6
ALKP (U/L)	97 ± 24	89 ± 113	110 ± 36	129 ± 33^c (55-183)	52 ± 15	53 ± 22	62 ± 21	86 ± 28^c (24-121)
BILI (mg/dL)	0.19 ± 0.03	0.19 ± 0.03	0.20 ± 0.04	0.20 ± 0.04	0.17 ± 0.03	0.17 ± 0.03	0.17 ± 0.03	0.16 ± 0.03
BUN (mg/dL)	14 ± 2	13 ± 2	14 ± 2	14 ± 2	12 ± 1	12 ± 2	12 ± 2	11 ± 2
CREA (mg/dL)	0.31 ± 0.4	0.28 ± 0.5	0.30 ± 0.04	0.27 ± 0.03	0.32 ± 03	0.31 ± 0.02	0.30 ± 0.04	0.27 ± 0.04^c (0.23-0.53)
CHOL (mg/dL)	82 ± 31	83 ± 16	84 ± 14	86 ± 17	80 ± 13	88 ± 14	101 ± 23	97 ± 21
TRIG (mg/dL)	81 ± 66	63 ± 25	54 ± 24	58 ± 23	37 ± 8^d	39 ± 9^b	51 ± 24	55 ± 17^e (16-199)
GLUC (mg/dL)	115 ± 13	112 ± 20	101 ± 13	91 ± 9^c (84-256)	106 ± 19	111 ± 12	96 ± 14	95 ± 16
TP (g/dL)	6.5 ± 0.5	6.7 ± 0.3	6.8 ± 0.3	6.5 ± 0.3	7.0 ± 7.3	7.3 ± 0.5	6.6 ± 0.4	6.5 ± 0.4^c (5.7-8.5)
ALB (g/dL)	3.1 ± 0.1	3.0 ± 0.2	3.0 ± 0.1	3.0 ± 0.1	3.4 ± 0.1	3.7 ± 0.3	3.2 ± 0.2	3.2 ± 0.2^c (3.0-5.0)
GLOB (g/dL)	3.5 ± 0.4	3.7 ± 0.3	3.7 ± 0.3	3.5 ± 0.2	3.5 ± 0.2	3.6 ± 0.3	3.4 ± 0.3	3.4 ± 0.2
CALC (mg/dL)	9.9 ± 0.5	9.9 ± 0.3	10.2 ± 0.3	10.2 ± 0.4	9.8 ± 0.2	10.0 ± 0.4	9.9 ± 0.4	10.1 ± 0.5
IPHS (mg/dL)	6.8 ± 0.4	6.4 ± 0.8	6.9 ± 0.4	6.8 ± 0.5	4.8 ± 0.6	4.6 ± 0.6	5.3 ± 0.5	5.3 ± 0.6
NA (mmol/L)	142.4 ± 7.2	138.8 ± 5.9	136.2 ± 3.5^e (127.6-166.1)	138.6 ± 4.0	141.7 ± 5.0	138.3 ± 5.1	140.1 ± 4.3	139.1 ± 4.2
K (mmol/L)	5.52 ± 0.66	5.44 ± 0.58	5.51 ± 0.32	5.63 ± 0.37	4.63 ± 0.36	4.56 ± 0.36	4.72 ± 0.38	4.61 ± 0.31
CL (mmol/L)	102.6 ± 4.6	100.9 ± 3.6	97.5 ± 2.5^c (91.9-120.1)	97.9 ± 3.0^c	103.2 ± 2.8	100.9 ± 2.7	100.8 ± 3.2	99.9 ± 3.8

Abbreviations: ALB, albumin; ALKP, alkaline phosphatase; ALT, serum alanine aminotransferase; AST, serum aspartate aminotransferase; BILI, total bilirubin; BUN, urea nitrogen; CALC, calcium; CHOL, total cholesterol; CL, chloride; CREA, blood creatinine; GLOB, globulin; GLUC, fasting glucose; IPHS, inorganic phosphorus; K, potassium; NA, sodium; SDH, sorbitol dehydrogenase; TP, total serum protein; TRIG, triglycerides.

^aLaboratory historical control values for this strain, age, and sex are given in parenthesis.

^bn = 9 for all group 3 males and females where indicated; all others n = 10.

^cTest: Dunnett 2-sided P < 0.05.

^dn = 8.

^eTest: Dunnett nonparametric 2-sided P < 0.05.

