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Genetic Toxicity

Justification for read-across

There are no data available on genetic toxicity of Reaction mass of 1-O-α-D-glucopyranosyl-D-fructose and 6-O-α-D-glucopyranosyl-D-fructose and fructose and glucose and sucrose. In accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5, read-across from structurally related substances is conducted to fulfill the standard information requirements set out in Regulation (EC) No 1907/2006, Annex VIII, 8.4.

According to Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across) “to avoid the need to test every substance for every endpoint”.

All substances contained in Reaction mass of 1-O-α-D-glucopyranosyl-D-fructose and 6-O-α-D-glucopyranosyl-D-fructose and fructose and glucose and sucrose represent mono- or disaccharides which all consist of glucose and/or fructose. Reaction mass of 1-O-α-D-glucopyranosyl-D-fructose and 6-O-α-D-glucopyranosyl-D-fructose and fructose and glucose and sucrose is the aqueous solution (syrup) of the reaction mass of isomaltulose (CAS 13718-94-0), trehalulose (CAS 51411-23-5), fructose (CAS 57-48-7), glucose (CAS 50-99-7), sucrose (CAS 57-50-1), isomaltose (CAS 499-40-1) and oligosaccharides.

All ingredients are substances naturally occurring in fruits, vegetables and other crops or honey.

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006, whereby physicochemical, toxicological and ecotoxicological properties may be predicted from data for reference substance(s) by interpolation to other substances on the basis of structural similarity, sucrose (CAS 57-50-1), isomaltulose (CAS 13718-94-0) and isomalt (CAS 64519-82-0) are selected as source substances for assessment of mutagenicity.

The read-across is based on the presence of common functional groups and common breakdown products via biological processes, which result in structurally similar chemicals. In general, disaccharides like isomaltulose, trehalulose and sucrose are enzymatically hydrolysed at the glycosidic bond between the monosaccharide units to equal parts in glucose and fructose (Cheetham, 1982; Goda and Hosoya, 1983; MacDonald and Daniel, 1983; Yamada et al., 1985; Ziesenitz, 1986; Goda et al., 1991; Würsch, 1991; Günther and Heymann, 1998), which subsequently enter well-characterized carbohydrate metabolic pathways (Lina et al ., 2002) as essential energy substrate or they are converted to storable glycogen (see Toxicokinetics). A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).


Gene mutation in bacteria

Data on gene mutation in bacteria are available for the source substances isomaltulose, sucrose and isomalt.

Mutagenicity of isomaltulose was tested in a bacterial reverse mutation assay equivalent to OECD 471 with a standard battery of Salmonella typhimurium tester strains including TA 1538, TA 1535, TA 1537, TA 98 and TA 100 at concentrations of up to 4 000 µg isomaltulose/plate (Asquith, 1986). Isomaltulose did not exhibit any mutagenic properties in the absence or presence of metabolic activation nor did it induce cytotoxicity after incubation for 48 hours in a plate incorporation assay.

The genotoxic potential of sucrose was tested in a toxicity screening assay performed in 17 different laboratories.Various S. typhimurium and E. coli strains (TA 1538, TA 1535, TA 1537, TA 98 and E. coli WP2 uvrA and 343) were exposed to concentrations ranging from 500 - 5000 µg sucrose/plate (Bridges et al., 1981). Each laboratory applied its own standard procedures and different protocols for the preparation of S9 mix were applied. In addition, co-culture with non-induced primary rat hepatocytes was used as metabolic activation system.No mutagenic properties of sucrose were determined in any of the 17 test laboratories in spite of the use of different test methods.

The genotoxic potential of isomalt was determined according to OECD guideline 471 in the Salmonella tester strains TA 1535, TA 1537, TA 98 and TA 100 with and without metabolic activation at concentrations ranging from 310 - 5000 µg isomalt/plate in 2 independent experiments (Lina and Woutersen, 1995).Isomalt did not increase the number of revertant colonies in any tester strain either in the absence or presence of metabolic activation and no cytotoxicity was observed. Therefore, isomalt was characterized as non-mutagenic and non-cytotoxic.

Gene mutation in mammalian cells

Data on gene mutation in mammalian cells are available for the source substances sucrose and isomalt.

The potential of sucrose to induce gene mutations on the thymidine kinase, TK +/-, locus was tested in mouse lymphoma L5178Y cells according to OECD guideline 476 (Mitchell et al., 1988). Cells were treated with concentrations ranging from 1311 to 5000 µg sucrose/mL in the presence and absence of metabolic activation. No increase in the mutation frequency was determined either in the absence or presence of metabolic activation. Further, sucrose did not induce cytotoxicity in the applied concentrations. Therefore, sucrose was considered as non-mutagenic in mammalian cells.

Further, mutagenic properties of isomalt were tested in a mammalian cell gene mutation assay according to OECD guideline 476 (Lina and Woutersen, 1995). Chinese hamster Ovary cells were exposed to concentrations up to the limit concentration of 5000 µg isomalt/mL in the absence and presence of metabolic activation.In contrast to the positive control substances ethylmethanesulfonate and dimethylbenzanthracene, isomalt did not induce an increase in the mutation frequency which assumes that isomalt is not mutagenic in the HPRT test.

