A 90-day preclinical toxicological evaluation in rats of a highly purified and concentrated mulberry leaf extract.

Mulberry (genus Morus) leaves have long been used as a human food, especially in Asia, and animal feed. More recently, mulberry leaf extracts have been introduced as a convenient way to consume mulberry for non-nutritional functional effects. Reducose® 5% is an Morus alba leaf extract that has been highly purified and standardized to a content of 5 ± 0.5% 1-deoxynojirimycin, a naturally present polyhydroxylated piperidine alkaloid analog of D-glucose. This extract has previously been evaluated in acute and subacute (28-day) oral toxicity studies in which no adverse effects of the test item were observed in mice or rats, respectively. Due to continued and growing interest in the extract in multinational markets, we have now further investigated potential toxic effects in subchronic (90-day) oral toxicity study in male and female Han:WIST rats. The test item was administered at doses of 850, 1700, and 2550 mg/kg bw/day, and did not cause adverse effects in clinical signs, body weight development, clinical pathology, gross pathology, or histopathology in comparison to the vehicle-control group. The no-observed-adverse-effect-level was determined to be 2550 mg/kg bw/day. These results add to the existing body of both preclinical and clinical work relevant to the safety of the extract and of interest to regulators in various global markets.

An evaluation of the genotoxicity and 90-day repeated-dose toxicity of a CBD-rich hemp oil.

Currently, there is much interest in the sales and study of consumable Cannabis sativa L. products that contain relatively high levels of cannabidiol (CBD) and low levels of Δ-9-tetrahydrocannabinol. While there are published safety evaluations for extracts containing low concentrations of CBD, toxicological assessments for those with higher concentrations are still scant in the public domain. In this paper, genotoxicity tests and a 90-day repeated-dose toxicity study of an ethanolic extract of C. sativa containing ~85% CBD were performed following relevant OECD guidelines. No increased gene mutations were observed in a bacterial reverse mutation assay compared to controls up to the maximum recommended concentration of the guideline. An in vitro chromosomal aberration assay showed no positive findings in the short-term (3 h) treatment assays. Long-term treatment (20 h) showed an increased number of cells containing aberrations at the highest dose of 2 µg/mL, which was outside of historical control levels, but not statistically significantly different from the controls. An in vivo micronucleus study showed no genotoxic potential of the test item in mice. A 90-day repeated-dose gavage study using 0, 75, 125, and 175 mg/kg bw/day showed several slight findings that were considered likely to be related to an adaptive response to consumption of the extract by the animals but were not considered toxicologically relevant. These included increases in liver and adrenal weights compared to controls. The NOAEL was determined as 175 mg/kg bw/day, the highest dose tested (equivalent to approximately 150 mg/kg bw/day of CBD).

A 90-day toxicity study of tripeptide, arginine-alanine-lysine.

There is a growing global interest in using peptides in the health industry for pharmaceuticals, cosmetics, and natural food products. Peptides contain two or more linked amino acids, whereas more than 50 amino acids are classified as polypeptides. Although there is a growing level of interest in the use of peptides in the health and wellness industry, there is a lack of literature pertaining to a specific tripeptide derived from arginine, alanine, and lysine (RAK) that is of interest for human dietary use. Therefore, a 90-day repeated-dose toxicity study was performed in rats to evaluate the subchronic oral toxicity of RAK. Eighty Han:WIST rats were administered RAK by gavage at doses of 0, 250, 500, or 1000 mg/kg bw/day. There were no mortalities or other treatment related effects, and no target organs were identified. A no-observed-adverse-effect-level (NOAEL) of 1000 mg/kg bw/day, the highest dose tested, was determined. This study will contribute to the body of research in regard to the safety of the use of RAK.

Slaughterhouse Visual and Palpation Method for Estimating the Economic Damage of Porcine Proliferative Enteropathy (PPE).

