Overview of potential adverse health effects of oral exposure to nanocellulose

Figures & data

Table 1. Typical characteristics of different types of nanocellulose and their production methods.

Properties	CNCs (cellulose nanocrystals)	CNFs (cellulose nanofibers)	BNC (bacterial nanocellulose)
Shape	Rod, spherical	Fibrils/fibers	Fibrils/fibers
Diameter	5–70 nm ^[1,2]	5–100 nm ^[1,2]	10–100 nm ^[2,3,4,5]
Length	50–350 nm ^[1,2]	0.1–2 µm ^[1,2]	Network of nanofibers. Individual fibers several to tens of µm ^[2]
Aspect ratio (ratio of length to width)	5–30 ^[1]	50–100 ^[1,2]	≥100 ^[2]
Alternative names	Nanocrystalline cellulose, cellulose nanowhiskers	Nanofibrillated cellulose, cellulose nanofibrils	Bacterial cellulose, biotech cellulose, biocellulose, microbial cellulose
Source material	Predominantly (various) plant sources. Alternative sources incl. tunicates, algae, bacteria	Predominantly (various) plant sources. Alternative sources incl. tunicates, algae, bacteria	De novo bacterial synthesis
Production method	Delamination of cellulose through chemical treatments (predominantly acid hydrolysis)	Delamination of cellulose through mechanical and optional chemical pretreatments	Biosynthesis using low molecular weight carbon sources (e.g. D-glucose)

^[1[Foster et al. 2018; ^[2]Klemm et al. 2018; ^[3]Jozala et al. 2016; ^[4]Klemm et al. 2011; ^[5]de Amorim et al. 2020.

Table 2. Nonexhaustive list of common modifications of the hydroxyl groups on the surface of nanocellulose and their function or application. Abbreviations are listed in a footnote.

Surface group (Chemical formula)	Cation/ anion	Typical treatment	Function/application
Carboxyl (Cel-COOH^-)	Anion	Carboxylation of hydroxyl groups via TEMPO oxidization of cellulose surfaces during pretreatment	Enhanced surface charge to facilitate the delamination process, dispersibility and solubility in water and reduced irreversible aggregation of the fibrils (hornification) ^[1,5,10]
Carboxymethyl (Cel-CH2-COOH^-)	Anion	Carboxymethylation of hydroxyl groups using e.g. sodium monochloroacetate
Sulfate half ester (Cel-O-SO3^2-)	Anion	Hydrolysis of cellulose using sulfuric acid resulting in grafting of sulfate half ester groups
Phosphate half ester (Cel-O-H2PO3^2-)	Anion	Phosphorylation of hydroxyl groups using phosphoric acid/salt ^[2]	Improved flame retardancy ^[4], enhanced viscosity ^[2] and dispersibility ^[3]
Acetyl (Cel-O-CH3CO)	Anion	Addition of acetic anhydride and acetic acid in combination with a catalyst such as sulfuric acid ^[5]	Enhanced hydrophobicity ^[5]
EPTMAC (Cel- C6H14NOCl)	Cation	Etherification of hydroxyl groups	Introduction of cationic groups to the surface ^[5]
poly(APMA)	Cation	surface-initiated single-electron transfer living radical polymerization method	Controlled drug release through delivery carriers ^[8, 9]
poly(NIPAAm)	Anion	surface-initiated single-electron transfer living radical polymerization method	Controlled drug release through delivery carriers ^[8, 9]
Metals (e.g. Ag)	Cation	Addition of metals to growth medium resulting in precipitation of metals on surface of BNC	Ag: production of nanocelluloses with antibacterial coating
Other polymers/ molecules such as fluorophores	Polymer/molecule dependent	Various methods including grafting of polymers using ring opening polymerization ^[5] and atom transfer radical polymerization ^[6]	Modification of various properties such as fluorescence, dispersibility in different mediums and nanocomposite strength
Biomolecule conjugation		Conjugation of peptide motifs (e.g RGD) ^[7]	Improved adhesion of cells for biomedical application (e.g. wound dressing)

^[1]Klemm et al. 2011; ^[2]Noguchi et al. 2017; ^[3]Camarero Espinosa et al. 2013; ^[4]Ghanadpour et al. 2015; ^[5]Habibi 2014; ^[6]Eyley and Thielemans, 2014; ^[7]Klemm et al. 2018; ^[8]Jimenez et al. 2017; ^[9]Zhang et al. 2016; ^[10]Lopez et al. 2015.

