3.2.1.1 Cellulose

Cellulose is a linear homopolymer consisting of repeating β-d-glucopyranosyl units linked via (1,4) glycosidic bonds. In its pure form, the straight chains are bound closely together by multiple intermolecular hydrogen bonds and van der Waals forces, producing a water-insoluble fibrous or crystalline substance which is relatively inert. The EFSA ANS Panel assessed recently the absorption, distribution, metabolism and excretion (ADME) of celluloses (EFSA, 2018) and draw the following conclusions concerning non-herbivore mammals: cellulose is not absorbed intact in the gastrointestinal tract of animals and humans but is fermented during its passage through the large intestine by the microbiota, with the limited production (9% of the administered dose in the rat) of short-chain fatty acids (mainly acetic acid and succinic acid), hydrogen, carbon dioxide and methane.

In ruminants, cellulose is first hydrolysed by ruminal microorganisms into cellobiose, then is fermented to pyruvate and finally volatile fatty acids. The changes of forage to concentrate ratios in the diet significantly affect the number and type of rumen microorganisms and then affect the end products of fermentation. Moreover, the extent of cellulose digestion is a compromise between the rate of hydrolysis and the retention time in the rumen related to the particle size of the forage. The intrinsic digestibility of cellulose depends on the origin and treatment of the forage. As far as cellulose is associated to lignin, hemicelluloses and cutin in natural forages, a wide range of digestibility is observed (30 to 90%). Crystallinity of cellulose decreases the rate but not the extent of digestibility that may reach 80% (Van Soest, 1994).

Marine and freshwater fish harbour an intestinal microbiota less abundant than in mammals, made of aerobic and facultative anaerobic bacteria. Limited and conflicting data have shown either the complete lack of cellulose degradation in the trout or tilapia, or a limited (13%) activity in the trout. A digestibility study carried out in the trout and the carp administered a purified (devoid of lignin and reduced amount of hemicelluloses) crystalline cellulose extracted from wood (fibre length ˂ 150 μm, diameter ˂ 45 μm), showed the practical absence of cellulose degradation in both species (Bergot and Breque, 1983).

In poultry, most data in the literature tend to demonstrate that the cellulose complex from plant feedingstuffs that exhibits high crystallinity and water insolubility is not digested (Janssen and Carré, 1985).

In the rabbit hindgut, fermentation occurs through a wide prevalence of Bacteroides that do not allow an extended digestibility of fibre. Digestibility of cellulose was shown to amount to 16% of the administered dose, whereas values of 14% and 18% were reported for fibre (cellulose being the main component) (review from the NRC, 1977). Later studies reported values comprised between 15 and 25% in rabbits administered different plant sources of cellulose (Gidenne and Perez, 1996; Chiou et al., 1998). In the horse, digestion of plant structural carbohydrates (including cellulose) occurs in the hindgut (colon and overall caecum). The microbiota of the caecum comprises bacteria similar to those of the rumen, but specific protozoa. The resulting digestibility is about two-third that measured in ruminants.

HPMC

3.2.1.2 Modified celluloses

The etherification of cellulose disrupts the hydrogen bonding and the resulting compounds are not ionised and more water soluble. The EFSA ANS Panel (2018) concluded that modified celluloses including ethyl and methyl, hydroxypropyl celluloses, would not be absorbed intact and not fermented in the gastrointestinal tract of animals (rat) or humans; they are excreted essentially unchanged mainly via the faeces (more than 90% of the administrated doses), while only minor amounts of metabolites and derived-products are excreted via urine or expired air (as ¹⁴CO₂) and there is no indication for accumulation in the body.

No data are available concerning the ADME of modified celluloses in the target animal species. However, the absence of significant metabolism in the rat and the human indicates that, despite an increase of water solubility, the etherification of cellulose would strongly limit the action of microbial cellulases. Consequently, the FEEDAP Panel considers that the lack of digestibility of these compounds in monogastric mammals, poultry and fish is likely to occur. In ruminants and hindgut animals (rabbit and horse), it cannot be excluded that the rich and complex microbiota would allow a limited enzymatic attack of these structures.

3.2.2.1 Genotoxicity

Overall, the data set for genotoxicity is not complete for all the substances and several studies were not in line with the current standard. However, it should be considered that the chemical structure of unmodified and modified cellulose does not show any alert for genotoxicity and that no indication of genotoxicity was found for any of these substances in several in vitro and in vivo genotoxicity studies.

Concerning modified celluloses, methyl cellulose was negative in the bacterial reverse mutation assay, in the in vitro chromosomal aberration test in mammalian cells and in host-mediated assays with yeast and bacteria. In vivo methyl cellulose was also negative in a chromosome aberration assay in rat bone marrow and in the dominant lethal assay in male rats. Carboxymethyl cellulose was negative in the bacterial reverse mutation assay, in the in vitro chromosomal aberration test in mammalian cells, performed only without metabolic activation, and in host-mediated assays.

