CELLULASE FROM TRICHODERMA REESEI EXPLANATION This substance has not been evaluated previously by the Joint FAO/WHO Expert Committee on Food Additives. Cellulase is produced extracellularly by T. reesei (QM6a), a mutant of T. viride. The enzyme preparation is characterized by two activities, exo-cellobiohydrolase (E.C. 3.2.1.1) and 1,4-endoglucanase (E.C. 3.2.1.4). Tests have been performed to show that the strain of T. reesei used for the production of this enzyme preparation does not produce any antibiotics, and it can be regarded as non-pathogenic. The enzyme preparation used in the toxicological studies was a spray dried product derived from a cruder preparation than the commercial product. The TOS of the tested product was 31%. The available safety data have been summarized by Hjortkjer et al., 1986. BIOLOGICAL DATA Biochemical aspects No information available. Toxicological studies Special studies on mutagenicity The mutagenicity of the enzyme preparation was tested using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538, both with and without metabolic activation by 5-9 fraction from rat liver. No mutagenic effects were observed at concentrations up to 10,0 mg/plate (Crichton & McGregor, 1977). A cytogenetic bone marrow study was performed using adult male Chinese hamsters. Groups of adult male hamsters received up to 5000 mg/kg b.w./day of the cellulase preparation for 5 consecutive days. The cellulase preparation did not produce an increase in chromosome aberrations in bone marrow cells (McGregor, 1979). In a dominant lethal assay, adult male CD-1 mice were dosed orally by gavage for 5 consecutive days at dose levels up to 5000 mg/kg b.w./day. No treatment-related effects were observed on implantation or fetal deaths (Cuthbert et al., 1980). Special study on reproduction Rats Four groups, each containing 20 male and 20 female 6-week old CD rats, were maintained on diets containing 0, 1, 2, or 5% of the enzyme preparation. The test diet was fed for 10 weeks prior to breeding and throughout mating, gestation, and lactation. Weanlings were maintained on the same diet as parents, until autopsied at 28 days of age. Parameters evaluated included body weights, feed consumption of F0 and F1 animals, and reproduction parameters (fertility index, gestation index, live birth index, litter size, viability index, and lactation index). Gross necropsies were conducted on all F0 and F1 animals, except pups dying before day 12 of lactation. Organ weights from 10 male and 10 female F1 animals from each treatment group were measured, and the principal tissues and organs from a similar number of F1 animals from the high-dose and control groups were microscopically examined. Clinical chemistry, haematology, and urinalysis were not performed. Compound-related mortality was not reported in the F0 generation. There were no treatment-related clinical signs. Body weights of males in the high-dose group were lower than those of controls, which was associated with decreased food intake. There were no treatment-related effects on reproductive parameters. In the F1 generation, there was a trend to increased mean body weights during the early period of lactation, but this effect was not significant toward the end of the treatment period. No significant treatment-related effects on absolute or relative organ weights in F1 males and females were observed, and no treatment-related gross or microscopic adverse effects were reported (Hazelden et al., 1982). Special study on teratogenicity Three groups of 6 pregnant CD rats each were dosed by gavage with 700, 2400, or 7000 mg/kg b.w./day of the enzyme preparation from days 6 to 16 of gestation. The rats were killed on day 20 of gestation. Reduced body-weight gain was observed in the high-dose group, which was associated with decreased food consumption during the dosing period. No compound-related differences were observed in placental weights, number of corpora lutea, implantations, resorptions, or live fetuses. The small number of visceral and skeletal abnormalities showed no treatment associations or trends (Hazelden & Everett, 1980). Acute toxicity Species Route LD50 Reference (g/kg b.w.) Mouse (NMRI) oral 16 Modeweg-Hansen, 1978a Rat (Wistar) oral 8 Modeweg-Hansen, 1978b Dog oral 5 Osborne & Chambers, 1977 Short-term studies Rats Four groups, each containing 15 male and 15 female CD rats 4 weeks of age, were maintained for 13 weeks on diets containing 0, 1, 2, or 5% of the enzyme preparation. Abnormal grooming in the high-dose groups was observed during the first 8 weeks of the study. Decreased weight gain from weeks 4 to 10 was observed in the high-dose group. Blood urea values were elevated in treated animals at weeks 4 and 13, but a consistent dose-response effect was not observed. There were no other compound-related effects on clinical chemistry or haematological measurements. No compound-related deaths were reported. At autopsy, male rats in the high-dose group had a significant increase in organ-to-body-weight ratios for the liver, prostate, and kidney when compared to control values, and females in the high-dose group had significantly higher spleen-to-body-weight ratios than controls. No compound-related gross or microscopic changes were observed, except in the case of the kidney of some rats in the high-dose group, where there was a small increase in