Reduced genotoxicity of lignin-derivable replacements to bisphenol A studied using in silico, in vitro, and in vivo methods.

Bisguaiacols, lignin-derivable bisphenols, are considered promising and possibly safer alternatives to bisphenol A (BPA), but comprehensive toxicity investigations are needed to ensure safety. Most toxicity studies of BPA and its analogues have focused on potential estrogenic activity, and only limited toxicological data are available on other toxicity aspects, such as genotoxicity at low exposure levels. In this study, the genotoxicity of six lignin-derivable bisguaiacols with varying regioisomer contents and degrees of methoxy substitution was investigated using a multi-tiered method, consisting of in silico simulations, in vitro Ames tests, and in vivo comet tests. The toxicity estimation software tool, an application that predicts toxicity of chemicals using quantitative structure-activity relationships, calculated that the majority of the lignin-derivable bisguaiacols were non-mutagenic. These results were supported by Ames tests using five tester strains (TA98, TA100, TA102, TA1535, and TA1537) at concentrations ranging from 0.5 pmol/plate to 5 nmol/plate. The potential genotoxicity of bisguaiacols was further evaluated using in vivo comet testing in fetal chicken livers, and in addition to the standard alkaline comet assay, the formamidopyrimidine DNA glycosylase enzyme-modified comet assay was employed to investigate oxidative DNA damage in the liver samples. The oxidative stress analyses indicated that the majority of lignin-derivable analogues showed no signs of mutagenicity (mutagenic index < 1.5) or genotoxicity, in comparison to BPA and bisphenol F, likely due to the methoxy groups on the lignin-derivable aromatics. These findings reinforce the potential of lignin-derivable bisphenols as safer alternatives to BPA.

Formation of pure nanoparticles with solvent-fractionated lignin polymers and evaluation of their biocompatibility.

This study aimed to determine the effects of lignin characteristics (mainly molecular weight, functional groups, and internal linkages) on nanoparticle formation. First, five different lignin fractions (Mw 1460-12,900) were obtained from commercial kraft lignin (KL) by sequential solvent extraction. Functional groups and internal linkages were determined in lignin fractions, each fraction consisting of different levels and ratios. Second, spherical lignin nanoparticles (i.d. 193-1039 nm) were synthesized by nanoprecipitation at different pre-dialysis concentrations (1, 2, 4, and 6 mg mL-1 THF) with the different fractions (F1, F2, F3, F4, and F5). The study revealed that larger particles consisted of lignin fractions of lower molecular weight and higher phenolic group content (KL-F1 and F2), while smaller but non-uniform particles were produced from fractions of higher molecular weight and lower phenolic group content (KLF4 and F5). Every zeta potential value of the particle exceeded -35 mV. The nanoparticles from raw kraft lignin exhibited no significant cytotoxicity, hemotoxicity, and hypersensitivity. This study revealed that molecular weight and hydroxyl group content in the lignin highly correlated with nanoparticle properties. The present kraft lignin nanoparticles have potential for use in various polymer-based nanotechnology.

Safety aspects of kraft lignin fractions: Discussions on the in chemico antioxidant activity and the induction of oxidative stress on a cell-based in vitro model.

Lignin is a complex phenolic biopolymer present in plant cell walls and a by-product of the cellulose pulping industry. Lignin has functional properties, such as antioxidant activity, that make it a potential natural active ingredient for health-care products. However, not all safety aspects of lignin fractions have been adequately investigated. Herein, we evaluated the antioxidant and genotoxic potential of two hardwood kraft lignins (F3 and F5). The chemical characterization of F3 and F5 demonstrated their thermal stability and the presence of different phenolic units, while the DPPH assay confirmed the antioxidant activity of these lignin fractions. Despite being antioxidants in the DPPH assay, F3 and F5 were capable of generating intracellular reactive oxygen species (ROS) and subsequently causing oxidative DNA damage (Comet assay) in HepG2 cells. The biological relevance of the DPPH assay might be uncertain in some cases; therefore, we suggest combining in chemico tests with biological system-based tests to determine efficacy and safety levels of lignins and define appropriate applications of lignins for consumer products. Moreover, kraft lignins obtained by acid precipitation may pose risks to human health; however, as genotoxicity is not the sole endpoint of toxicity required in hazard assessments, additional toxicological evaluations are needed.

Lignin-based fluorescence hollow nanoparticles: Their preparation, characterization, and encapsulation properties for doxorubicin.