Macroscopic observations, histopathology, and organ weights

Macroscopic examination of surviving animals revealed no gross abnormalities related to treatment with nature-identical ergothioneine. Incidental findings, confirmed on microscopic analysis, of mild dermal fibrosis in 1 low-dose male, moderate polycystic kidney in 1 mid-dose male, marked auricular chondritis in 1 high-dose female associated with bilaterally enlarged ears at sacrifice, and fluid-filled uterus attributable to variation in the estrus cycle in individual animals of both control and treated rats.

There were no absolute or relative organ weight findings in male rats attributable to the administration of the nature-identical ergothioneine. Decreases in absolute and relative thymus weights along with increases in liver and kidney relative to body weight changes observed in high-dose female rats unassociated with hematological and histological changes were interpreted to be nonadverse and incidental (Tables 6 -8). There were no microscopic findings related to the exposure to the nature-identical ergothioneine (Table 9). All microscopic findings were incidental, associated with those observed normally in this age and strain of rat.

Table 6.

Mean Terminal Body and Organ Weights (Mean ± Standard Deviation, g) Days 95 to 96 of Sprague Dawley Rats Following EGT+ Gavage for 90 Days.

Group	1	2	3	4	1	2	3	4
Concentration (mg/kg/d)
	0	400	800^a	1,600	0	400	800^a	1,600
	Male				Female
Terminal BW
Mean	512.6	496.3	500.7	476.5	272.7	273.2	272.2	252.7
SD	52.6	47.8	63.9	53.3	10.7	21.4	18.1	17.5
Adrenal
Mean	0.062	0.066	0.063	0.061	0.083	0.084	0.082	0.086
SD	0.009	0.013	0.011	0.007	0.013	0.008	0.006	0.016
Brain
Mean	2.22	2.22	2.28	2.2	2.1	2.1	2.12	2.09
SD	0.11	0.16	0.14	0.14	0.09	0.08	0.1	0.08
Epididymis
Mean	1.535	1.544	1.593	1.517	-	-	-	-
SD	0.109	0.154	0.114	0.117	-	-	-	-
Heart
Mean	1.47	1.45	1.55	1.34	0.97	1.03	0.95	0.87
SD	0.23	0.22	0.25	0.14	0.08	0.13	0.09	0.08
Kidneys
Mean	3.26	3.43	3.68	3.36	2.05	2.04	2.06	2.14
SD	0.29	0.58	0.78	0.47	0.16	0.09	0.15	0.25
Liver
Mean	13.34	12.62	13.7	13.07	7.9	7.85	8.69	8.58
SD	1.97	2.34	2.15	2.09	1.12	0.67	0.79	0.9
Ovaries
Mean	-	-	-	-	0.081	0.082	0.085	0.079
SD	-	-	-	-	0.015	0.017	0.025	0.017
Spleen
Mean	0.99	0.85	0.88	0.86	0.66	0.56	0.6	0.55
SD	0.22	0.13	0.17	0.25	0.18	0.08	0.08	0.09
Testes
Mean	3.53	3.58	3.73	3.7	-	-	-	-
SD	0.12	0.3	0.35	0.31	-	-	-	-
Thymus
Mean	0.283	0.308	0.28	0.258	0.318	0.322	0.351	0.237^b
SD	0.074	0.062	0.084	0.047	0.05	0.053	0.072	0.047
Uterus and oviduct
Mean	-	-	-	-	0.9	0.8	0.69	0.68
SD	-	-	-	-	0.39	0.34	0.24	0.24

Abbreviation: SD, standard deviation.

^an = 9 for all group 3 males and females where indicated; all others n = 10/sex/group.

^bTest: Dunnett 2-sided P < 0.01.

Table 7.

Mean Organ-to-Body Weights (Mean ± Standard Deviation, g) Days 95 to 96 of Sprague Dawley Rats Following EGT+ Gavage for 90 Days.