BACKGROUND: Ileitis is a wasting disease of pigs. Clinical symptoms are diarrhea in growing pigs, wasting and reduced performance. Ileitis is ubiquitous in pig producing countries all around the world. It is estimated that the economic losses caused by the disease are USD 4.65 per fattening pig, and American pig farmers lose USD 56.1 million annually. It has become necessary to develop a slaughterhouse ileitis monitoring method that is simple, feasible to perform at modern slaughter lines, leads to immediate results and is cost effective. The practical experiences of applying the method are presented below. METHODS: Our studies were performed on pig herds and slaughterhouses in Central European countries (Hungary, Romania, Poland, Croatia, and Slovakia). Experiences were evaluated based on visual and palpation. The results of our investigations were evaluated by a scoring method. Authors made histological and immunohistochemical examinations of investigated ileums. The hypothetic economic losses due to the disease in each farm were determined by estimating the loss of profit according to Holtkamp's presentation in 2019. RESULTS: The essentials of the method we have developed are: it can be performed during slaughterhouse processing, it does not interfere with or make it impossible to carry out normal technological processes, and the results of the procedure are correlated with the results of other laboratory diagnostic tests for ileitis (histology, immunohistochemistry, herd serology, fecal PCR). It is noteworthy that the results of the method can be used to immediately estimate the impact of Lawsonia intracellularis infection on the performance of the herd from which the slaughter animals come. CONCLUSION: Using the results of the slaughtered pigs' visual and tactile examination at the slaughterhouse, the magnitude of the loss caused by Lawsonia intracellularis infection can be estimated quickly and accurately, and the return on investment of the strategy to be applied can be accurately planned.

Toxicological evaluation of protein powder derived from Cupriavidus necator.

Microorganisms have the potential to produce nutrient-rich products that can be consumed as food or feed. The protein-rich powder derived from heat treatment of the whole-cell biomass of polyhydroxybutyrate-deficient Cupriavidus necator, a metabolically versatile organism that uses elements found in the air, is an example of such a product. To assess the safety of the protein powder for use as a nutritional ingredient in human food, in accordance with internationally accepted standards, its genotoxic potential and repeated-dose oral toxicity were investigated. A bacterial reverse mutation test, an in vitro mammalian chromosomal aberration test, and an in vivo mammalian micronucleus test were performed. No evidence of mutagenicity or genotoxicity was found. Additionally, a 90-day repeated-dose oral toxicity study in rats was completed, in which a total of 100 male and female Wistar rats were exposed by gavage to daily doses of 1000, 2000, or 3000 mg/kg bw/day of the test material. Following 90 days of continuous exposure, no mortality or treatment-related adverse effects were observed and no target organs were identified. Therefore, a no observed adverse effect level was determined at 3000 mg/kg bw/day, the highest dose tested.

Toxicology and digestibility of Chlamydomonas debaryana green algal biomass.

There is an economic interest, both for food security and for the non-meat-eating population, in the development of novel, sustainable sources of high-quality protein. The green algae Chlamydomonas reinhardtii has already been developed for this purpose, and the closely related species, Chlamydomonas debaryana, is a complementary source that also presents some additional advantages, such as reduced production cost. To determine whether C. debaryana may have a similar safety profile to that of C. reinhardtii, a wild type strain was obtained, designated TS04 after confirmation of its identity, and subjected to a battery of preclinical studies. Genetic toxicity was evaluated using a bacterial reverse mutation test, an in vitro mammalian chromosomal aberration test, and an in vivo mammalian micronucleus test in a mouse model. No genotoxic potential (e.g., mutagenicity and clastogenicity) was observed in these tests under the employed conditions up to maximum recommended concentrations or doses. To assess general toxicity, a 90-day repeated-dose oral toxicity study was conducted in rats. No mortality or adverse effects were observed, and no target organs were identified up to the maximum feasible dose, due to solubility, of 4,000 mg/kg bw/day. The no-observed-adverse-effect level was determined as the highest dose tested. A digestibility study in simulated gastric fluid was conducted and determined that TS04 has low allergenic potential, exhibiting rapid digestion of proteins. Due to the negative results of our evaluation, it is reasonable to proceed with further development and additional investigations to contribute towards a safety assessment of the proposed use in food for human consumption.

Safety evaluation of Veillonella atypica FB0054 with genotoxicity and subchronic toxicological studies.