Lignin shows strong adsorption, biodegradability and non-toxicity, and has opened a research hotspot in the design and manufacture of controllable nanomaterials for drug delivery. However, lignin-based materials, with both diagnostic and therapeutic functions, have yet to be developed. In this work, enzymatically hydrolysable lignin (EHL) was used to prepare blue fluorescent lignin copolymer by grafting 1-Pyrenebutyric acid onto lignin via amidation reaction and then formed self-assembled nanoparticles. The results show that such lignin-based hollow nanoparticles exhibit characteristics of fluorescent functions, size controlled and stable structure within 15 days. For anticancer drug Doxorubicin, the encapsulation efficiency and drug loading reached, respectively, 50% and 10%. This encapsulation had no cytotoxicity, and sustained-release effect on the drug. The aim of this study was to develop the multifunctional bio-nanomaterials for medical applications, through simple, environmentally friendly, low-cost methods.

Systematic in vitro biocompatibility studies of multimodal cellulose nanocrystal and lignin nanoparticles.

Natural biopolymer nanoparticles (NPs), including nanocrystalline cellulose (CNC) and lignin, have shown potential as scaffolds for targeted drug delivery systems due to their wide availability, cost-efficient preparation, and anticipated biocompatibility. As both CNC and lignin can potentially cause complications in cell viability assays because of their ability to scatter the emitted light and absorb the assay reagents, we investigated the response of bioluminescent (CellTiter-Glo®), colorimetric (MTT® and AlamarBlue®), and fluorometric (LIVE/DEAD®) assays for the determination of the biocompatibility of the multimodal CNC and lignin constructs in murine RAW 264.7 macrophages and 4T1 breast adenocarcinoma cell lines. Here, we have developed multimodal CNC and lignin NPs harboring the radiometal chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid and the fluorescent dye cyanine 5 for the investigation of nanomaterial biodistribution in vivo with nuclear and optical imaging, which were then used as the model CNC and lignin nanosystems in the cell viability assay comparison. CellTiter-Glo® based on the detection of ATP-dependent luminescence in viable cells revealed to be the best assay for both nanoconstructs for its robust linear response to increasing NP concentration and lack of interference from either of the NP types. Both multimodal CNC and lignin NPs displayed low cytotoxicity and favorable interactions with the cell lines, suggesting that they are good candidates for nanosystem development for targeted drug delivery in breast cancer and for theranostic applications. Our results provide useful guidance for cell viability assay compatibility for CNC and lignin NPs and facilitate the future translation of the materials for in vivo applications.

Cellulose nanofibers from lignocellulosic biomass of lemongrass using enzymatic hydrolysis: characterization and cytotoxicity assessment.

BACKGROUND: The lemongrass (LG) leaves could be a useful source of cellulose after its oil extraction, which is still either dumped or burned, not considered as a cost-effective approach. The synthesis of cellulose nanofibers (CNF) from LG waste has emerged as a beneficial alternative in the value-added applications. The non-toxicity, biodegradability, and biocompatibility of CNF have raised the interest in its manufacturing. METHOD: In the present study, we have isolated and characterized CNFs using enzymatic hydrolysis. We also explored the cytotoxic properties of the final material. The obtained products were characterized using dynamic light scattering (DLS), fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), x-ray diffraction (XRD), and thermogravimetric/differential thermal gravimetric analysis (TG/DTG). The cytotoxicity of CNF was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay against three different cancer cell lines NCIH460, PA1, and L132 cells. RESULTS: The FT-IR results showed that the resulting sample was of cellulose species, and CNF was found free from the non-cellulosic components like lignin and hemicellulose. The SEM micrographs of the cellulose showed a bundle like structure. The TEM micrographs of CNF showed diverse long fibers structure with 105.7 nm particle size analysed using DLS. The TGA analysis revealed that the thermal stability was slightly lower, compared to cellulose. Additionally, CNF did not show the cytotoxic effect at the tested concentrations (~10-1000 µg/ml) in any of the cell lines. CONCLUSION: Overall, the results concluded that LG waste-derived CNF is a potential sustainable material and could be employed as a favourable reinforcing agent or nanocarriers in diverse areas, mainly in food and drug delivery sectors. Graphical abstract Systematic representation of the synthesis of the cellulose nanofibers: The lignocellulosic waste of lemongrass (after oil extraction) was pretreated for the isolation of raw cellulose, followed by enzyme hydrolysis for the synthesis of pure cellulose nanofibers.