BNC: bacterial nanocellulose; EPTMAC: epoxypropyltrimethylammonium chloride; poly(APMA): poly(N-3-aminopropylmethacrylamide); poly(NIPAAm): poly(N-isopropylacrylamide); RGD: arginine-glycine aspartic acid; TEMPO: 2,2,6,6-tetramethylpiperidin-1-oxyl.

Table 3. Overview in vivo oral toxicity studies performed with nanocellulose.

Reference	Study design (type of study, dose, duration)	Test material	Effects observed	Remarks regarding quality of study
Subchronic studies
Ede et al., 2020	Repeated dose 90 day oral toxicity study (OECD 408). Animals: Sprague-Dawley CD IGS rats; males and females; n = 10/sex/group (total: n = 120). Exposure: 90 days Route: via diet Diet: OpenStandard Diet with no added fiber (D11112219N; Research Diets Inc.) Dose: 0, 2, 3 and 4% CNC in diet, equal to 1056, 1584 and 2085 mg/kg bw/day (males) and 1278, 1930 and 2683 mg/kg bw/day (females). Control: conventional cellulose (CC); 2, 3 and 4%. Dispersion: sample dispersion by using a Vortex for 10 min prior to analyses. Parameters: body weight, food consumption, ophthalmology, clinical pathology, hematology, serum chemistry, urinalysis, anatomic pathology, histopathology.	CNC: - Impurities: low levels of metal impurities (0.01-0.1 ppb range) - Endotoxins: no data - Appearance: spray-dried powder (100% wt.) - Particle size: average HDD in water 893 ± 251 nm (DLS); average DI 0.51 ± 0.02. Light and electron microscopy: widths <10 nm; lengths 25-250 nm - Surface treatment: sulfate groups - Shape: rod-shaped - Surface area: no data - Zeta potential: −50.8 ± 6 mV - Analytical method: DLS, light microscopy, TEM, SEM, EDXS - Production: produced through sulfuric acid hydrolysis of wood pulp, purification, and subsequent neutralization with sodium hydroxide	Early effects: Vacuolation of periportal hepatocytes was observed at 4% CNC. Incidence and severity was the same as in the CC group. Adverse effects: No pathology findings or other treatment-related adverse effects were observed. NOAEL = 2085.3 (m) and 2682.8 (f) mg/kg bw/day. No internal concentrations measured. CNC versus CC: Food consumption and body weight were higher in the 4% CNC group (females) compared with the 4% CC group (females).	Unclear if dispersion by Vortex is adequate. HDD is determined.
		CC: - Food-grade Solka Floc - Impurities: no data - Appearance: no data - Particle size: average HDD 26.7 ± 6 µm; average DI 0.65 ± 0.07; Microscopy: length and width in microns - Surface treatment: none - Shape: amorphous - Surface area: no data - Zeta potential: −2.1 ± 0.9 mV - Analytical method: DLS, light microscopy, TEM, SEM, EDXS - Production: no data	Early effects: Vacuolation of periportal hepatocytes was observed at 4% CC. Incidence and severity was the same as in the CNC group. Adverse effects No pathology findings or other treatment-related adverse effects were observed. No internal concentrations measured.	No data provided on impurities.
Ong et al., 2020	Repeated dose 90 day oral toxicity study (OECD 408). Animals: Sprague-Dawley rats; males and females; 6-7 weeks old; n = 10/group. Exposure: 90 days Route: via diet. Diet: OpenStandard Diet with no added fiber (D11112219N; Research Diets Inc.) Dose groups: 6 - CNF: 2%, 3% and 4%, equal to 1044, 1550, and 2194 mg/kg bw/day for males and 1302, 1886, and 2667 mg/kg bw/day for females. - Cellulose: 2%, 3% and 4%, equal to 1070, 1536, and 2119 mg/kg bw/day for males and 1311.7, 1920.2, and 2597.5 mg/kg bw/day for females. Dispersion: a Disruptor Genie (60 kHz; 240 W; 3000 rpm) was used to vortex the solution for 10 min during dilution; no other data. Parameters: Survival, clinical observations, body weight, food consumption, ophthalmologic evaluations, hematology, serum chemistry, urinalysis, postmortem anatomic pathology, and histopathology.	