Group	1	2	3	4	1	2	3	4
	Concentration (mg/kg/d)
	0	400	800^a	1,600	0	400	800^a	1,600
	Male				Female
Adrenal
Mean	0.121	0.134	0.127	0.129	0.304	0.311	0.303	0.338
SD	0.019	0.03	0.023	0.019	0.046	0.05	0.026	0.05
Brain
Mean	4.36	4.5	4.59	4.66	7.73	7.72	7.8	8.29
SD	0.48	0.43	0.4	0.48	0.46	0.51	0.53	0.6
Epididymis
Mean	3.024	3.12	3.221	3.203	-	-	-	-
SD	0.399	0.256	0.407	0.251	-	-	-	-
Heart
Mean	2.86	2.92	3.1	2.83	3.56	3.76	3.51	3.45
SD	0.24	0.2	0.38	0.2	0.24	0.29	0.29	0.22
Kidneys
Mean	6.4	6.9	7.41	7.05	7.5	7.51	7.56	8.44^b
SD	0.65	0.87	1.6	0.5	0.45	0.55	0.4	0.57
Liver
Mean	26.01	25.26	27.37	27.42	28.9	28.83	31.94	34.01^c
SD	2.6	2.36	2.8	3.04	3.29	2.84	2.18	3.31
Ovaries
Mean	-	-	-	-	0.298	0.3	0.312	0.314
SD	-	-	-	-	0.055	0.05	0.084	0.062
Spleen
Mean	1.91	1.71	1.75	1.83	2.41	2.04	2.21	2.18
SD	0.27	0.16	0.26	0.62	0.6	0.27	0.18	0.29
Testes
Mean	6.95	7.26	7.55	7.82	-	-	-	-
SD	0.63	0.79	1.07	0.85	-	-	-	-
Thymus
Mean	0.555	0.62	0.553	0.544	1.168	1.178	1.287	0.947^d
SD	0.145	0.11	0.134	0.092	0.177	0.175	0.231	0.219
Uterus and oviduct
Mean	-	-	-	-	3.32	2.94	2.53	2.65
SD	-	-	-	-	1.51	1.25	0.77	0.83

Abbreviation: SD, standard deviation.

^an = 9 for all group 3 males and females where indicated; all others n = 10/sex/group.

^bTest: Dunnett 2-sided P < 0.001.

^cTest: Dunnett 2-sided P < 0.01.

^dTest: Dunnett 2-sided P < 0.05.

Table 8.

Mean Organ-to-Brain Weights (Mean ± Standard Deviation, g) Days 95 to 96 of Sprague Dawley Rats Following EGT+ Gavage for 90 Days.

Group	1	2	3	4	1	2	3	4
	Concentration (mg/kg/d)
	0	400	800^a	1,600	0	400	800^a	1,600
	Male				Female
Adrenal
Mean	0.028	0.03	0.028	0.028	0.039	0.04	0.039	0.041
SD	0.004	0.007	0.004	0.004	0.006	0.005	0.003	0.007
Epididymis
Mean	0.693	0.697	0.701	0.691	-	-	-	-
SD	0.044	0.066	0.066	0.063	-	-	-	-
Heart
Mean	0.67	0.66	0.68	0.61	0.46	0.49	0.45	0.42
SD	0.11	0.1	0.09	0.07	0.05	0.05	0.05	0.04
Kidneys
Mean	1.47	1.55	1.62	1.53	0.98	0.97	0.97	1.03
SD	0.14	0.27	0.34	0.19	0.91	0.06	0.09	0.13
Liver
Mean	6.04	5.7	6	5.94	3.77	3.74	4.12	4.12
SD	0.97	1.01	0.82	1.03	0.6	0.34	0.44	0.51
Ovaries
Mean	-	-	-	-	0.039	0.039	0.04	0.038
SD	-	-	-	-	0.008	0.008	0.012	0.008
Spleen
Mean	0.45	0.39	0.39	0.39	0.31	0.27	0.29	0.26
SD	0.1	0.07	0.07	0.12	0.08	0.03	0.04	0.04
Testes
Mean	1.6	1.62	1.64	1.69	-	-	-	-
SD	0.08	0.19	0.17	0.21	-	-	-	-
Thymus
Mean	0.129	0.139	0.122	0.117	0.152	0.153	0.166	0.113^b
SD	0.036	0.029	0.034	0.019	0.024	0.026	0.031	0.021
Uterus and oviduct
Mean	-	-	-	-	0.43	0.38	0.33	0.32
SD	-	-	-	-	0.19	0.17	0.12	0.11

Abbreviation: SD, standard deviation.

^an = 9 for all group 3 males and females where indicated; all others n = 10/sex/group.

^bTest: Dunnett 2-sided P < 0.01.

Table 9.

Incidence and Severity of Microscopic Findings for EGT+ in Sprague Dawley Rats Following EGT+ Gavage for 90 Days.