Cytotoxicity and biological capacity of sulfur-free lignins obtained in novel biorefining process.

Lignin is one of the most promising and versatile products obtained in biorefineries due to its diverse therapeutic properties such as antimicrobial, antioxidant and anti-inflammatory activity. However, these properties depend on the source of lignin and the way it was isolated from the biomass. In this study, four different lignins are compared (extracted with Aquasolv (ASL1, ASL2), Organosolv (OSL) and Alkali (ALK) processes) for their cellular antioxidant capacity, anti-diabetic activity, free radical scavenging and cytotoxicity. Alkali and Organosolv lignins showed the highest antioxidant capacity 1159.815 µmol TE g-1 and 1463.415 µmol TE g-1, respectively, in agreement with their highest amount of free -OH groups. Additionally, OSL showed the highest inhibition in the antidiabetic assay followed by ASL1 with values for α-amylase of 3.6 mg/ml and 4.3 mg/ml respectively, and α-glucosidase 1.6 mg/ml and 2.5 mg/ml correspondingly. Nevertheless, cell-based assays revealed that ASL has the lowest cytotoxic effect in Caco-2 cells and, thus, is 10 times less cytotoxic than Alkali and OSL. This work suggests the applicability of ASL for high value applications such as cosmetics or pharmaceuticals. INDUSTRIAL RELEVANCE: Industrially, Liquid Hot Water (LHW) and Organosolv processes may appear as promising biorefining technologies in the following years. Lignin produced is free of sulfur, can be labelled as clean and environmentally-friendly and in this study, was proven that LHW lignin is non cytotoxic. The findings in this paper showed that different sources of lignin can be used in product formulation for life science purposes, thus opening a broad spectrum of possibilities for lignin valorisation in biorefineries.

Transformation of lignosulfonate into graphene-like 2D nanosheets: Self-assembly mechanism and their potential in biomedical and electrical applications.

Facile and controllable synthesis of graphene or graphene-like 2D nanosheets from plentiful and biocompatible materials still remains a great challenge. Herein, a bottom-up and controllable approach was firstly reported to transform earth-abundant lignosulfonate into graphene-like materials, in which lignosulfonate-based 2D nanosheets were fabricated via self-assembly in water/acetone dual solvent system, and then the nanosheets materials were transformed into graphene-like materials by carbonization. The physical properties of obtained lignosulfonate-based nanosheets were characterized, and the formation mechanism of these nanosheets was also elucidated. The thickness of the nanosheets was in the range of 5-20 nm depending on the concentration of lignosulfonate in water. Directed by π - π interactions and hydrogen bonds, the evolution of layered nanosheets seemed to experience from nano-sized rodlikes, a flake with defect holes, and smooth lignosulfonate-based nanosheets. Because of the relatively lower resistance, nano-sized structures and good cytocompatibility, the lignosulfonate-based graphene-like materials exhibited great potential in biomedical energy-related applications.

Proteome response of two natural strains of Saccharomyces cerevisiae with divergent lignocellulosic inhibitor stress tolerance.

Strains of Saccharomyces cerevisiae with improved tolerance to plant hydrolysates are of utmost importance for the cost-competitive production of value-added chemicals and fuels. However, engineering strategies are constrained by a lack of understanding of the yeast response to complex inhibitor mixtures. Natural S. cerevisiae isolates display niche-specific phenotypic and metabolic diversity, encoded in their DNA, which has evolved to overcome external stresses, utilise available resources and ultimately thrive in their challenging environments. Industrial and laboratory strains, however, lack these adaptations due to domestication. Natural strains can serve as a valuable resource to mitigate engineering constraints by studying the molecular mechanisms involved in phenotypic variance and instruct future industrial strain improvement to lignocellulosic hydrolysates. We, therefore, investigated the proteomic changes between two natural S. cerevisiae isolates when exposed to a lignocellulosic inhibitor mixture. Comparative shotgun proteomics revealed that isolates respond by regulating a similar core set of proteins in response to inhibitor stress. Furthermore, superior tolerance was linked to NAD(P)/H and energy homeostasis, concurrent with inhibitor and reactive oxygen species detoxification processes. We present several candidate proteins within the redox homeostasis and energy management cellular processes as possible targets for future modification and study. Data are available via ProteomeXchange with identifier PXD010868.

Design, characterization and preliminary biological evaluation of new lignin-PLA biocomposites.

