1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl 4-chloro-4-deoxy-α-D-galactose

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1984 version of OECD 474

GLP compliance:

not specified

Remarks:

Predates GLP

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl 4-chloro-4-deoxy-α-D-galactose

EC Number:

259-952-2

EC Name:

1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl 4-chloro-4-deoxy-α-D-galactose

Cas Number:

56038-13-2

Molecular formula:

C12H19Cl3O8

IUPAC Name:

1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl 4-chloro-4-deoxy-α-D-galactose

Test material form:

solid: crystalline

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: From Sponsor / Batch Number 163003
- Purity: 99.4% (HPLC)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 0-4 degrees C in the dark
- Solubility and stability of the test substance in the solvent/vehicle: Soluble in distilled water up to maximum concentration of 330 mg/ml
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not reactive

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: Dissolved in water

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Preparations of test compound were mixed with dosing vehicle (distilled water) to achieve appropriate concentrations

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK
- Age at study initiation: 4-5 weeks
- Weight at study initiation: 17-24g
- Housing: Polypropylene cages, single sex, groups of 5
- Diet: ad libitum
- Water:ad libitum
- Acclimation period: minimum of 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 (+/-2)
- Humidity (%): 55% (+/-20%)
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Distilled, sterile water.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

Test compound dissolved in sterile distilled water on the day of use.

Duration of treatment / exposure:

Single dose, termination and sampling after 24, 48 and 72 hours.

Frequency of treatment:

single dose

Post exposure period:

24, 48 and 72 hours.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

5 000 mg/kg bw/day

No. of animals per sex per dose:

5 animals per sex per dose per time point (24, 48 & 72 Hrs)

Control animals:

yes

yes, concurrent vehicle

Positive control(s):

Chlorambucil
- Route of administration: oral
- Doses / concentrations: 30 mg/kg in 10% ethanol

Examinations

Tissues and cell types examined:

Both femurs of each mouse were dissected out following termination by carbon dioxide gassing and cervical dislocation.
Marrow cells were flushed into clean centrifuge tubes with 2.5 ml foetal calf serum, the suspension was centrifuged (1000 RPM, 5 minutes) and the bulk of supernatant removed.

Three smear slides were prepared from the cells of each animal and stained manually using the Schmid staining technique.

Slides were evalulated blind to eliminate bias, approximately, 2000 mature and polychromatic erythrocytes were scored for each animal, at least 1000 of each cell type will be scored for the presence of micronuclei.

Details of tissue and slide preparation:

Both femurs of each mouse were dissected out following termination by carbon dioxide gassing and cervical dislocation.
Marrow cells were flushed into clean centrifuge tubes with 2.5 ml foetal calf serum, the suspension was centrifuged (1000 RPM, 5 minutes) and the bulk of supernatant removed.

Three smear slides were prepared from the cells of each animal and stained using the Schmid staining technique.

Slides were evalulated blind to eliminate bias, approximately, 2000 mature and polychromatic erythrocytes were scored for each animal, at least 1000 of each cell type will be scored for the presence of micronuclei.

Evaluation criteria:

A total of ~2000 mature and polychromatic erythrocytes per animal were scored for the presence or abcence of micronuclei, the study aimed to score 1000 each of polychromatic and mature erythrocytes where possible, only deviating where there was a significant shift in the ratio of polychromatic to mature cells.

The counts for mature erythrocytes provide a check on the results of the younger cells, the ratio of mature cells to polychromatic cells was calculated for each animal as an indicator of gross toxicity.

Statistics:

Comparisons of the number of micronuclei scored in polychromatic cells between the control amd treatment groups were made using the Mann-Whitney U test.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

No Treated animals showed any adverse reactions to treatment, all animals survived to termination.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Frequencies of micronucleated polychromatic erythrocytes in treated animals at 24,48 and 72 hours post exposure were similar to those in concurrent controls, there were no significant differences observed between sexes in any group.

Statistical analysis indicated that there was a significant increase in the frequency of micronucleated polychromatic erythrocytes at a dosage of 1000mg/kg and 72hrs post exposure. This was caused by an unusually low occurance of micronuclei in the concurrent control animals (9/10 mice showed no micronuclei), no sucralose treated animals gave values in excess of 3.2 micronuclei per 1000 cells scored, therefore this apparent increase is not considered to to be of biological significance.

Statistically and biologically significant increases in the number of micronucleated polychromatic erythrocytes over control values were seen in positive control group animals given chlorambucil at 30mg/kg, demonstrating the sensitivity of the system.

Any other information on results incl. tables

Exposure period	24 hrs				48 hrs			72 hrs
Group	1	2	3	4	1	2	3	1	2	3
Mean Micronucleated cells / 1000 polychromatic cells (S.D)	0.7	0.7	0.4	23.7	0.7	1	0.5	0.1	1.2	0.6
Range	0.0-2.0	0.0-2.8	0.0-1.7	10.3-32.0	0.0-2.0	0.0-2.0	0.0-1.8	0.0-0.9	0.0-3.3	0.0-2.7
Ratio Polychromatic cells:mature cells	1	1	1	1	1	0.9	1	1	0.9	1

Group 1	distilled water
Group 2	1000mg/kg Sucralose
Group 3	5000mg/kg Sucralose
Group 4	Chlorambucil

Applicant's summary and conclusion

Conclusions:

The genetic toxicity of the test item on mammalian cells was determined in a study according to the OECD guideline 474. It is concluded that, under the conditions of the test, there was no biologically significant evidence of treatment induced damage to chromosomes or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice after 24, 48, or 72 hours of exposure to sucralose.