Veillonella atypica is a nonmotile, nonsporulating anaerobic bacteria commonly found in various human biofilms. V. atypica FB0054 was isolated from the gastrointestinal tract of marathon runners, who have increased amounts of this species after athletic events. Interestingly, the consumption of this strain by rodents has been shown to increase their treadmill endurance, leading to the hypothesis that consumption of this species may improve athletic performance in humans as well. Further evaluation, in humans, of the usefulness of this strain should be preceded by safety studies. Therefore, the genotoxic and subchronic toxicological potential was evaluated as a contribution to this effort. Genotoxicity investigation was performed using the in vivo comet assay and in vivo mammalian micronucleus assay due to the anaerobic characteristic of the strain. A 90-day, repeated-dose oral toxicity study was performed in rats up to 2200 mg/kg bw/d to investigate general toxicity and identify any target organs. Mitsuoka buffer, a solution shown to preserve the viability of anaerobic bacteria, was used as the vehicle. All three studies revealed no toxicological effects from exposure to FB0054 was isolated from the gastrointestinal tract of marathon runners, who have increased amounts of this species after athletic events. Interestingly, the consumption of this strain by rodents has been shown to increase their treadmill endurance, leading to the hypothesis that consumption of this species may improve athletic performance in humans as well. Further evaluation, in humans, of the usefulness of this strain should be preceded by safety studies. Therefore, the genotoxic and subchronic toxicological potential was evaluated as a contribution to this effort. Genotoxicity investigation was performed using the in vivo comet assay and in vivo mammalian micronucleus assay due to the anaerobic characteristic of the strain. A 90-day, repeated-dose oral toxicity study was performed in rats up to 2200 mg/kg bw/d to investigate general toxicity and identify any target organs. Mitsuoka buffer, a solution shown to preserve the viability of anaerobic bacteria, was used as the vehicle. All three studies revealed no toxicological effects from exposure to FB0054 at the highest doses tested.

A toxicological evaluation of 8-28 nm gold nanocrystals.

Gold nanocrystals (AuNC) are gold nanoparticles (AuNP) relatively homogenous in size at 8-28 nm with clean surfaces and crystalline structures. There are concerns and a lack of consensus in the scientific literature and major regulatory bodies regarding not only the safety of nanoparticles when consumed by humans, but exactly how to determine their safety and whether evidence from a nanoparticle with one set of physiochemical properties extends to one with a different set. Additionally, there are few general long-term toxicity data on AuNP. To our knowledge, the potential toxicity of AuNC specifically, with the above characteristics, or otherwise, has not been investigated in preclinical studies; thus, we conducted a battery of genetic toxicity tests and an oral repeated-dose toxicity test to further explore their safety. AuNC were not mutagenic or clastogenic in bacterial reverse mutation and in vitro mammalian chromosomal aberration tests, respectively, and did not exhibit in vivo genotoxicity in a micronucleus test in mice. In a 60-day, repeated-dose oral toxicity study, rats were administered 0, 2.5, 5, or 10 mg/kg bw/day of AuNC by gavage. No toxicity was identified. Therefore, a no observed adverse effect level was determined as 10 mg/kg body weight/day.

Evaluation of the genotoxic potential of protoporphyrin IX and the safety of a protoporphyrin IX-rich algal biomass.

Chlamydomonas reinhardtii is a nonpathogenic, nontoxigenic green algae used as a sustainable source of protein in foods. In order to mimic meat-like qualities, a strain rich in protoporphyrin IX (PPIX), an endogenous heme/chlorophyll precursor, was developed using an evolution and selection strategy, and investigations were carried out to evaluate the safety of the novel strain, C. reinhardtii (red), strain TAI114 (TAI114). Digestibility and proteomic evaluations were conducted to determine whether any potentially allergenic or toxic proteins occurred as the result of the mutation process. The genotoxic potential of pure PPIX was evaluated using a bacterial reverse mutation test, an in vitro mammalian chromosomal aberration test, and an in vivo mammalian micronucleus test. Finally, the novel TAI114 biomass was evaluated for general toxicity and identification of target organs in a 90-day repeated-dose oral toxicity study in rats. All proteins were rapidly degraded in pepsin at pH 2.0 suggesting low allergenic potential. The proteomic evaluation indicated that TAI114 biomass contains typical C. reinhardtii proteins. PPIX was unequivocally negative for genotoxic potential and no target organs or adverse effects were observed in rats up to the maximum feasible dose of 4000 mg/kg bw/day TAI114 biomass, which was determined to be the no-observed-adverse-effect-level (NOAEL). These results support the further development and risk characterization of TAI114 biomass as a novel ingredient for use in the meat analogue category of food.