The ANS Panel considered that the read-across from methyl cellulose and carboxymethyl cellulose to other modified celluloses bearing similar simple substituents (including HPMC) was justified.

Moreover, the FEEDAP Panel also noted that methyl cellulose and carboxymethyl cellulose have been used for a long time as vehicles for non-water-soluble substances in several in vivo genotoxicity assays and are recommended for this use by the current OECD test guidelines (e.g. TGs 474, 475, 478 and 483). Based on the available experimental data, neither microcrystalline cellulose nor modified cellulose (including HPMC) raise concern for genotoxicity.

3.2.2.2 Short-term and subchronic toxicity

The majority of the available studies have been performed in rats, just few of them in rabbits and dogs. Among those meeting the current criteria for toxicological testing, the no observed adverse effect level (NOAEL) values for the different modified celluloses were identified most often corresponding to the highest tested level or changes in body or organ weights. For microcrystalline cellulose (E 460) the identified NOAELs in rats ranged from 3,769 mg/kg body weight (bw) per day to 9,000 mg/kg bw per day and in all cases corresponded to the highest levels of the test substance. For methyl cellulose (E 461), the dose level of 3% in rats (equivalent to 2,700 mg/kw bw per day) was selected as the NOAEL based on a decrease in body and organ weight displayed in male rats administered with the top additive level (10%, equivalent to 9,000 mg/kg bw per day and day).

For hydroxypropyl cellulose (E 463), the identified NOAEL corresponded to the highest dose 6,000 mg hydroxypropyl cellulose/kg bw per day (by gavage). The most relevant feeding studies with HPMC (E 464) were performed in rats which tolerated up to 10%, corresponding to 9,000 mg test item/kg bw per day. Rabbits tolerated up to 7,500 mg HPMC/kg bw per day administered via the diet (30 day exposure) and dogs up to 1,500 mg HPMC/kg bw per day, in either case being the highest tested dosages. More studies were conducted using sodium carboxy methylcellulose (E 466). The most relevant ones were conducted in rats, with NOAEL values ranging from 4,500 to 9,000 mg test item/kg bw per day (in all cases the highest dose tested). In these studies, some effects (caecum and colonic enlargement, urothelial hyperplasia, nephrocalcinosis, diffuse epithelial hyperplasia in the urinary bladder) were observed, however, not considered of toxicological concern: the findings in the gastrointestinal tract were considered to be a consequence of the accumulation of poorly absorbed water-soluble material and the findings in kidneys and urinary bladder were attributed to the up to fourfold higher concentration of sodium in the test diet compared with the basal diet. In one further study, rats were daily exposed (gavage) to doses equivalent to 0, 500, 2,500 or 5,000 mg/kg bw per day. Animals treated with ≥ 2,500 mg/kg bw per day had soft and pale faeces, which was attributed to the presence of test material and not considered of toxicological relevance. In the absence of any other adverse effects, also for this study, the identified NOAEL was the top dose (5,000 mg/kg bw per day).

3.2.2.3 Chronic toxicity and carcinogenicity

Data on chronic toxicity and carcinogenicity are available for microcrystalline cellulose (E 460), methyl cellulose (E 461) hydroxypropyl cellulose (E 463), HPMC (E 464) and sodium carboxymethyl cellulose (E 466). Some studies were unfit for evaluation due to methodological shortcomings. In the only relevant study, the dietary administration of even high doses of microcrystalline cellulose (E 460) (30%, 15,000 mg/kg bw) to rats for 72 weeks did not affect survival, feed efficiency or haematology. Apart from some dystrophic calcification in renal tubules, no other relevant lesions were noted and tumour incidence did not differ with that of controls. Several studies were conducted in rats with methyl cellulose (E 461) via feed or drinking water or by gavage at concentrations up to 5% (2,500 mg methyl cellulose/kg bw per day) and for up to 2 years. For all examined parameters, no adverse effects were reported and also the observed tumours did not differ in type and number in treated and control groups. In the only identified study, the daily dosing of male and female rats (0, 1,500, 3,000 or 6,000 mg hydroxypropyl cellulose/kg bw) via gavage for 6 months did not cause adverse effects (including carcinogenicity) apart from a decrease in body weight in high-dosed rats (statistically significant in females only). Apart from a decrease in body weights of high-dosed males, no other significant adverse findings were reported and there was no indication of a carcinogenic effect in rats of either sex dietary exposed to HPMC (E 464) up to 20% (10,000 mg/kg bw per day) for 1 year. Carboxy methylcellulose (E 466) was tested in mice and rats at dosages of 0, 10,000 or 100,000 mg/kg diet (equivalent to 0, 1,500 or 15,000 mg/kg bw per day for mice and to 0, 500 or 5,000 mg/kg bw per day for rats) for up to 104 weeks. Despite the increase in feed intake, a treatment related decrease in body weight was noted at the end of the treatment. Histological examination revealed no intestinal abnormality or evidence of the passage of the additive across the intestinal wall in either species and the tumour incidences were comparable among groups.