	1	2	3	4	1	2	3	4
	Concentration (mg/kg/d)
	0	400	800^a	1,600	0	400	800^a	1,600
Group	Male				Female
Number examined	10	1	1	10	10	5	1	10
Bone marrow, femur					10	0	0	10
Hypercellularity
Moderate					1	0	0	0
Bone marrow, sternum					10	0	0	10
Hypercellularity
Moderate					1	0	0	0
Ear					0	0	0	1
Chondritis, marked					0	0	0	1
Esophagus	10	0	0	10	10	0	0	10
Degeneration, myofiber
Mild	1	0	0	2	1	0	0	1
Minimal					1	0	0	0
Eyes	10	0	0	10	10	0	0	10
Rosettes, retina
Mild	1	0	0	0
Minimal					1	0	0	0
Harderian gland	10	0	0	10	10	0	0	10
Inflammation, mild	1	0	0	0
Minimal	1	0	0	0	1	0	0	0
Kidneys	10	0	1	10	10	0	0	10
Chronic progressive
Mild	1	0	0	0
Minimal	3	0	0	2
Polycystic, moderate	0	0	1	0
Cystic
Mild					0	0	0	1
Minimal					0	0	0	1
Larynx	10	0	0	10	10	0	0	10
Foreign body
Single granuloma					1	0	0	0
Inflammatory, granuloma
Mild					0	0	0	1
Minimal					1	0	0	0
Moderate					2	0	0	0
Degeneration, cartilage
Mild	0	0	0	1
Hemorrhage, mild	0	0	0	1
Liver	10	0	0	10	10	0	0	10
Bile duct hyperplasic
Minimal	0	0	0	1
Hematopoiesis, mild					1	0	0	0
Lipidosis, mild					1	0	0	0
Mononuclear infiltrate
Minimal	0	0	0	1
Necrosis, minimal	2	0	0	1	1	0	0	0
Lungs	10	0	0	10	10	0	0	10
Alveolar macrophages
Minimal	0	0	0	1	1	0	0	0
Inflammatory chronic
Minimal	0	0	0	1
Granuloma, minimal	0	0	0	3
Lymph node, mesenteric					10	0	0	10
Hemorrhage, mild					1	0	0	0
Nose and nasal turbin	10	0	0	10
Degeneration, tooth
Moderate	0	0	0	1
Inflammatory, acute, mild	1	0	0	0
Purulent exudate, mild	0	0	0	1
Pharynx	10	0	0	10
Hemorrhage, mild	0	0	0	1
Spleen					10	0	0	10
Hematopoiesis
Moderate					1	0	0	0
Pituitary gland	10	0	0	10
Cysts, pars distalis
Minimal	1	0	0	0
Prostate gland	10	0	0	10
Inflammatory
Mild	3	0	0	1
Mild chronic	1	0	0	0
Minimal	0	0	0	6
Skin, nose	0	1	0	0
Fibrosis, dermis
Mild	0	1	0	0
Testes	10	0	0	10
Hypospermatogen
Minimal	0	0	0	1
Multinucleated germ
Minimal	0	0	0	1
Uterus					10	5	1	10
Dilation					4	3	1	2

^an = 9 for all group 3 males and females where indicated; all others n = 10/sex/group.

The highest concentration tested of 1,600 mg/kg bw/d produced no treatment-related adverse effects and was regarded as the no observed adverse effect level (NOAEL). Therefore, under the conditions of this study, the clinical and anatomic pathology, the NOAEL for nature-identical ergothioneine, EGT+ was 1,600 mg/kg bw/d following daily oral gavage to male and female Sprague Dawley rats for at least 90 days.

Discussion

The present study examined l-ergothioneine, a naturally derived thiol amino acid and manufactured as Mironova EGT+, in mutagenicity and 28-and 90-day safety studies for its suitability as a dietary supplement. In a genotoxicity study to qualify this nature-identical ergothioneine under a Generally Recognized as Safe proprietary manufacturing process, a bacterial reverse mutation assay using S typhimurium and E coli strains with and without metabolic activation showed the nature-identical ergothioneine to be nonmutagenic at plate concentrations of up to 5,000 µg/mL without precipitation and no cytotoxic effects or biologically relevant increases of revertant colony numbers. The absence of genotoxic potential is in agreement with the findings of Schauss et al for corresponding bacterial strains.⁴²

The short-term, range-finding oral toxicity study further supported that the test substance, EGT+, the highly pure (>99%), biomimetic compound was well tolerated by male and female rats at doses of 500 mg/kg bw/d. There were no test substance-related mortalities during the course of the study. There were no clinical signs, body weight, body weight gain, food consumption, food efficiency, or macroscopic observations at necropsy attributable to the nature-identical ergothioneine administration. Significant increases in mean weekly body weight and mean daily body weight gain in low-dose males over the course of the 28-day study were consistent and associated with increased food consumption and without dose dependency.