the size of proteinaceous globules in the epithelial cells lining the renal convulated tubules (Ben-Dyke et al., 1977). Dogs Four groups, each containing 3 male and 3 female dogs, were dosed by gavage once a day, seven days a week, for 13 weeks with the enzyme preparation (30% dispersion in water) at doses equivalent to 0, 750, 1500, or 3000 mg/kg b.w./day. Vomiting was reported after dosing in the high-dose groups during the first 3 to 4 weeks of the study, and diarrhea was observed in these groups during the first two weeks of the study. There were no significant differences in body weight and food consumption between dosed and control animals during the course of the study. Haematology, clinical chemistry, and urinalysis at weeks 6 and 12 of the study showed no treatment-related effects. Ophthalmoscopic examinations at weeks 6 and 12 showed no treatment-related effects. At termination of the study, gross necropsies, organ weight analyses, and microscopic examinations of the principal organs and tissues showed no treatment-related effects (Osborne et al., 1977). Long-term studies No information available. Observations in man No information available. COMMENTS This cellulase preparation from T. reesei was not mutagenic in bacterial or in mammalian systems. The preparation caused no adverse effects in a reproduction study in rats at levels up to 5% in the diet, and it was not teratogenic in a rat study at doses up to 7 g/kg b.w./day. Short-term studies are available in dogs and rats, the no-adverse-effect levels being 3 g/kg b.w./day in dogs and 2 g/kg b.w./day in rats. The Committee was also informed that tests have been performed to show that the strain of T. reesei used for the production of this enzyme does not produce any antibiotics and it is not known to be a human pathogen. Based on the available information, the Committee established a temporary ADI for this enzyme preparation. Because this enzyme is derived from a microorganism that is neither a normal constituent of food nor a common contaminant in food, in accordance with Annex III of "Principles for the Safety Assessment of Food Additives and Contaminants in Food" (Annex 1, reference 76), this preparation requires the submission of the results of a long-term study in a rodent species. EVALUATION Level causing no toxicological effect Rat: 20,000 ppm in the diet, equivalent to 2000 mg/kg b.w./day (600 mg TOS/kg b.w./day). Estimate of temporary acceptable daily intake for man 0-0.3 mg TOS/kg b.w. Further work or information Required (by 1992) Long-term feeding study in a rodent species. REFERENCES Ben-Dyke, R., Strachen, E., Kiss, I., & Finn, J.P. (1977). Cellulase: toxicity in dietary administration to rata for thirteen weeks. Unpublished report No. 77/NTL-33/382 from Life Science Research, Stock, England. Submitted to WHO by Novo Industri A/S, Bagsvaerd, Denmark. Crichton, C., & McGregor, D.B. (1977). Testing for mutagenic activity in cellulase(R) (SP-122). Unpublished IRI project No. 40849 from Inveresk Research International, Edinburgh, Scotland. Submitted to WHO by Novo Industri A/S, Bagsvaerd, Denmark. Cuthbert, J.A., McGregor, D.B., & Willins, M.J. (1980). Dominant lethal study in mice of acid cellulase. Unpublished IRI project No. 702021, report No. 1699, from Inveresk Research International, Edinburgh, Scotland. Submitted to WHO by Novo Industri A/S, Bagsvaerd, Denmark. Hazelden, K.P. & Everett, A. (1980). Teratogenicity testing in rats of acid cellulase. Unpublished IRI project No. 702016 from Inveresk Research International, Edinburgh, Scotland. Submitted to WHO by Novo Industri A/S, Bagsvaerd, Denmark. Hazelden, K.P., Maddock, S.M., & Rushton, A.K.A. (1982). Acid cellulose dietary toxicity study in rats with in utero exposure. Unpublished IRI project No. 704851, report No. 2350, from Inveresk Research International, Musselburgh, Scotland. Submitted to WHO by Novo Industri A/S, Bagsvaerd, Denmark. Hjortkjer, R.K., Bille-Hansen, V., Hazelden, K.P., McConville, M., McGregor, D.B., Cuthbert, J.A., Greenough, R.J., Chapman, E., Gardner, J.R., & Ashby, R. (1966). Safety of celluclast(R), an acid cellulase derived from Trichoderma reesei. Fd. Chem. Tox., 24, 53-63. McGregor, D.B. (1979). Cytogenetic study in chinese hamsters of acid cellulase. Unpublished IRI project No. 702042, report No. 1585, from Inveresk Research International, Edinburgh, Scotland. Submitted to WHO by Novo Industri A/S, Bagsvaerd, Denmark. Modeweg-Hansen, L. (1978a). Acute oral toxicity of cellulase SP-122 to rats. Unpublished report from Novo Industri A/S. Submitted to WHO by Novo Industri, Bagsvaerd, Denmark. Modeweg-Hansen, L. (1978b). Acute oral toxicity of cellulase SP-122 to mice. Unpublished report from Novo Industri A/S. Submitted to WHO by Novo Industri, Bagsvaerd, Denmark. Osborne, B.E. & Chambers, P.R. (1977). Cellulase SF-122, acute oral toxicity study in dogs. Unpublished IRI project No. 408473 from Inveresk Research International, Edinburgh, Scotland. Submitted to WHO by Novo Industri A/S, Bagsvaerd, Denmark. Osborne, B.E., Rushton, A.K.A., & Dent, N.J. (1977). Cellulase SP-122, toxicity study in beagle dogs (oral administration for 13 weeks). Unpublished IRI project No. 408489, report No. 919, from Inveresk Research International, Edinburgh, Scotland. Submitted to WHO by Novo Industri A/S, Bagsvaerd, Denmark.
See Also: Toxicological Abbreviations