CNF - Impurities: assessed, but no data - Endotoxins: no data - Appearance: cellulose in distilled water to 2 % wt. - Particle size: average width of the finest fractions: 25.06 nm; aggregates, average size: 227.7 µm; hydrodynamic diameter: 3330 nm (DLS) - Density: no data - Surface treatment: none - Shape: entangled network of fibers and fibrils of varying widths - Surface area: no data - Zeta potential: −37.5 mV - Analytical method: light microscopy, TEM, SEM, DLS, EDXS - Production: produced through mechanical homogenization of a wood pulp starting material	Early effects: not measured. Adverse effects: Vacuolation of periportal hepatocytes was present in the 4% CNF group, but was also observed in the group treated with 4% conventional cellulose. No adverse observations were noted in relation to the administration of fibrillated cellulose.	Unclear if dispersion by Vortex is adequate. HDD is determined. No data provided on impurities. No negative control group without treatment was included.
		CC - Food-grade Solka Floc - Impurities: provided by manufacturer, no data - Appearance: cellulose in distilled water to 2% wt. - Particle size: average width: 3.72 µm; aggregates, average size: 58.6 µm; hydrodynamic diameter: 625 nm (DLS) - Density: no data - Surface treatment: none - Shape: amorphous morphology, not an entangled network of fibers, lower aspect ratio than fibrillated cellulose - Surface area: no data - Zeta potential: −24.3 mV - Analytical method: light microscopy, TEM, SEM, DLS, EDXS - Production: no data	Early effects: not measured. Adverse effects: Vacuolation of periportal hepatocytes was present in the 4% CC group.	No data were provided on impurities.
Subacute studies
Adewuyi et al., 2018	14-day oral toxicity study. Animals: Wistar rats; males; n = 20/group. Route: oral, no details provided. Diet: commercial pelleted diet (Ladokun Feeds, Ibadan, Nigeria); no data on fibers. Exposure: 14 days, once, daily. Dose groups: controls (olive oil), 50, 75 and 100 mg/kg bw/day CNC. Dispersion: ultrasonication in a Cole Parmer sonicator (model CV334) for 15 min to disperse the nanocrystals and break any agglomerates formed. Parameters: biomarkers of renal oxidative damage, inflammation, immunohistochemical expressions of selected genes, histopathology.	CNC - Impurities: no data - Endotoxins: no data - Appearance: suspension in deionized water - Particle size: mean distribution size of 0.0149 μm - Density: no data - Surface treatment: sulfated (in the publication named as ‘sulfonated’ by the authors) - Shape: monomodal, flaky surface with agglomerations - Surface area: no data - Zeta potential: ∼ −30 mV at pH 1.5; ∼ −5 mV at pH 7; ∼ 5 at pH 12 - Analytical method: SEM, EDX, FTIR, XRD, TG, PSD - Production: Sulfonated nanocellulose was obtained by acid hydrolysis with 65% sulfuric acid solution (v/v) at 50 °C	Early effects: Body weight was not affected. CNC had no effect on markers of kidney function. Calcium and potassium levels were significantly decreased at 50 mg/kg bw/day (calcium and potassium) and 75 mg/kg bw/day (potassium), but not at the other dose levels. Sodium was increased at the low and high dose level, but without a dose-response relationship. Several antioxidant enzymes were changed in kidney tissue upon treatment: - SOD was increased in the low and mid dose group (P < 0.01). - GPx was increased in the mid dose group (P < 0.01). - GSH was increased in the low dose group (P < 0.05) and decreased in the high dose group (P < 0.05). - MPO was decreased in all dose groups (P < 0.01). CAT, GST, MDA, hydrogen peroxide and nitric oxide were not significantly changed. Immunohistochemical staining showed more intense expression of iNOS and COX-2 upon treatment, indicative of inflammation. Adverse effects: Histopathology of the kidney demonstrated moderate cortical congestion at 50 mg/kg bw/day and interstitial hemorrhage and presentation of protein casts in the tubules of rats at 75 and 100 mg/kg bw/day.	The study was limited to kidney; effects on other organs were not examined. No data were provided on impurities. The description of the methods lacks details on the treatment of the animals.