A toxicological evaluation of lithium orotate.

Lithium orotate, the salt of lithium and orotic acid, has been marketed for decades as a supplemental source of lithium with few recorded adverse events. Nonetheless, there have been some concerns in the scientific literature regarding orotic acid, and pharmaceutical lithium salts are known to have a narrow therapeutic window, albeit, at lithium equivalent therapeutic doses 5.5-67 times greater than typically recommended for supplemental lithium orotate. To our knowledge, the potential toxicity of lithium orotate has not been investigated in preclinical studies; thus, we conducted a battery of genetic toxicity tests and an oral repeated-dose toxicity test in order to further explore its safety. Lithium orotate was not mutagenic or clastogenic in bacterial reverse mutation and in vitro mammalian chromosomal aberration tests, respectively, and did not exhibit in vivo genotoxicity in a micronucleus test in mice. In a 28-day, repeated-dose oral toxicity study, rats were administered 0, 100, 200, or 400 mg/kg body weight/day of lithium orotate by gavage. No toxicity or target organs were identified; therefore, a no observed adverse effect level was determined as 400 mg/kg body weight/day. These results are supportive of the lack of a postmarket safety signal from several decades of human consumption.

A toxicological evaluation of geranylgeraniol.

Geranylgeraniol (GGOH) is an isoprenoid compound found in annatto seeds and an intermediate of the mevalonate pathway found within organisms serving various functions. Toxicological studies on its safety profile are not readily available. To assess the safety of GGOH, a molecularly distilled, food grade annatto oil, consisting of approximately 80% trans-GGOH, was subjected to a bacterial reverse mutation test, an in vitro mammalian chromosomal aberration test, and an in vivo mammalian micronucleus test in order to investigate its genotoxic potential and a 90-day repeated-dose oral toxicity study in rats in order to investigate its potential subchronic toxicity and identify any target organs. No evidence of mutagenicity or genotoxic activity was observed under the applied test systems. In the 90-day study, male and female Hsd. Han Wistar rats were administered daily doses of 0, 725, 1450, and 2900 mg/kg bw/day by gavage. Treatment-related adverse effects were observed in the forestomach at all dose levels and in the liver at the intermediate- and high-dose levels. Based on these results, the lowest observed adverse effect level (LOAEL) for local effects and the no observed adverse effect level (NOAEL) for systemic effects were determined as 725 mg/kg bw/day.

Assessment of toxicological potential of sodium carboxymethyl beta-glucan, a novel beta-glucan.

In this experimental work, sodium carboxymethyl beta-glucan (CMBG), a chemically altered beta-glucan, is evaluated for mutagenicity and sub-acute oral toxicity. Specifically, the tested material was CM-Glucan Nu, a food grade powder ≥90% CMBG derived from Saccharomyces cerevisiae. A bacterial reverse mutation test was performed and resulted in no mutagenicity. A 28-day, repeated-dose, oral (gavage) toxicity test on rats was performed at dose levels of 0, 500, 1000, and 2000 mg/kg bw/day. No mortality, target organs or other treatment related effects were observed. The no observed adverse effect level (NOAEL) was 2000 mg/kg bw/day, the highest dose tested, for both male and female Han:WIST rats.

A toxicological evaluation of a fulvic and humic acids preparation.

Humic substances are ubiquitous in soils and waters. These complex superstructures are derived from the decomposition of dead plant and animal matter and are vital to soil health. Their heterogenous composition is specific to their site of origin and is comprised of weakly bound aggregates of small organic compounds that can sequester minerals and make them available to plants. As such, they may possess potential nutritional value for humans, and extractions of fulvic and humic acids can be produced that could be suitable for such purposes. For this reason, we evaluated the toxicological profile of a specific preparation (blk. 333) of fulvic and humic acids derived from a lignite deposit in Alberta, Canada and found it to lack genotoxic potential in a bacterial reverse mutation test, in vitro mammalian chromosomal aberration test, and in vivo mammalian micronucleus test. No general or organ toxicity was observed in Wistar rats following 90 days of continuous exposure, and a no observed adverse effect level (NOEAL) was determined at 2000 mg/kg bw/day, the highest tested dose. Our results suggest the feasibility of further evaluation for development of the preparation as a nutritional supplement in food.