Based on the 28-day range-finding study, dose selection of 400, 800, and 1,600 mg/kg bw/d for the 90-day study, equivalent to a substantial safety margin over the estimated human exposure, was well tolerated by the animals. In the longest and highest dosage study of a synthetic ergothioneine to be reported to date, the test substance was determined to be stable, homogeneously distributed, and at targeted dose levels in the distilled water vehicle under the conditions of the study. Plasma analysis at the end of the study confirmed the dose proportionality of the received test substance and its systemic exposure. These results differ with those of Heath et al who reported steady l-ergothioneine concentration increases in whole blood, but not in plasma, following a 14-day 0.1% dietary level (approximately 83 mg/kg bw/d) in rats⁴⁰ and with those of Forster et al who, upon finding no dose proportionality of l-ergothioneine in Sprague Dawley Rj Han: SD rat blood plasma at dietary administration of up to approximately 700 mg/kg bw/d for 13 weeks, attributed the result to saturation of uptake mechanisms in the liver.⁴⁴ Since the present study included doses as high as 1,600 mg/kg bw/d with verifiable dose-proportional plasma levels and no plateau, the complete mode of l-ergothioneine distribution remains yet to be determined. Nonetheless, these results lend support to the assertion that l-ergothioneine accumulates in tissues at a variable rate, dependent upon concentration (dose level) and genetic background (rat strain), and may require baseline correction factors for exogenous individual bioavailability.^59,60 Clinical observations of soft feces/diarrhea attributed to oral administration of the nature-identical ergothioneine were observed in high-dose males at the end of the study only. These findings, while nonadverse, appear associated with intestinal disorder. These changes may point to a possible deleterious impact of excessive l-ergothioneine uptake in chronic inflammatory disease.⁹ Although gender-related differences in blood and kidney ergothioneine levels have been reported, including higher levels in males,^29,61 no consensus for these responses exist, though age-related declines are found to be more consistent.^9,61
-63 There were no ophthalmologic, body weight, body weight gain, food consumption, food efficiency, macroscopic, absolute or relative organ weight, or clinical or histopathological changes associated with the administration of nature-identical ergothioneine. Any body weight and food consumption changes were considered transient and, without correlate, nonadverse. Decreases in female food consumption overall were of small magnitude and largely attributable to reductions considered resulting from acclimation of the test substance at the beginning of the study. Changes in hematology and coagulation parameters for both males (absolute monocytes and prothrombin time) and females (hemoglobin, hematocrit, mean corpuscular volume, red cell width, absolute eosinophil and reticulocyte count, and prothrombin time) were unassociated with any histopathological or clinical findings and were within historical control values (Table 4) for all but the absolute eosinophilic levels in high-dose females. Without corresponding findings, the minimally reduced eosinophil levels were not considered toxicologically significant. Serum chemistry changes observed in high-dose females (alkaline phosphatase, creatinine, albumin, total protein, and triglycerides) and males (alkaline phosphatase, glucose, sodium, and chloride) unassociated with correlating pathological or clinical findings were also considered nonadverse due to their overall small magnitude, within the range of historical control for males and females (Table 5). Decreased thymus weights observed in high-dose female rats were considered incidental in the absence of hematological or histopathological correlates. Other findings were unrelated to the nature-identical ergothioneine administration and were considered incidental as can be observed in the age and strain of rats used in this study.

Oral administration of up to 1,600 mg/kg bw/d nature-identical ergothioneine to CD IGS Sprague Dawley rats for 90 days did not produce any adverse effects under the conditions of the present study. Since the NOAEL represents a large multiple of expected human exposure and a highly suitable safety margin from the intended use of 5 to 10 mg/d for an adult, it is concluded that from a critical evaluation of the available information on EGT+, a nature-identical ergothioneine, the results are supportive of appropriate levels for the safety of the intended use of this product as a dietary supplement.