In conclusion, based on a limited data set, the chronic toxicity studies revealed growth retardation for some modified celluloses mostly at the highest dosage level. There was no indication for carcinogenic effects for all tested compounds.

3.2.2.4 Reproductive and developmental studies

There are data for microcrystalline cellulose (E 460), methyl cellulose (E 461), hydroxypropyl cellulose (E 463) and sodium carboxymethyl cellulose (E 466), which were tested in mice, rats, hamsters and/or rabbits with oral dosing or via gavage. As regards microcrystalline cellulose (E 460) studies have been conducted in rats (dietary exposure) with a mixture including guar gum or sodium carboxymethylcellulose (E 466) (15% in either case). The NOAEL for both maternal and developmental toxicity were the highest experimental dosages, i.e. 4,500 mg/kg bw (for mixture with guar gum) and 4,600 mg/kg bw (for mixture with sodium carboxymethyl cellulose). Methyl cellulose (E 461) was examined in mice, rats, hamsters and rabbits. In two different studies, pregnant mice were exposed via gavage (vehicle corn oil) to a dose range of 16-1,600 mg methyl cellulose (E 461)/kg bw per day from day 6 to 15 of gestation, followed by a caesarean section at day 17 of gestation. In the first study, maternal toxicity (increase in mortality and reduced pregnancy rate in the survivors) as well as retarded ossification in fetuses were noticed at the highest tested level, pointing to a NOAEL of 345 mg methyl cellulose (E 461) mg/kg bw per day (the last but one highest dosage) in mice. In the second study, no maternal toxicity and fetal abnormalities were observed in mice exposed up to 700 mg methyl cellulose (E 461) mg/kg bw per day. Rat studies (n = 2) were performed in pregnant dams exposed via gavage (vehicle corn oil) to a dose range of 16-1,320 mg methyl cellulose (E 461) mg/kg bw per day from day 6 to 15 of gestation followed by a caesarean section at day 20. In the first study (0, 13, 51, 285 or 1,320 mg methyl cellulose (E 461)/kg bw per day) the highest tested dosage resulted in no maternal toxicity but also in increased incidence of extra centres of ossification in vertebrae of fetuses from high dose dams; in a second rat study, the incidence of such alteration slightly increased in fetuses from the highest dosed group (1,200 mg methyl cellulose (E 461)/kg bw per day). Based on the above results, a NOAEL of 285 mg methyl cellulose (E 461) mg/kg bw per day could be identified in rats. No maternal or fetal toxicity was detected in Golden hamsters exposed via gavage (vehicle corn oil) up to 1,000 mg methyl cellulose (E 461) mg/kg bw per day from day 6 to 10 of gestation followed by a caesarean section at day 20. The study on rabbits was discarded due to poor experimental design. The only relevant developmental toxicity study with hydroxypropyl cellulose (E 463) (dissolved in 1% gum arabic solution) was performed in pregnant rats exposed via gavage from day 7 to 17 of gestation to 0, 200, 1,000 or 5,000 mg/kg bw test item and some of them subjected to caesarean sections at day 20. No treatment-related adverse effects were detected in dams or in the examined fetuses. A number of dams were allowed to deliver and no clinical, behavioural or morphological changes were observed in the examined pups. Their reproductive ability was seemingly not affected and no abnormalities were found in the F1-derived fetuses. The in utero exposure to the highest dose (5,000 mg/kg bw per day) may be considered as the NOAEL of methyl cellulose (E 461) for this study. No mortality, and no adverse effects were observed on implantation or on fetal survival in pregnant mice or rats dosed via gavage with up to 1,600 mg sodium carboxymethyl cellulose (E 466)/kg bw per day.

3.2.2.5 Conclusions on toxicological properties of celluloses

The FEEDAP Panel agrees with the approach of the ANS Panel that, although the data set available for the different celluloses is not complete and most of the studies were old and do not meet the current requirements of toxicological testing, the physico-chemical, structural, biological and kinetic similarities between the modified celluloses make it possible to apply a read-across approach among the different celluloses. Overall, the available information allows to conclude that the celluloses (as a group) are of low toxicological concern.