Registration Dossier

Administrative data

Endpoint:
developmental toxicity
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:
1997

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
An embryotoxicity/teratogenicity study was conducted in which the substance was administered orally in the diet from day 0 to day 21 of pregnancy.
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, a white crystalline powder, was 98.5% TS.

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Twelve-week old, specific-pathogen-free-bred Wistar rats (Hsd/Cpb:WU) were obtained from Harlan-CPB, Zeist, The Netherlands, and acclimated to the animal facilities for 5 days before mating. The rats were housed in suspended stainless-steel wire-screen cages. Mated females were housed individually. The room was ventilated with 10 air changes/hour and maintained at 22-24 degrees C, a relative humidity of 50-60%, and a 12-hour light/dark cycle.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
The rats were fed the Institute's cereal-based powdered stock diet, the composition of which was 11% soybean oil meal, 7% fish meal, 4% meat and bone scraps, 36% whole ground wheat, 29.7% whole ground maize, 3% grass meal, 2% whey powder, 0.4% defatted bone meal, 3% soybean oil, 0.5% trace elements and salt, 3% brewers yeast, 0.1% vitamin B preparation, and 0.3% vitamin ADEK mixture. During the treatment period the stock diet was supplemented with 2.5% casein, 0.1% DL-methionine and 0-10% isomaltulose at the expense of the maize starch. Fresh portions were fed to the rats every 4-6 days. Feed and water were provided ad libitum.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All diets were analyzed for isomaltulose content (after extraction in water and protein precipitation) by HPLC using a Nucleosil NH2 column. Five batches were analyzed to evaluate the mixing procedure. The stability of isomaltulose was examined after storage for 7 days at room temperature.
Details on mating procedure:
Young adult female rats were mated with males (male/female ratio 1:2). The day of detection of sperm cells in the vaginal smear was considered day 0 of pregnancy. Mated females were then assigned randomly to four groups of 24 animals each, which were then fed diets containing 0, 2.5, 5 or 10% isomaltulose from day 0 to day 21 of pregnancy.
Duration of treatment / exposure:
Mated females were exposed to isomaltulose in the diet from day 0 to day 21 of pregnancy.
Frequency of treatment:
Daily in the feed.
Duration of test:
21 days
Doses / concentrations
Remarks:
Doses / Concentrations:
o, 2.5%, 5%, 10%
Basis:
nominal in diet
No. of animals per sex per dose:
24 female animals per dose
Control animals:
yes, plain diet

Examinations

Maternal examinations:
The general condition and behavior of all animals were checked daily. During gestation, body weight gain and food and water consumption were measured. At autopsy, the pregnant rats were evaluated by macroscopy of major abdominal and thoracic maternal organs,and weights of the ovaries taken.
Ovaries and uterine content:
The number and weights of gravid and empty uterus, the number of corpora lutea, implantation sites, live and dead fetuses and early or late resorptions, the sex of the live fetuses, fetal and placental weights, fetal length and the presence of external malformations were measured.
Fetal examinations:
Besides that already noted, half of the fetuses of each litter were fixed in 70% ethanol, eviscerated, partly skinned, cleared and stained with Alizarin Red S for examination of the skeleton. The remaining fetuses were fixed in Bouin's fluid for visceral examination. Both skeletal and visceral screening were performed blindly in all groups.
Statistics:
Data on maternal body weights, food and water intake, organ weights, fetal weights and lengths, and placental weights were subjected to one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. Maternal survival and pregnancy status, maternal and fetal external macroscopic observations, and skeletal and visceral examinations were evaluated by Fisher's exact probability test. The percentages of pre- and post-implantation loss were subjected to Kruskal-Wallis one-way ANOVA followed by the Mann-Whitney U test.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No clinical signs attributable to the treatment were observed. None of the dams died and no gross abnormalities of the tissues or organs were observed. Body weight gain and food and water intake showed no treatment-related differences during pregnancy.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
> 7 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
> 7 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no signfiicant differences in fertility and gestation indices, the numbers of corpora lutea and implantations, live and dead fetuses, fetal sex ratio and early and late resorptions. Weights of ovaries and gravid and empty uterus were comparable between treatment and control groups, as was placental weight, fetal weight, and fetal length in both sexes. The few fetal external findings observed were randomly distributed among all groups. No treatment-related changes in fetal soft tissues of visceral malformations were observed. No fetal skeletal malformations occurred and there were no changes of toxicological significance in fetal skeletal anomalies or fetal skeletal variations.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

A summary of the reproductive performance of rats is shown in the table below.