Footnotes

Author Contributions

Palma Ann Marone contributed to design, contributed to analysis and interpretation, drafted the manuscript, and critically revised the manuscript. Jan Trampota contributed to conception and design, contributed to acquisition and analysis, and critically revised the manuscript. Steven Weisman contributed to conception and design, contributed to acquisition and interpretation, and critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of work ensuring integrity and accuracy.

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. Drs Marone and Weisman received payment from Mironova Labs Inc. for preparation of this manuscript.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: All research reported was funded by Mironova Labs Inc., Fairfield, New Jersey.

References

Uttara

Singh

Zamboni

Mahajan

. Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options. Curr Neuropharmacol. 2009;7(1):65–74.

Reuter

Gupta

Chaturvedi

Aggarwal

. Oxidative stress, inflammation, and cancer: how are they linked? Free Radic Biol Med. 2010;49(11):1603–1616.

Wruck

Fragoulis

Gurzynski

. Role of oxidative stress in rheumatoid arthritis: insights from the Nrf2-knockout mice. Ann Rheum Dis. 2011;70(5):844–850.

Bennett

Griffiths

. Regulation of T-cell functions by oxidative stress. Chapter 2. In: Alcaraz

Guallillo

Sanchez-Pernaute

, eds. Studies on Arthritis and Joint Disorders. New York, NY: Humana Press, Springer; 2013:33–48.

Oxis International, Inc. Compound Monograph l-ergothioneine. Web site. http://www.oxisresearch.com/ergo/l-ergothioneine_monograph.pdf. Accessed December 16, 2015.

Snyder

Paul

. The unusual amino acid l-ergothioneine is a physiologic cytoprotectant. Cell Death Different. 2010;17(7):1134–1140.

Hand

Honek

. Biological chemistry of naturally occurring thiols of microbial and marine origin. J Nat Prod. 2005;68(2):293–308.

Pfieffer

Bauer

Surek

Schomig

Grundemann

. Cyanobacteria produce high levels of erogthioneine. Food Chem. 2011;129(4):1766–1769.

Cheah

Halliwell

. Erogthioneine: antioxidant potential, physiological function ad role in disease. Biochim Biophys Acta. 2012;1822(5):784–793.

10.

Melville

Ergothioneine. Harris

Marrian

Thimann

, eds. Vitamins and Hormones: Advances in Research and Applications. New York, NY: Academic Press; 1959:55–204.

11.

Grundemann

. The ergothioneine transporter controls and indicates ergothioneine activity-a review. Prev Med. 2012;(suppl 54):S71–S74.

12.

Melville

Horner

Lubschez

. Tissue ergothioneine. J Biol Chem. 1954;206(1):221–228.

13.

Mann

Leone

. Studies on the metabolism of semen VIII. Ergothioneine as a normal constituent of boar seminal plasma: purification and crystallization: site of formation and function. Biochem J. 1953;53(1):140–148.

14.

Kawano

Otani

Takeyama

Kawai

Mayumi

Hama

. Studies on ergothioneine. VI. Distribution and fluctuations of ergothioneine in rats. Chem Pharm Bull. 1982;30(5):1760–1765.

15.

Markova

Karaman-Jurukovska

Dong

Damaghi

Smiles

Yarosh

. Skin cells and tissue are capable of using l-ergothioneine as an integral component of their antioxidant defense system. Free Radic Biol Med. 2009;46(8):1168–1176. doi:10.1016/j.freeradbiomed.2009.01.021.

16.

Yang

Sit

. Uptake and protective effects of ergothioneine in human endothelial cells. J Pharmacol Exp Ther. 2014;350(3):691–700.

17.

Peltekova

Wintle

Rubin

. Functional variants of OCTN cation transporter genes are associated with Crohn disease. Nat Genet. 2004;36(5):471–475.

18.

Fisher

Hampe

Onnie

. Direct or indirect association in a complex disease: the role of SLC22A4 and SLC22A5 functional variants in Crohn disease. Hum Mut. 2006;27(8):778–785.

19.

Santiago

Martinez

de la Calle

. Evidence for the association of the SLC22A4 and SLC22A5 genes with Type I diabetes: a case control study. BMC Med Genet. 2006;7:54–60.

20.

Taubert

Lazar

Grimberg

. Association of rheumatoid arthritis with ergothioneine levels in red blood cells: a case control study. J Rheumatol. 2006;33(11):2139–2145.