The study focuses on the obtainment of new poly (lactic acid)-lignin biocomposites. The effect of lignin loading on the morphology and mechanical properties, as well as the water uptake behaviour of the obtained biocomposites, was investigated in order to elucidate the influence of lignin incorporation into a poly (lactic acid) matrix. The addition of 7% lignin improved the Young modulus and led to a decrease in the tensile strength in comparison with the corresponding values of the poly (lactic acid) matrix, while the water sorption capacity slowly decreased. A subsequent increment in lignin loading from 7 to 15wt% resulted in an increase in tensile strength, as well as in a decline in the water sorption capacity. These results show the importance of the lignin content in controlling the properties of such composites. Furthermore, the behaviour of the PLA-lignin biocomposites in SBF was another concern for evaluation of mechanical performance and biological activity. The mechanical performance declined after immersion in simulated body fluid, but the properties of the biomaterials remained sufficiently high for the perspective of their use in medical applications. In-vitro biocompatibility studies evidenced that the addition of lignin to a poly (lactic acid) matrix can allow tailoring the final properties of the composites without inducing any significant change in cell metabolic activity (compared to poly (lactic acid) itself).

Toxicity evaluation of lignocellulose-derived phenolic inhibitors on Saccharomyces cerevisiae growth by using the QSTR method.

Quantitative Structure-toxicity Relationship (QSTR) models were built to evaluate the toxicity of lignocellulose-derived phenolic inhibitors on the growth of Saccharomyces cerevisiae in a bioethanol production process. The established models were proved to be reliable after rigorous validation and showed values of R2 > 0.6 and Q2LOO > 0.5. They could provide accurate guidance for alleviating the most toxic inhibitors in pretreated lignocellulosic hydrolysates, thus facilitating bioethanol production. The results showed that the inhibitors that possessed unsaturated bonds, formyl groups and carbonyl group substituents showed obvious toxicity effects. The toxicity of the inhibitors with ortho-electron-withdrawing substituents was stronger than that of metra- or para-electron-donating substituents. Ferulic acid was chosen to analyze its toxicity in practical alkali-pretreated rice straw hydrolysates because of its strong toxicity and high concentration. The results showed that its toxicity was up to 82%, which was suggested to be dominantly detoxified in the bioethanol production process.

Genome-wide association across Saccharomyces cerevisiae strains reveals substantial variation in underlying gene requirements for toxin tolerance.

Cellulosic plant biomass is a promising sustainable resource for generating alternative biofuels and biochemicals with microbial factories. But a remaining bottleneck is engineering microbes that are tolerant of toxins generated during biomass processing, because mechanisms of toxin defense are only beginning to emerge. Here, we exploited natural diversity in 165 Saccharomyces cerevisiae strains isolated from diverse geographical and ecological niches, to identify mechanisms of hydrolysate-toxin tolerance. We performed genome-wide association (GWA) analysis to identify genetic variants underlying toxin tolerance, and gene knockouts and allele-swap experiments to validate the involvement of implicated genes. In the process of this work, we uncovered a surprising difference in genetic architecture depending on strain background: in all but one case, knockout of implicated genes had a significant effect on toxin tolerance in one strain, but no significant effect in another strain. In fact, whether or not the gene was involved in tolerance in each strain background had a bigger contribution to strain-specific variation than allelic differences. Our results suggest a major difference in the underlying network of causal genes in different strains, suggesting that mechanisms of hydrolysate tolerance are very dependent on the genetic background. These results could have significant implications for interpreting GWA results and raise important considerations for engineering strategies for industrial strain improvement.

Individual and binary mixture effects of bisphenol A and lignin-derived bisphenol in Daphnia magna under chronic exposure.

In recent years, many new chemicals have been synthesized from biomass with an aim for sustainable development by replacing the existing toxic chemicals with those having similar properties and applications. However, the effects of these new chemicals on aquatic organisms remain relatively unknown. In this study, the effects of bisphenol A (BPA) and lignin-derived bisphenol (LD-BP, a BPA analogue) on Daphnia magna were evaluated. The animals were exposed to BPA, LD-BP, and their binary mixture at concentrations (2-2000 µg L-1) for 21 days. The expression of various biochemical markers and the effects on growth, molting, and reproduction parameters were examined. The results showed that the weight of daphnids significantly increased after exposure to BPA, LD-BP, and the binary mixture relative to that of the control animals. The activity of superoxide dismutase was significantly inhibited by LD-BP and the binary mixture. At the highest exposure concentration of the binary mixture, the activities of acetylcholinesterase and α-glucosidase, fecundity, and the number of neonates per brood were significantly altered. Our results showed that the effects of BPA and LD-BP on D. magna were generally comparable, except for the effect on the weight at their environmentally relevant concentrations (e.g., <20 µg L-1). The effects on the reproduction of D. magna could be mainly due to the shift in energy redistribution under BPA and LD-BP exposures. Our results implied that exposures to both BPA and LD-BP could potentially cause deleterious effects at the population level in D. magna.