DeLoid et al., 2019	Five-week oral toxicity study. Animals: Wistar-Han rats; males; n = 13/group. Route: via oral gavage (10 ml/kg). Diet: PicoLab Rodent Diet 5053; contains 4.7% fiber (cellulose, hemi-cellulose and lignin). Exposure: 5 weeks, twice a week gavage. Suspension in water or cream (20% fat) Dose groups: water, cream, 1% CNF, or cream + CNF-50. Dispersion: by using a disintegrator, which consists of an agitator with a variable rotation setting. The disintegrator was set for 10,000 revolutions. Parameters: analysis of whole blood and serum markers, histology of lung, liver, kidney and small intestine.	CNF-50 - Impurities: no data - Endotoxins: free of endotoxins (all below LOD of 0.5 EU/mg) - Appearance: 2.5% w/w cellulose, in water (stock) - Particle size: mean diameter 64 ± 29 nm; length 6.71 ± 5.61 µm - Density: 1.312 ± 0.016 g/cm^3 - Surface treatment: not mentioned - Shape: not specified, only microscopy data - Surface area: 34 m^2/g - Zeta potential: no data - Analytical method: SEM - Production: mechanical grinding of dried sheets of softwood bleached kraft fiber	Early effects: blood differential counts, hematological parameters and serum markers showed no significant effects. Adverse effects: Histopathology, showed no significant adverse effects. A moderate but statistically insignificant reduction in weight gain was observed between rats receiving CNF-50 alone and all other groups. Rats receiving CNF-50 alone gained on average 30–40% less weight than other groups during the five weeks of treatment. No internal concentrations measured.	The description of analytical methods is missing in the publication by error. More methods than SEM may have been used. Unclear if dispersion by a disintegrator is adequate. No data were provided on impurities. Only one dose tested.
Khare et al., 2020	Five-week oral toxicity study. Animals: Wistar-Han rats; males; 12 weeks old; n = 4-5/group (controls: n = 3). Route: oral gavage (10 ml/kg). Diet: PicoLab Rodent Diet 5053; contains 4.7% fiber (cellulose, hemi-cellulose and lignin). Exposure: 5 weeks, twice a week gavage. Dose groups: control (water), 1% CNF, 20% cream, or 1% CNF + 20% cream Dispersion: by using a disintegrator, which consists of an agitator with a variable rotation setting. The disintegrator was set for 10,000 revolutions. Parameters: fecal microbiome and metabolome, intestinal epithelial expression of cell junction genes, and ileal cytokine production.	CNF 50: - Cellulose nanofibers - Impurities: no data - Endotoxins: endotoxin level determined, data not provided - Appearance: 2.5% w/w cellulose in sterile deionized water - Particle size: length 6710 ± 5611 nm (SEM); diameter 64 ± 29 nm (SEM); aspect ratio 107.6 ± 54.5 - Density: 1.312 ± 0.0185 g/cm^3 - Surface treatment: no - Shape: fibrils in a web-like structure - Surface area: no data - Zeta potential: no data - Analytical methods: TEM, SEM - Production: mechanical grinding of dried sheets of softwood bleached kraft fiber	Early effects: - CNF altered microbial diversity. The population Bificobacterium was 38% decreased compared to the control group (p < 0.05). - CNF diminished specific species that produce short chain fatty acids, and that are associated with increased serum insulin and IgA production. - CNF had few effects on the fecal metabolome, with significant changes in only ten metabolites of 366 measured. - Exposure to CNF also altered expression of genes involved in intestinal epithelial cell junction integrity and permeability, and increased production of cytokines that modulate proliferation of CD8 T cells. Adverse effects: not measured.	Unclear if dispersion by a disintegrator is adequate. No data were provided on impurities. Only one dose tested.