21.

Waller

Tremelling

Bredin

Godfrey

Howson

Parkes

. Evidence for association of OCTN genes and IBD5 with ulcerative colitis. Gut. 2006;55(6):809–814.

22.

Hartman

PE.

Ergothioneine as antioxidant. Oxygen radicals in biological systems. Part B: oxygen radicals and antioxidants. Meth Enzymol. 1990;186:310–318.

23.

Chaudiere

Ferrari-Iliou

. Intracellular antioxidants: from chemical to biochemical mechanisms. Food Chem Toxicol. 1999;37(9-10):949–962.

24.

Arouma

Spencer

JPE

Mahmood

. Protection against oxidative damage and cell death by the natural antioxidant ergothioneine. Food Chem Toxicol. 1999;37(11):1043–1053.

25.

Schömig

Taubert

. Dietary sources and antioxidant effects of ergothioneine. J Agric Food Chem. 2007;55(16):6466–6474.

26.

Paul

Snyder

. The unusual amino acid l-ergothioneine is a physiologic cytoprotectant. Cell Death Different. 2010;17(7):1134–1140.

27.

Kato

Kubo

Iwata

. Gene knockout and metabolome analysis of carnitine/organic cation transporter OCTN1. Pharm Res. 2010;27(5):832–840.

28.

Gründemann

Harlfinger

Golz

. Discovery of the ergothioneine transporter. Proc Nat Acad Sci U S A. 2005;102(14):5256–5261.

29.

Epand

Wong

. Study of the ergothioneine concentration in the blood of individuals with diabetes mellitus. J Clin Chem Clin Biochem. 1988;26(10):623–626.

30.

Weigand-Heller

Kris-Etherton

Beelman

. The bioavailability of ergothioneine from mushrooms (Agaricus bisporus) and the acute effects on antioxidant capacity and biomarkers of inflammation. Prev Med. 2012;(suppl 54):S75–S78.

31.

Zembron-lacny

Gajewski

Naczk

Siatkowski

. Effect of shiitake (Lentinus edodes) extract on antioxidant and inflammatory response to prolonged eccentric exercise. J Physiol Pharmacol. 2013;64(2):249–254.

32.

Sotgia

Zenellu

Mangoni

. Clinical and biochemical correlates of serum l-ergothioneine concentrations in community-dwelling middle-aged and older adults. PloS One. 2014;9(1):e84918.

33.

Obayashi

Kurihara

Okano

Masaki

Yarosh

. l-Ergothioneine scavenges superoxide and singlet oxygen and suppresses TNF-α and MMP-1-expression in UV-irradiated human dermal fibroblasts. J Cosmet Sci. 2005;56(1):17–27.

34.

Dong

Damaghi

Kibitel

Canning

Smiles

Yarosh

. A comparison of the relative antioxidant potency of l-ergothioneine and idebenone. J Cosmet Dermatol. 2007;6(3):183–188.

35.

Hseu

Korivi

. Dermato-protective properties of ergothioneine through induction of Nrf2/ARE-mediated antioxidant genes in UVA-irradiated human keratinocytes. Free Rad Biol Med. 2015;86(September, 2015):102–117.

36.

Tainter

. Notes on the pharmacology of l-ergothioneine. Proc Coc Exp Biol Med. 1926;24:621.

37.

Hartman

. Interception of toxic agents/mutagens/carcinogens: some of nature’s novel strategies. In: Shankel

Hartman

Kada

, eds. Antimutagenesis and Anticarcinogenesis Mechanisms. New York, NY: Plenum Press; 1986:169–179.

38.

Hartman

. Interception of some directing acting mutagens by l-ergothioneine. Environ Mol Mutagen. 1987;10(1):3–15.

39.

Hartman

Citardi

. Ergothioneine, histidine, and two naturally occurring histidine dipeptides as radioprotectors against γ-Irradiation inactivation of bacteriophages T4 and P22. Rad Res. 1988;114(2):319–330.

40.

Heath

Rimington

Searle

Lawson

. Some effects of administering ergothioneine to rats. Biochem J. 1952;50(4):530–533.

41.

Deiana

Rosa

Casu

Piga

Dessi

Aruoma

. l-Ergothioneine modulates oxidative damage in the kidney and liver of rats in vivo: studies upon the profile of polyunsaturated fatty acids. Clin Nutr. 2004;23(2):183–193.