Lignin model compound in alginate hydrogel: a strong antimicrobial agent with high potential in wound treatment.

Nowadays bacterial resistance to known antibiotics is a serious health problem. In order to achieve more efficient treatment, lately there is an effort to find new substances, such as certain biomaterials, that are non-toxic to humans with antibiotic potential. Lignins and lignin-derived compounds have been proposed to be good candidates for use in medicine and health maintenance. In this study, the antibacterial activity of the lignin model polymer dehydrogenate polymer (DHP) in alginate hydrogel (Alg) was studied. The obtained results show that DHP-Alg has strong antimicrobial activity against several bacterial strains and biofilms and does not have a toxic effect on human epithelial cells. These results strongly suggest its application as a wound healing agent or as an adjunct substance for wound treatments.

Artemia salina as a new index for assessment of acute cytotoxicity during co-composting of sewage sludge and lignocellulose waste.

Considering the necessity to constantly monitor the safety of use of sewage sludge, we have focused on evaluating the toxicity of raw sludge and sludge treated by co-composting with date palm waste using an in vitro assessment of cytotoxicity based on Artemia salina larvae as a simple new sensitive and reliable routine test. The efficiency of co-composting in decreasing sludge toxicity was evaluated in terms of cytotoxicity abatement reaching 100% by the second month of composting for mixture A (1/3 sludge+2/3 date palm waste) and the third month for mixture B (1/2 sludge+1/2 date palm waste). Cytotoxicity abatement was confirmed by the increase of germination index, which reached over 100% with positive correlation for lettuce (R(2)=0.81 and 0.86) and for turnip (R(2)=0.87 and 0.74) for mixtures A and B respectively. A strong correlation between the proposed cytotoxicity test and the evolution of regulatory physical-chemical approaches was found, (R(2)=0.88 and 0.89) for NH4(+)/NO3(-) and (R(2)=0.80 and 0.88) for C/N respectively for mixture A and B. These findings allow the inexpensive bioassay reported to be used as a highly sensitive test to determine the cytotoxicity and maturity of composts.

Hydrogen peroxide sensing and cytotoxicity activity of Acacia lignin stabilized silver nanoparticles.

The study is aimed at detection of hydrogen peroxide (H2O2) using Acacia lignin mediated silver nanoparticles (AGNPs). The synthesis of AGNPs was achieved at conditions optimized as, 3 ml of 0.02% lignin and 1mM silver nitrate incubated for 30 min at 80°C and pH 9. Initial screening of AGNPs was performed by measuring the surface plasmon resonance peak at 410-430 nm using UV-vis spectrophotometer. Transmission electron microscopy, atomic force microscopy, X-ray diffraction and particle size analysis confirmed the spherical shaped face centered cubic structure and 10-50 nm size of AGNPs. The infrared spectroscopy study further revealed that the active functional groups present in lignin were responsible for the reduction of silver ions (Ag(+)) to metallic silver (Ag(0)). Lignin stabilized silver nanoparticles showed good sensitivity and a linear response over wide concentrations of H2O2 (10(-1) to 10(-6)M). Further, the in vitrocytotoxicity activity of the lignin mediated AGNPs (5-500 µg/ml) demonstrated toxicity effects in MCF-7 and A375 cell lines. Thus, lignin stabilized silver nanoparticles based optical sensor for H2O2 could be potentially applied in the determination of reactive oxygen species and toxic chemicals which further expands the importance of lignin stabilized silver nanoparticles.

An environmentally benign antimicrobial nanoparticle based on a silver-infused lignin core.