Chen et al., 2020	4-6 week oral toxicity study. Animals: C57BL/6 mice; males and females; 8-10 weeks old; n = 4-6/group. Route: via oral gavage (0.01 ml/g body weight). Diet: PicoLab Rodent Diet 5053; contains 4.7% fiber (cellulose, hemi-cellulose and lignin). Western diet: Rodent Western diet (D12079B, Research Diets Inc.); contains 5% fiber (Solka Floc, FCC 200). Exposure: 4-6 weeks. Dose groups: - normal diet + water; - Western diet + water; - Western diet + CNF; 30 mg/kg bw/day - Western diet + cellulose; 30 mg/kg bw/day (Western diet: high in fat and sugars). Dispersion: solutions were mixed well immediately before use. No additional data. Parameters: body weight, food consumption, body composition, microscopy liver and GI-tract, serum triglyceride, glucose levels, glucose and insulin tolerance test, D-xylose absorption test.	CNF - Impurities: no data - Endotoxins: no data - Appearance: white, odorless, slurry form, 3% w/w. 98 w/% dry powder, 3.0 w/% aqueous gel - Particle size: nominal fiber width 50 nm; lengths up to several hundred microns - Density: 1.5 g/cm^3 dry powder - Surface treatment: none - Shape: no data - Surface area: 31-33 m^2/g - Zeta potential: −48 to −5 mV - Analytical method: no data, characterization was done by manufacturer - Production: produced by an ultrafine grinder to reduce the fiber bundles of softwood kraft pulp to nanoscale	Early effects: Summarized, CNF decreased the intestinal absorption compared with the control group and caused disturbance of glucose homeostasis. - Less severity of fatty liver in the CNF group, compared with the Western diet control group. Fat globules were seldomly seen in the CNF group. This is contrary to what is observed in the cellulose group, see below. - Significant increase of body fat and a decrease in lean body mass in the CNF group, compared to the normal diet control group. Slight increase was also observed in the control and cellulose Western diet groups, but not significant. - Disturbance of glucose homeostasis in the CNF group, including a decreased glucose metabolism and increased insulin resistance. - Decreased GI tract weight in the CNF group. No overt toxicity of the GI tract. - The D-xylose absorption test to examine the small bowel mucosal function showed a decrease in D-xylose concentrations compared to the control, indicative of a lower intestinal absorption. Late adverse effects: not examined.	It is noted that most characteristics are obtained from a manufacturers report. No data were provided on impurities. Only one dose tested. Dispersion by mixing well only, no further information. Unclear if dispersion was adequate; no characterization performed after dispersion.
		Cellulose No data reported.	Early effects: Similar of more fatty liver was observed as compared to the Western diet control group. More fat globules were found in the jejunum than in the Western diet control group. The body fat was slightly increased when compared to the normal diet control group, but comparable to the Western diet control group. Late adverse effects: not examined.	No data on characterization provided. Only one dose tested. Dispersion by mixing well only.