A toxicological evaluation of monomethylsilanetriol (MMST) stabilized in acacia gum, a novel silicon preparation.

Monomethylsilanetriol (MMST), a silicon-containing compound, has been sold in dietary supplements. However, toxicological studies on its safety profile are not readily available. To assess the safety of MMST stabilized in acacia gum, a novel delivery form of MMST, in accordance with internationally accepted standards, the genotoxic potential and repeated-dose oral toxicity of Living Silica® Acacia Gum Stabilized Monomethylsilanetriol (formerly known as Orgono Acacia Gum Powder®), a food grade product consisting of 80 ± 10% acacia gum and 2.8% (SD ± 10%) elemental silicon from MMST, was investigated. A bacterial reverse mutation test, an in vitro mammalian chromosomal aberration test, an in vivo mammalian micronucleus test, and a 90-day repeated-dose oral toxicity study in rats were performed. No evidence of mutagenicity or genotoxic activity was observed under the applied test systems. In the 90-day study, male and female Hsd.Han Wistar rats were administered daily doses of 0, 500, 1000, and 2000 mg/kg bw/day by gavage. No mortality or treatment-related adverse effects were observed, and no target organs were identified. Therefore, the no observed adverse effects level (NOAEL) was determined as 2000 mg/kg bw/day (201 mg MMST/kg bw/day), the highest dose tested.

Prenatal developmental toxicity study of an alkaloid-free Ageratum conyzoides extract powder in rats by oral administration.

A prenatal developmental toxicological study was conducted to evaluate the safety of an alkaloid-free Ageratum conyzoides extract powder administration on pregnant female Wistar rats and on the development of the conceptus in accordance with OECD test guideline (no. 414). Pyrrolizidine alkaloids (PAs) naturally present in A. conyzoides have been shown to induce toxicity in past studies, particularly towards hepatic cells. Therefore our test item preparation of A.conyzoides extract (aerial part of the plant) consisted of the removal of PAs. There were no treatment related adverse effects found during maternal examinations (body weights, food consumption, numbers of pregnant and non-pregnant female rats, endocrine evaluation, gravid uterine weights, and number of corpora lutea), maternal/fetal examinations (numbers of implantation sites, pre-and post-implantation loss (%), dead and live fetuses (%), resorption sites), or fetal examinations (litter size and weights, number of fetuses, sex ratio, or external, visceral, and skeletal variations and malformations) in the Ageratum conyzoides extract powder groups at doses of 500, 1000 and 2000 mg/kw bw/day compared to vehicle control group. The no observed adverse effect level (NOAEL) determined for both maternal and developmental toxicity was 2000 mg/kg bw/day, which was the highest dose tested.

A Toxicological Evaluation of Germanium Sesquioxide (Organic Germanium).

A battery of OECD- and GLP-compliant toxicological studies was performed to assess the safety of a highly purified germanium sesquioxide, an organic form of the naturally occurring, nonessential trace element germanium. Germanium dioxide and germanium lactate citrate (inorganic germaniums) have been shown to induce renal toxicity, whereas germanium sesquioxide (an organic germanium) has been shown to have a more favorable safety profile. However, past toxicity studies on germanium sesquioxide compounds have not clearly stated the purity of the tested compounds. In the studies reported herein, there was no evidence of mutagenicity in a bacterial reverse mutation test or an in vitro mammalian chromosomal aberration test. There was no genotoxic activity observed in an in vivo mammalian micronucleus test at concentrations up to the limit dose of 2000 mg/kg bw/day. In a 90-day repeated-dose oral toxicity study in Han:WIST rats conducted at doses of 0, 500, 1000, and 2000 mg/kg bw/day by gavage, there were no mortalities, treatment-related adverse effects, or target organs identified. The no-observed-adverse-effect-level (NOAEL) was determined to be 2000 mg/kg bw/day.

Porcine Hepatic Response to Fumonisin B1 in a Short Exposure Period: Fatty Acid Profile and Clinical Investigations.

