Constitutional isomers of penta-O-allyl-β-D-fructofuranosylα-D-glucopyranoside; Constitutional isomers of hexa-O-allyl-β-D-fructofuranosylα-D-glucopyranoside; Constitutional isomers of hepta-O-allyl-β-D-fructofuransoylα-D-glucopyranoside

Endpoint summary

Additional information

OECD471 Bacterial Mutation test

Introduction The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline 870.5100 - Bacterial Reverse Mutation Test.

Methods Salmonella typhimurium strains TA1535, TA1537, TA98 an d TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 ug/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended, following the results of Experiment 1, and was 150 to 5000 ug/plate.

Six test item dose levels were selected in the main test in order to confirm whether a statistically significant increase in TA1535 revertant colony frequency, noted in the range-finding test, was real or spurious.

Results The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 ug/plate . There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the first mutation test and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the second mutation test. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

The test item induced a dose-related, reproducible and statistically significant increase in the frequency of TA1535 revertant colonies from 1500 ug/plate in both the absence and presence of S9-mix. The increases observed for TA1535 were large and well above the in-house historical control ranges for the strain with a maximum fold increase in excess of 10-fold over the concurrent vehicle control in the main test at 5000 ug/plate in the presence of S9-mix. Small, statistically significant increases were also noted for TA100. No significant increases in the frequency of revertant colonies were recorded for any of the remaining bacterial strains, with any dose of the test item, either with or without metabolic activation.

Conclusion The test item was considered to be mutagenic to the bacterial strains TA1535 and TA100 under the conditions of this test.

OECD476 Gene mutation test in mammalian cells

Introduction The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

Method Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24 hour exposure group in the absence of metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The maximum dose level used in the main test was limited by test item induced toxicity.

Results The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system. The test item did not induce any toxicologically significant or dose-related (linear-trend) increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment.

Conclusion The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.

OECD474 Micronucleus test in the mouse

Introduction. The study was performed to assess the potential of the test item to induce damage to chromosomes or aneuploidy when adminstered to mice. The method was designed to be compatible with the 1997 OECD Guidelines for Testing of Chemicals No. 474 "Mammalian Eryuthrocyte Micronucleus Test".

Methods. A range-finding test was performed to find suitable dose levels of the test item, route of adminstration and to investigate if there was a marked difference in toxic response between the sexes.There was no marked diofference in toxicity between the sexes and therefore the main test was performed in males only. The micronucleus test was conducted using the oral route in gropups of seven male mice at the maximum tolerated dose of 800 mg/kg, with 400 and 200 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted and smear preparation made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei.

Results. There were no premature deaths seen in any of the dose groups. The following clinical signs were observed at 800 mg/kg in both the 24 and 48 -hour exposure groups: hunched posture and ptosis. No statistically significant decreases in the PCE/NCE ratio were observed in any test item dose group when compared to the vehicle control group. There was no evidence of any significant increases in the incidence on MNPCE in animals dosed with the test item when compared to the vehicle control group.The positive control group showed a marked increase in the incidence of MNPCE hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Conclusion. The test item was considered to be non-genotoxic under the conditions of the test.

Overall Conclusion on Genetic Toxicity

The substance has shown weak mutagenic activity in two strains of Salmonella that are designed to detect base-pair substitution. mutagenic events. The response was almost identical in both the presence and absence of S9, indicating that the mutagenic component was direct acting and was not mitigated by the metabolism of S9. However, a mutagenicity assay in mammalian cells, the L5178Y TK assay, that can detect both point mutations and clastogenic events, was clearly negative in all exposure conditions and up to dose levels that caused marked toxicity. Consequently, it may be concluded that the mutagenic activity observed in bacteria was not reproduced in mammalian cells. In addition, an in vivo study in the mouse examining bone marrow cells for micronuclei was performed. The dose levels used achieved significant toxicity and clinical signs were observed, which were taken as evidence that systemic absorption and exposure to the target tissue had been achieved. The study showed no evidence of clastogenic or aneugenic potential in vivo. It is concluded that the substance is of no concern for mutagenicity because the weak bacterial mutagenic effects were not observed in mammalian systems and the substance is considered to be bacterial specific mutagen.

Justification for selection of genetic toxicity endpoint
A positive result was observed in the bacterial mutagenicity test, both with and without metabolic activation. However, no mutagenic or clastogenic effects were observed in a mammalian cell assay (mouse lymphoma L5178Y cells). Furthermore, a negative result was obtained in an in vivo mouse micronucleus study. Therefore, the result of the bacterial assay is considered to be of no concern to humans.

Short description of key information:
GLP compliant Bacterial mutation test according to OECD 471, GLP compliant gene mutation assay in mouse lymphoma L5178Y cells according to OECD 476, a GLP compliant micronucleus test in the mouse according to OECD 474.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No positive in vivo genotoxicity results therefore no classification is warranted or required.