Additional studies without sufficient information on characterization are not included in the table but mentioned in the text. Abbreviations are listed below the table.

CC: conventional cellulose; CNC: cellulose nanocrystal; CNF: cellulose nanofiber; DI: dispersity index; DLS: Dynamic Light Scattering; EDXS: Energy Dispersive X-ray Spectroscopy; FTIR: Fourier transformed infrared; HDD: Hydrodynamic Diameter; PSD: particle size distribution; SEM: scanning electron microscope; TEM: Transmission Electron Microscopy; TG: thermogravimetric analysis; XRD: X-ray diffraction.

Table 4. Overview of in vitro studies that tested for effects of different types (shape, size or surface modification) of nanocellulose.

Study	Cellulose types tested and key experimental procedures	Are there differences in effects between different nanocellulose types?				Summary of findings and comments relevant for the risk assessment
		Cyto-toxicity	Geno-toxicity	ROS	Immuno-toxicity
Alexandrescu et al., 2013	Eight types of CNFs from various plant sources and different production methods and surface modifications.	Yes	–	–	–	- Decreased cell viability only for CTAB modified CNFs - CTAB is a antimicrobial substance which explains the toxic effects of these CNCs - Exposure was done as films instead of dispersed particles. This may affect the actual exposure of cells to individual particles compared to studies that used dispersed fibers. - Sizes of individual fibers not reported and no information about impurities
	Size method: not measured Dispersion: NA (exposure as film) Endotoxins: not measured Impurities: no removal procedure, not measured
Bhattacharya et al., 2017	Four types of CNFs and cellulose of different sizes and zeta-potentials, but ranges were large and medium dependent.	No	–	–	Yes	- CNFs induced pro-inflammatory responses - Cytokine production was CNF dependent - Effects could not be explained by endotoxins - One CNF type did not induce cytokine production but this was not further explained in the publication
	Size method: DLS in exposure medium Dispersion: sonication Endotoxins: most below LOD Impurities: no removal procedure, not measured					- Dispersion well described, endotoxin measured, other impurities not reported. - Size of materials measured with DLS in exposure medium
Catalán et al., 2015	Two crystals: CNCs and CMCs (crystalline micro crystals).	No	No	–	Yes	- CMC induced cytokines, CNC did not
	Size method: AFM, TEM Dispersion: sonication Endotoxins: not measured Impurities: removal procedure, not measured					- No reported impurities removal procedure for CMCs which could explain immunotoxicological effects - Clear dispersion and impurities removal procedure for CNCs - Endotoxins not measured
Chen et al., 2020	One type of CNFs of commercial source and cellulose.	No	–	–	–	- Effects reported only at the highest CNF dose, likely caused by covering of cells by CNFs
	Size method: not measured Dispersion: mixing, no further details Endotoxins: not measured Impurities: no removal procedure, not measured					- CNF size was not measured, only (minimal) data from supplier is provided - No details provided on dispersion, endotoxins or other impurities
DeLoid et al., 2019	One CNC type, one CNF type and cellulose (MC).	No	–	Yes	–	- Marginal effect (1.1 fold increase) at the highest CNC dose, but ROS production was higher in cellulose and TiO2 controls
	Size method: TEM Dispersion: vortexing, no further details Endotoxins: Below LOD or low Impurities: <1% trace metals. CNCs: 0.08% w/w sulfur^a					- (Nano)celluloses were digested in food matrix to mimic passage through stomach. Cells were exposed to digesta (up to 1.5 % w/w nanocellulose) which may impact exposure - Few details on dispersion/mixing procedure reported
Despres et al., 2019	Two types of CNCs (anionic and cationic).	–	–	Yes	Yes	- Surface modification had an effect on mitochondrial function and mitochondria derived ROS formation - Cationic CNCs induced stronger immune responses than anionic CNCs
	Size method: measured but not available Dispersion: not reported Endotoxins: not measured Impurities: removal procedure in place					- Incomplete size characterization (no data provided) - Details dispersion procedure missing - CNCs were modified with two polymers which may be used in drug delivery - Not clear if these surface modifications are expected for food applications
Ede et al., 2020	One CNC type and cellulose.	No	–	No	No	- No differences in cytotoxicity, ROS formation and immunotoxicity between cellulose and CNC
	Size method: DLS Dispersion: vortex Endotoxins: not measured, but no microbiological contamination Impurities: according to international standard (FCC)					- Dispersion of materials through vortexing only - Detailed particle characterization and impurities assessment