Scarce studies have investigated the impact of fumonisin B1 (FB1) on the hepatic tissue fatty acid (FA) profile, and no study is available on piglets. A 10-day in vivo experiment was performed on seven piglets/group: control and FB1-fed animals (diet was contaminated with fungal culture: 20 mg FB1/kg diet). Independent sample t-test was carried out at p < 0.05 as the significance level. Neither growth, nor feed efficiency, was affected. The hepatic phospholipid (PL) fatty acids (FAs) were more susceptible for FB1, while triglyceride (TG) was less responsive. The impact of FB1 on hepatic PL polyunsaturated fatty acids (PUFAs) was more pronounced than on saturated fatty acids. Among all PUFAs, predominant ones in response were docosapentaenoicacid (DPA) (↓), docosahexaenoic DHA (↓) and arachidonic acids (↑). This led to a higher omega-6:omega-3 ratio, whereas a similar finding was noted in TGs. Neither total saturation (SFA) nor total monousaturation (MUFA) were affected by the FB1 administration. The liver showed an increase in malondialdehyde, as well as antioxidant capacity (reduced glutathione and glutathione peroxidase). The plasma enzymatic assessment revealed an increase in alkaline phosphatase (ALP), while alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH), and gamma-glutamyltransferase (GGT) were not influenced. The microscopic sections provided evidence of vacuolar degeneration of the hepatocytes' cytoplasm, but it was not severe. Furthermore, the lung edema was developed, while the kidney was not affected. In conclusion, regarding FB1-mediated hepatotoxicity in piglets, the potential effect of slight hepatotoxicity did not compromise growth performance, at least at the dose and exposure period applied.

A Toxicological Evaluation of Mango Leaf Extract (Mangifera indica) Containing 60% Mangiferin.

A battery of OECD- and GLP-compliant toxicological studies was performed on mango leaf extract (Mangifera indica) containing 60% mangiferin (MLE). No evidence of genotoxicity was found in a bacterial reverse mutation test (Ames). While evidence of clastogenic activity was noted in an in vitro chromosomal aberration test, an in vivo mammalian micronucleus test showed no findings up to the limit dose (2000 mg/kg bw). A 90-day repeated dose oral toxicity study was conducted in rats using doses of 0 (vehicle control), 500, 1000, and 2000 mg/kg bw/day. Based on the lack of mortality or toxic effects in the 90-day study, the NOAEL for MLE in Han:Wist male and female rats was determined to be 2000 mg/kg bw/day, the highest dose tested.

Toxicological Evaluation of a Mixture of Astragalus membranaceus and Panax notoginseng Root Extracts (InnoSlim®).

Astragalus spp. and Panax spp. have a long history of traditional human use. A blend, InnoSlim®, of highly purified and fractionated root extracts from Astragalus membranaceus and Panax notoginseng has now been developed for human consumption; however, the unique constituent content of this blend has not been specifically evaluated with respect to safety. Therefore, the toxicological potential of the blend was formally investigated in a series of studies-genetic toxicity was evaluated in a bacterial reverse mutation test followed by an in vivo mammalian micronucleus test, and general toxicity was evaluated in a 28-day repeated-dose oral toxicity study in rats. No evidence of mutagenicity was observed in the bacterial tester strains used, and no evidence of in vivo chromosomal damage resulting in increased frequency of micronucleated cells was observed in male Crl:NMRI BR mice. No mortality or toxic effects were observed, and no target organs were identified, in male and female Han:WIST rats exposed to 0, 400, 800, or 1200 mg/kg bw/day of the blend by gavage for 28 consecutive days. The highest dose-1200 mg/kg bw/day-was determined to be the NOAEL. Based on these results, extrapolation towards a safe human consumption level can be explored.

Toxicological evaluations of colostrum ultrafiltrate.

Colostrum has been consumed safely for many years as a food collected directly from cows. More recently, an ultrafiltrated bovine colostrum product has been developed; however, its safety in toxicology studies has not been extensively evaluated. To assess the safety of bovine colostrum ultrafiltrate, in accordance with internationally accepted standards, the genotoxic potential was investigated in a bacterial reverse mutation test, an in vitro chromosomal aberration test, and an in vivo mammalian micronucleus test. No mutagenicity or genotoxic activity was observed in these three tests. A 90-day repeated-dose oral toxicity study in Hsd.Han Wistar rats was conducted at doses of 0, 1050, 2100, and 4200 mg/kg bw/day by gavage. After 90 days of continuous exposure, no mortality or treatment-related adverse effects were observed, and no target organs were identified. The no-observed-adverse-effect level (NOAEL) was determined to be 4200 mg/kg bw/day, the highest dose tested.