Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented journal publication of a GLP study conducted at a respected contract laboratory.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2002

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
A subchronic toxicity test was conducted in which the substance was administered orally in the diet to male and female rats
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Isomaltulose (6-O-(alpha-D-glucopyranosyl)-D-fructofuranose), CAS No. 13718-94-0, is a reducing disaccharide that occurs natually in honey and sugar cane juice. Purity of the tested substance was 97.8%.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Specific-pathogen-free-bred male and female Wistar rats (Crl:(WI)WU BR) were obtained by Charles River Deutschland (Sulzfeld, Germany) and acclimated to the laboratory conditions for nearly 2 weeks. At the start of the treatment period they were about 6 weeks old [mean body weights: males 176 g (range 157-204 g), females 141 g (range 125-156 g)]. They were housed in macrolon cages (one rat per cage) with stainless steel grid covers and wood shavings as bedding material, in a controlled environment (temperature 21-23 degrees C; humidity 30-60%; 12-h light/dark cycle; about 10 air changes per hour). The animals were housed individually to enable monitoring of individual food intake and food conversion efficiency in comparison with individual body weight development.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
A cereal-based, closed formula diet (Rat & Mouse No. 3 Breeding Diet, obtained from Special Diet Services, Witham, UK) was used as the basal diet. The experimental diets were prepared by supplementing the basal diet with isomaltulose and/or sucrose. Isomaltulose was incorporated at dietary levels of 2.5% (low-dose), 5% (mid-dose), and 10% (high-dose). The supplement in the low- and mid-dose diet was made up to 10% with sucrose. The control diet was made up of basal diet supplemented with 10% sucrose.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The diets were analyzed for isomaltulose using an HPLC method and found to be acceptable with respect to stability, homogeneity and achieved concentration of isomaltulose in the diet.
Duration of treatment / exposure:
The animals were fed the treatment diets daily for 13-weeks.
Frequency of treatment:
Daily in the diet
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 2.5, 5 or 10% isomaltulose
Basis:
nominal in diet
No. of animals per sex per dose:
Four groups of 20 rats per sex per dose
Control animals:
yes, concurrent no treatment
Details on study design:
Four groups of 20 animals per sex were given the experimental diet at each dose from the start of the study until necropsy early in week 14.

Examinations

Observations and examinations performed and frequency:
The animals were observed daily for abnormal clinical signs. Body weights were recorded weekly from day 0 and on the day of necropsy. Food consumption was measured over successive 1-week periods, for each animal individually, by weighing the feeders. Food conversion efficiency in successive weeks was calculated as g weight gain per g food consumed. The intake of isomaltulose per kg body weight was calculated from the nominal dietary levels of isomaltulose, the food intake and the body weight. Water consumption was measured per animal by weighing the drinking bottles daily during 5-day periods in weeks 1, 6 and 12. Ophthalmoscopic observations were made before initiation of treatment in all rats and in week 13 in the control and high-dose group using an ophthalmoscope after induction of mydriasis by a solution of atropine sulphate.
Sacrifice and pathology:
At the end of the treatment period, the rats were sacrificed by exsanguination from the abdominal aorta under light ether anaesthesia, and a thorough necropsy was performed. The adrenals, brain , full and empty caecum, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus and uterus of the rats were weighed (paired organs together). The relative organ weights were calculated on the basis of the terminal body weight of the rats. Samples of all organs and other tissues were examined for gross lesions and preserved in a neutral aqueous phosphate buffered at 4% solution of formaldehyde. Samples of the preserved organs from all rats of the control group and the high-dose group were processed and examined. The kidneys, liver and gross lesions were also examined in all rats of the intermediate dose groups.
Other examinations:
Examination of blood and urine was conducted on 10 rats per sex per group. Routine haematological examination was conducted in blood of non-fasted rats, collected from the abdominal aorta at necropsy early in week 14. The examination included haemoglobin, packed cell volume, red blood cell count, mean corpuscular volume, mean corpuscular concentration, total white blood cell count, thrombocyte count, differential white blood cell counts, and prothrombin time. Routine clinical chemistry was conducted in the same 10 rats per sex per group as used for haemotology. At necropsy, blood was collected from the abdominal aorta in heparinized tubes and plasma was prepared by centrifugation, then examined for alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, total protein, albumin, albumin/globulin ratio, urea, creatinine, total bilirubin, total cholesterol, triglycerides, phospholipids, calcium, inorganic phosphate, sodium, potassium and chloride. Additional blood and glucose examinations were also measured. Neurobehavioral functioning was evaluated in week 13 in those 10 rats per sex per group that were not used for collection of blood and urine. The FOB consisted of non-invasive observational and interactive measures designed to assess the neurobehavioral and functional integrity of the rat, using measures taken from different functional domains including autonomic and neuromuscular function, sensorimotor reactivity, arousal and excitability.
Statistics:
Body weights were evaluated by a one-way analysis of covariance (covariate: body weight at initiation of treatment) followed by Dunnett's multiple comparison tests. Other parameters were evaluated by ANOVA follwed by Dunnett's. Other standard statistical methods were used as appropriate.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
All rats survived until scheduled necropsy and appeared healthy throughout the study. All parameters measured were normal throughout the study. No effects or signs of toxicity were observed in any animal in any dose. The test substance is not toxic to rats at doses up to 10% in the diet given daily for 13 weeks.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
> 8 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Equivalent to 10% in the diet
Dose descriptor:
NOAEL
Effect level:
> 7 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Equivalent to 10% in the diet

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The test substance is not toxic.
Executive summary:

A subchronic toxicity test was conducted in which the substance was administered orally in the diet to male and female rats. Four groups of 20 rats per sex per dose were fed the treatment diets daily for 13-weeks at doses of 0, 2.5%, 5%, and 10%. All of the usual clinical parameters were measured. All rats survived until scheduled necropsy and appeared healthy throughout the study. All parameters measured were normal throughout the study. No effects or signs of toxicity were observed in any animal in any dose. The test substance is not toxic to rats at doses up to 10% in the diet given daily for 13 weeks.