Hanif et al., 2014	Four types of CNCs of different size and each a different chemical treatment.	Yes	–	–	–	- Effects on cytotoxicity only at high concentrations (500 or 1000 µg/ml) - At higher concentrations, different CNCs showed slightly different cytotoxic effects, but overall dose-response trends were similar for all types
	Size method: TEM Dispersion: no details Endotoxins: not measured Impurities: removal procedure, not measured					- Details dispersion procedure missing
Hua et al., 2015	Three types of CNFs, either unmodified or cationic of anionic surface treatment.	–	–	–	Yes	- Cationic CNFs promoted inflammation response more than anionic CNFs and activity monocytes dependent on surface treatment
	Size method: NA (exposure as film) Dispersion: NA (exposure as film) Endotoxins: not measured, but UV treated or autoclaved Impurities: not measured, HCl and NaOH removed through washing					- Exposure using films instead of dispersed fibers - Endotoxins not measured and limited impurities removal step. Hence, it cannot be ruled out that immunological differences are due to different levels of impurities
Jimenez et al, 2017	Four types of CNCs, with either anionic (3 types) or cationic (1 type) surface treatment.	No	–	–	–	- Two cell lines tested using MTT and LDH assays at 24h and 48h - Different effects of different CNCs on cell viability were only reported in the 48h exposures but effects were dependent on cell line and assay - Overall, no clear link between CNC type and cytotoxic effects
	Size method: DLS Dispersion: no details Endotoxins: not measured Impurities: removal procedure, not measured					- Details dispersion procedure missing
Lopes et al., 2017	Three CNFs, either unmodified or cationic of anionic surface treatment.	No	–	No	Yes	- Pro-inflammation response was reported for unmodified CNFs but not for the surface modified CNFs
	Size method: TEM Dispersion: sonication Endotoxins: not measured Impurities: biocide free, no bacterial contamination					- Addition of a cytokine suppressor reduced cytokine secretion which indicated endotoxin contamination of CNF suspensions - Endotoxins were not measured and therefore it cannot be excluded that differences between types are due to endotoxins
Lopes et al., 2020	Five types of CNFs, either unmodified, or cationic, anionic, phosphorylated, or sulfo-ethylated.	No	–	–	–	- No effects of CNFs on cell metabolic activity of Caco-2-cells
	Size method: not measured, sizes not reported Dispersion: sonication Endotoxins: not measured, CNFs were autoclaved Impurities: no procedure, not measured					- Detailed description of exposure procedure with Caco-2-cells missing - Size characterizations of CNFs are missing
Mahmoud et al., 2010	Two types of CNCs labeled with different fluorescent molecules.	No	–	–	–	- No significant cytotoxicity in human embryonic kidney cells in 24h exposures
	Size method: TEM Dispersion: sonication Endotoxins: not measured Impurities: removal procedure, not measured					- FITC labeled CNCs had negative zeta potentials, whereas RBITC labeled CNCs had positive zeta potentials
Wang et al., 2019	Two CNF and seven CNC types, each with different sizes and source materials.	No	–	Yes	Yes	- None of the tested nanocelluloses had an effect on cytotoxicity - Use of fluorescently labeled nanocellulose showed that different nanocellulose types were taken up different by THP-1 cells - Different types induced different levels of cytokine production (IL-1β) and generated different levels of ROS and glutathione
	Size method: TEM and DLS Dispersion: vortexing and sonication Endotoxins: measured, below 1 endotoxin unit per ml Impurities: no metal, alkaline or salt impurities (ICP-OES measurements)					- All assays included a positive controls and where relevant also LPS controls - Good characterization and dispersion procedure - Impurities thoroughly assessed
Weiss et al., 2021	Twelve types of CNCs of different sizes, source materials, chemical treatments and surface modifications.	No	–	–	No	- No effects of any type on cytotoxicity - Different CNCs did not induce different immune responses
	Size method: TEM Dispersion: no clear procedure Endotoxins: measured, <5 endotoxin units per ml Impurities: removed (dialysis) and measured (NMR)					- Dispersion of the particles not described in detail - Detailed characterization - Impurities and endotoxins successfully removed and measured

Hyphens (-) indicate that the respective endpoint was not tested. More details of these studies, including nanocellulose specifications and the study outcomes are reported in the Supplemental Table S1 (Supplementary information).

^aInformation on impurities taken from Pyrgiotakis et al. (2018).

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