42.

Schauss

Vértesi

Endres

. Evaluation of the safety of the dietary antioxidant ergothioneine using the bacterial reverse mutation assay. Toxicology. 2010;278(1):39–45. doi:10.1016/j.tox.2010.07.015.

43.

Schauss

Béres

Vértesi

. The effect of ergothioneine on clastogenic potential and mutagenic activity: genotoxicity evaluation. Int J Toxicol. 2011;30(4):405–409. doi:10.1177/1091581811405856.

44.

Forster

Spézia

Papineau

. Reproductive safety evaluation of l-ergothioneine. Food Chem Toxicol. 2015;80:85–91. doi:10.1016/j.fct.2015.02.019.

45.

Ramirez-Martinez

Wesolek

Yadan

Moutet

Roudot Professor

. Intake assessment of l-ergothioneine in some European countries and in the United States [published online October 29, 2015]. Human Ecol Risk Assess. 2015. Web site. http://www.researchgate.net/publication/283118982. Accessed December 17, 2015. doi:10.1080/10807039.2015.1104241.

46.

Dubost

Beelman

Peterson

Royse

. Identification and quantification of ergothioneine in cultivated mushrooms by liquid chromatography–mass spectroscopy. Int J Med Mush. 2006;8(3):215–222.

47.

Dubost

Beelman

. Quantification of polyphenols and ergothioneine in cultivated mushrooms and correlation to total antioxidant capacity. Food Chem. 2007;105(2):727–735.

48.

Nguyen

Giri

Ohshima

. A rapid HPLC post-column analysis for the quantification of ergothioneine in edible mushrooms and in animals fed a diet supplemented with extracts from the processing waste of cultivated mushrooms. Anal Meth. 2012;133(2):585–591.

49.

Office of Economic Cooperative Development. Guidelines for the Testing of Chemicals, Section 4. No. 408, “Repeated Dose 90-Day Oral Toxicity Study in Rodents”. 1998.

50.

Food and Drug Administration. GLP Regulations (Management Briefings, 8/79) and GLP Regulations (Questions/Answers, 6/81). Post conference report. Center for Drug Evaluation and Research. Special Interest Topic. 1979.

51.

Food and Drug Administration. Code of Federal Regulations (CFR), 1987. 21 CFR58 Good laboratory practice for nonclinical laboratory studies. Revised April 2015.

52.

Office of Economic Cooperative Development. Ninth Addendum to OECD Guidelines for the Testing of Chemicals, Section 4, No. 471, “Bacterial Reverse Mutation Test”. 1997.

53.

Office of Economic Cooperative Development. Guidelines for the Testing of Chemicals, Section 4. No. 407, “Repeated Dose 28-Day Oral Toxicity Study in Rodents”. 2008.

54.

Environmental Protection Agency (EPA). Health Effects Test Guidelines, OPPTS 870.5100 Bacterial Reverse Mutation Assay EPA 712-C-98-247. 1998.

55.

National Research Council. Guide for the Care and Use of Laboratory Animals. 8th ed. Washington, DC: National Academies Press; 2011.

56.

Ames

Durston

Yamasaki

Lee

. Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proc Natl Acad Sci U S A. 1973;70(8):2281–2285.

57.

Ames

Lee

Durston

. An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc Natl Acad Sci. 1973;70(8):782–786.

58.

Maron

Ames

. Revised methods for the Salmonella mutagenicity test. Mutat Res. 1983;113(3-4):173–215.

59.

Kawano

Otani

Takeyama

Kawai

Mayumi

Hama

. Studies on ergothioneine. VI. Distribution and fluctuations of ergothioneine in rats. Chem Pharm Bull. 1982;30(5):1760–1765.

60.

Davit

Braddy

Dale

Conner

. International guidelines for bioequivalence of systemically available orally administered generic drug products: a survey of similarities and differences. AAPS J. 2013;15(4):974–990.

61.

Mackenzie

. The effect of age, sex and androgen on blood ergothioneine. J Biol Chem. 1957;225(2):651–657.

62.

Kumosani

. l-ergothioneine level in red blood cells of healthy human males in the western province of Saudi Arabia. Exp Mol Med. 2001;33(1):20–22.

63.

Alnouti

Petrick

Klaassen

. Tissue distribution and ontogeny of organic cation transporters in mice. Drug Met Dispos. 2006;34(3):477–482.