Cytogenicity in vivo

Data on cytogenicity is available for the source substance isomalt.

The clastogenic potential of isomalt was studied in an in vivo mammalian erythrocyte micronucleus test performed according to OECD guideline 474 (Lina and Woutersen, 1995).10 Wistar rats/sex/dose group were exposed to 2.5, 5 and 10% isomalt (corresponding to 1 250, 2 500 and 5000 mg/kg bw/day) over 13 consecutive weeks via feed. Negative and positive control groups were included in the study which received 10% sucrose or 10% starch supplemented diets, respectively. The positive control group received a single intraperitoneal injection of 1.5 mg/kg bw mitomycin C 24 hours before sacrifice. Isomalt did not induce an increase in the incidence of micronucleated polychromatic erythrocytes compared to the respective negative control group and hence was shown to be non-genotoxic to bone marrow cells after in vivo exposure. Moreover, exposure to isomalt did not induce cytotoxicity which was evaluated via a comparison of the incidence of polychromatic erythrocytes per 1000 erythrocytes in control and test animals (Lina and Woutersen, 1995).



In summary, neither isomaltulose nor sucrose were shown to exhibit mutagenic properties in bacteria or mammalian cells. Moreover, the read-across analogue isomalt did not show any genotoxic potential under in vitro and in vivo conditions. Based on the available data, Reaction mass of 1-O-α-D-glucopyranosyl-D-fructose and 6-O-α-D-glucopyranosyl-D-fructose and fructose and glucose and sucrose is not considered as mutagenic or clastogenic.


References not included in IUCLID:

Blainey M, Avila Cd, van der Zandt P. Review of REACH Annex IV--establishing the minimum risk of a substance based on its intrinsic properties. Regul Toxicol Pharmacol. 2010 Feb;56(1):111-20.

Cheetham, P.S.J. 1982. The human sucrase-isomaltase complex: Physiological, biochemical, nutritional and medical aspects. In: Lee, C.K.; Lindley, M.G. (Eds.). Developments in Food Carbohydrate - 3. Disaccharidases. Applied Science Publishers; London, Engl./Englewood, New Jersey, pp. 107-140.

Goda, T.; Hoyosa, N. 1983. Hydrolysis of palatinose by rat intestinal sucrase-isomaltase complex. Nihon Eiyo Shokuryo Gakkaishi 36:169-173. Cited In: Würsch, 1991.

Goda, T.; Takase, S.; Hosoya, N. 1991. Hydrolysis of palatinose condens ates by rat intestinal disaccharidases. Nihon Eiyo Shokuryo Gakkaishi 44(5):395-398.

Günther, S.; Heymann, H. 1998. Di- and oligosaccharide substrate specificities and subsite binding engergies of pig intestinal glycoamylase-maltase.Arch Biochem Biophys 354(1):111-116.

Lina, B.A.R.; Jonker, D.; Kozianowski, G. 2002.Isomaltulose (Palatinose®): A review of biological and toxicological studies. Food Chem Toxicol 40(10):1375-1381

MacDonald, I.; Daniel, J.W. 1983. The bioavailability of isomaltulose in man and rat. Nutr Rep Int 28(5):1083-1090.

Würsch, P. 1991. Metabolism and tolerance of sugarless sweeteners. In: Rugg-Gunn, A.J.(Ed.). Sugarless: The Way Forward. Else vier Applied Science; New York, pp. 32-51.

Yamada, K.; Shinohara, H.; Hosoya, N. 1985. Hydrolysis of 1-O-α-D-glucopyranosyl-D-fructofuranose (Trehalulose) by rat intestinal surcrase-isomaltase complex.Nutrition Reports International 32 (5): 1211 - 1220

Ziesenitz, S.C. 1986. Carbohydrasen aus jejunalmucosa des Menschen = [Carbohydrases from the human jejunal mucosa]. Z Ernährungswiss 25(4):253-258. Cited In: Würsch, 1991.

Justification for selection of genetic toxicity endpoint

Hazard assessment is conducted by means of read-across from structural surrogates. All available in vitro and in vivo genetic toxicity studies

were negative. All available studies are adequate and reliable based on the identified similarities in structure and intrinsic properties between source and target substances and overall quality assessment (refer to the endpoint discussion for further details).

Short description of key information:

The following studies on genetic toxicity of read-across analogues are available for a weight-of-evidence approach:

Ames Test: negative; CAS 13718-94-0, isomaltulose (Asquith, 1986)

Ames Test: negative; CAS 57-50-1, sucrose (Bridges et al., 1981)

Ames Test: negative; CAS 64519-82-0, isomalt (Lina and Woutersen, 1995)

HPRT Test: negative; CAS 64519-82-0, isomalt (Lina and Woutersen, 1995)

MLA Test: negative; CAS 57-50-1, sucrose (Mitchell et al., 1988)

MNT: negative; CAS 64519-82-0, isomalt (Lina and Woutersen, 1995)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on read-across, the available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.