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

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 April 2014 to 27 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD test guideline study performed under GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

419-640-0

EC Name:

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

Cas Number:

68784-14-5

Molecular formula:

C3H4O to C36H54O11

IUPAC Name:

2-{[3,4-bis(prop-2-en-1-yloxy)-2,5-bis[(prop-2-en-1-yloxy)methyl]oxolan-2-yl]oxy}-3,4,5-tris(prop-2-en-1-yloxy)-6-[(prop-2-en-1-yloxy)methyl]oxane

Test material form:

other: straw coloured viscous liquid

Details on test material:

- Substance type: UVCB
- Physical state: straw coloured viscous liquid
- Analytical purity: not applicable
- Expiration date of the lot/batch: 04 April 2016
- Storage condition of test material: room temperature in the dark

Test animals

Species:

mouse

Strain:

ICR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon, UK
- Age at study initiation: 6 to 10 weeks
- Weight at study initiation: 21 to 28g
- Assigned to test groups randomly: yes
- Fasting period before study: None
- Housing: solid-floor polypropylene cages
- Diet (e.g. ad libitum): ad libitum Harlan Teklad 2014C diet
- Water (e.g. ad libitum): ad libitum mains drinking water
- Acclimation period: five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25C
- Humidity (%): 30 to 70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23 April 2014 To: 27 June 2014

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Arachis oil

Details on exposure:

Oral route by gavage.

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

Single

Post exposure period:

24 and 48 hours

No. of animals per sex per dose:

7 males per group

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide 50 mg/kg

Examinations

Tissues and cell types examined:

Bone marrow polychromatic and normochromatic cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:The maximum tolerated dose level was used as the highest dose (800 mg/kg). The two lower doses were 400 and 200 mg/kg.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Dosed once at time 0 and sampled either 24 or 48 hours later.

DETAILS OF SLIDE PREPARATION: Femurs were aspirated with foetal bovine serum and bone marrow smears prepared folowing centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grunwald/Giemsa, allowed to air-dry and a cover slip applied using mounting medium.

METHOD OF ANALYSIS: Stained bone marrow smear were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue staind immature cells) per animal was scored. Micronuclei ar enormally circular is shape, although occassionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for the incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviation.

Evaluation criteria:

A comparison was made between the number of MNPCE occuring in each of the test item groups and the number occuring in the vehicle control group. A positive mutagenic response was demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of MNPCE was observed for either the 24 or 48 hour time points when compared to the vehicle control group. If these criteria were not fulfilled, then the test item was considered to be non-gentoxic under the conditions of the test. A positive response for bone marrow toxicity was demonstrated when the dose group mean PCE to NCE ratio was shown to be statistically significantl y lower than the vehicle control group.

Statistics:

The data were analyzed following a square root (x+1) transformation using Student's t-test (two tailed) and any significant results were confirmed using one-way analysis of variance.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The test item was considered to be non-genotoxic under the conditions of the test.

Executive summary:

Introduction. The study wasa 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 difference 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 groups 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.