Reproductive performance of rats fed isomaltulose from day 0 to day 21 of pregnancy

Values for rats fed isomatulose at dietary levels (%) of

Parameters

Statistics

0

2.5

5

10

No. of mated females

 

24

24

24

24

No. of pregnant females

(f)

23

22

23

24

Fecundity index C/011

 

96

92

96

100

No. of females bearing live foetuses

 

23

22

23

24

No. of corpora lutea per dam

(k)

13.1 ± 0.4

13.3 ± 0.3

13.3 ± 0.3

13.4 ± 0.4

No. of implantations per dam

(k)

11.9 ± 0.5

12.1 ± 0.6

11.7 ± 0.5

12.0 ± 0.3

Preimplantation loss (%)2

(k)

8.9 ± 2.5

9.6 ± 4.1

12.0 ± 3.3

10.1 ± 2.1

No. of resorptions per litter

(k)

0.4 ± 0.1

0.5 ± 0.1

0.5 ± 0.2

0.5 ± 0.2

No. of dead foetuses per litter

(k)

0.1 ± 0.1

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

Postimplantation loss (%)3

(k)

4.3 ± 1.5

4.0 ± 1.2

4.5 ± 1.4

4.1 ± 1.6

No. of live foetuses per litter

(k)

11.4 ± 0.5

11.6 ± 0.6

11.1 ± 0.5

11.5 ± 0.4

No. of males per litter

(k)

5.7 ± 0.5

5.7 ± 0.5

5.0 ± 0.4

5.7 ± 0.4

No. of females per litter

(k)

5.7 ± 0.4

5.8 ± 0.4

6.2 ± 0.5

5.8 ± 0.4

Ovary weight (mg)

(a)

101.2 + 4.1

98.0 + 4.0

99.4 + 4.8

94.4 + 2.3

Gravid uterus weight (g)

(a)

73.4 ± 2.9

74.8 ± 3.6

73.6 ± 2.0

75.0 ± 2.0

Empty uterus weight (g)

(a)

5.0 ± 0.2

5.1 + 0.3

4.8 + 0.2

5.0 ± 0.2

Placental weight (g)

(a)

0.52 ± 0.01

0.54 ± 0.02

0.54 ± 0.02

0.56 ± 0.03

Foetal weight (g)

(a)

4.85 ± 0.04

4.87 ± 0.06

4.92 ± 0.05

4.94 ± 0.05

Foetal length (cm)

(a)

4.1 ± 0.0

4.1 ± 0.0

4.1 ± 0.0

4.0 ± 0.0

 

Values (other than percentages) are means ± SEM.

Statistical analyses did not reveal any significant differences between the treatment groups and the controls; a = ANOVA/Dunnett's test; f = Fisher's exact probability test; k= Kruskal-Wallis/Mann-WhitneyUtest.

'Fecundity index (%) = (no. of pregnant females/no. of mated females) x100.

2Preimplantation loss (%) = [(no. of corpora lutea-no. of implantation sites)/no. of corpora lutea] x100.

3Postimplantation loss (%) = [(no. of implantation sites-no. of live foetuses)/no. of implantation sites] x100.

Applicant's summary and conclusion

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

An embryotoxicity/teratogenicity study was conducted in which the substance was administered orally in the diet from day 0 to day 21 of pregnancy. Doses were control, 2.5%, 5% and 10% isomaltulose in the feed. No maternal toxicity occurred during the study, and no effects on reproductive performance, embryotoxicity, fetal development, or teratogenicity were observed. Therefore the dietary level of 10% isomaltulose, which is equivalent to approximately 7000 mg/kg bw/day, is considered a no effect level.