Silver nanoparticles have antibacterial properties, but their use has been a cause for concern because they persist in the environment. Here, we show that lignin nanoparticles infused with silver ions and coated with a cationic polyelectrolyte layer form a biodegradable and green alternative to silver nanoparticles. The polyelectrolyte layer promotes the adhesion of the particles to bacterial cell membranes and, together with silver ions, can kill a broad spectrum of bacteria, including Escherichia coli, Pseudomonas aeruginosa and quaternary-amine-resistant Ralstonia sp. Ion depletion studies have shown that the bioactivity of these nanoparticles is time-limited because of the desorption of silver ions. High-throughput bioactivity screening did not reveal increased toxicity of the particles when compared to an equivalent mass of metallic silver nanoparticles or silver nitrate solution. Our results demonstrate that the application of green chemistry principles may allow the synthesis of nanoparticles with biodegradable cores that have higher antimicrobial activity and smaller environmental impact than metallic silver nanoparticles.

SUMO expression shortens the lag phase of Saccharomyces cerevisiae yeast growth caused by complex interactive effects of major mixed fermentation inhibitors found in hot-compressed water-treated lignocellulosic hydrolysate.

The complex inhibitory effects of inhibitors present in lignocellulose hydrolysate suppress the ethanol fermentation of Saccharomyces cerevisiae. Although the interactive inhibitory effects play important roles in the actual hydrolysate, few studies have investigated glycolaldehyde, the key inhibitor of hot-compressed water-treated lignocellulose hydrolysate. Given this challenge, we investigated the interactive effects of mixed fermentation inhibitors, including glycolaldehyde. First, we confirmed that glycolaldehyde was the most potent inhibitor in the hydrolysate and exerted interactive inhibitory effects in combination with major inhibitors. Next, through genome-wide analysis and megavariate data modeling, we identified SUMOylation as a novel potential mechanism to overcome the combinational inhibitory effects of fermentation inhibitors. Indeed, overall SUMOylation was increased and Pgk1, which produces an ATP molecule in glycolysis by substrate-level phosphorylation, was SUMOylated and degraded in response to glycolaldehyde. Augmenting the SUMO-dependent ubiquitin system in the ADH1-expressing strain significantly shortened the lag phase of growth, released cells from G2/M arrest, and improved energy status and glucose uptake in the inhibitor-containing medium. In summary, our study was the first to establish SUMOylation as a novel platform for regulating the lag phase caused by complex fermentation inhibitors.

By-products resulting from lignocellulose pretreatment and their inhibitory effect on fermentations for (bio)chemicals and fuels.

Lignocellulose might become an important feedstock for the future development of the biobased economy. Although up to 75 % of the lignocellulose dry weight consists of sugar, it is present in a polymerized state and cannot be used directly in most fermentation processes for the production of chemicals and fuels. Several methods have been developed to depolymerize the sugars present in lignocellulose, making the sugars available for fermentation. In this review, we describe five different pretreatment methods and their effect on the sugar and non-sugar fraction of lignocellulose. For several pretreatment methods and different types of lignocellulosic biomass, an overview is given of by-products formed. Most unwanted by-products present after pretreatment are dehydrated sugar monomers (furans), degraded lignin polymers (phenols) and small organic acids. Qualitative and quantitative effects of these by-products on fermentation processes have been studied. We conclude this review by giving an overview of techniques and methods to decrease inhibitory effects of unwanted by-products.

Calcium lignosulphonate: re-evaluation of relevant endpoints to re-confirm validity and NOAEL of a 90-day feeding study in rats.

A 90-day feeding study in Han/Wistar rats with calcium lignosulphonate was evaluated by the EFSA. The study was considered to be inadequate due to potentially impaired health status of the animals based upon a high incidence of minimal lymphoid hyperplasia in mesenteric/mandibular lymph nodes and Peyer's patches, and minimal lymphoid cell infiltration in the liver in all animals. The EFSA Panel further disagreed with the conclusion that the treatment-related observation of foamy histiocytosis in mesenteric lymph nodes was non-adverse and asked whether this observation would progress to something more adverse over time. A PWG was convened to assess the sections of lymph nodes, Peyer's patches and liver. In addition, all lymphoid tissues were re-examined. The clinical pathology and animal colony health screening data were re-evaluated. The question whether the foamy histiocytosis could progress to an adverse finding with increasing exposure duration was addressed by read-across. In conclusion, the animals on the 90-day feeding study were in good health, the study was adequate for safety evaluation, and the foamy histiocytes in the mesenteric lymph nodes were not considered adverse, but rather an adaptive response that was considered unlikely to progress to an adverse condition with time. The NOAEL was re-affirmed to be 2000 mg/kgbw/d.