Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Reference 2

Reference Type:

publication

Title:

Embryotoxicity/Teratogenicity Of Isomalt In Rats And Rabbits

Author:

Waalkens-Berendsen, D.H., et al.

Year:

1990

Bibliographic source:

Fd Chem. Toxic. 28: 1-9

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted in 2001

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol

EC Number:

908-700-6

Cas Number:

64519-82-0

Molecular formula:

6-O-alpha-D-Glucopyranosyl-D-sorbitol: C12H24O11 1-O-alpha-D-Glucopyranosyl-D-mannitol: C12H24O11

IUPAC Name:

Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol

Test material form:

solid: crystalline

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Cpb:WU (Wistar, random)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Central Institute for the Breeding of Laboratory Animals TNO, Zeist, The Netherlands
- Weight at study initiation: 153 to 204 g
- Fasting period before study: no
- Housing: in supsended steel cages fitted with wire-mesh floors (4 females/cage during acclimatization, individually during pregnancy)
- Diet: stock diet for rats, modified with casein, d1-methionine and 10% maize food starch. The modification allowed the incorporation of up to 10% test item at the expense of maize starch (see Table 1). Food was offered ad libitum, to the control and all test item groups. A restricted feeding control group received 80% of the amount consumed by the control group animals from day 1 onwards.
- Water: source not specified (ad libitum)
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): ≥ 40
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 Oct 1981 To: 25 Nov 1981

Administration / exposure

Route of administration:

oral: feed

Vehicle:

not specified

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): not specified
- Mixing appropriate amounts with (Type of food): not specified
- Storage temperature of food: not specified

TEST ITEM INTAKE
- Test item intake was not specified in the report.
- Test item intake was calculated retrospectively based on mean body weights and food consumption at the end of the period of organogenesis. The calculation of test item intake is detailed in Table 3.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The actual levels of test item in the diet were analysed once during the study and were found to equal or close to the target concentrations (see Table 2).

Details on mating procedure:

- Impregnation procedure: co-housed
- If co-housed:
- M/F ratio per cage: 1/2
- Length of cohabitation: overnight
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

from gestation day 0 to 21

Frequency of treatment:

daily, 7 days/week

Duration of test:

throughout pregnancy (day 0 to 21)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2.5 other: %

Remarks:

corresponding to 25 000 ppm (ca. 1970 mg/kg bw/day, calculated from mean bw and food consumption)

Doses / concentrations 2

Dose / conc.:

5 other: %

Remarks:

corresponding to 50 000 ppm (ca. 4120 mg/kg bw/day, calculated from mean bw and food consumption)

Doses / concentrations 3

Dose / conc.:

10 other: %

Remarks:

corresponding to 100 000 ppm (ca. 7970 mg/kg bw/day, calculated from mean bw and food consumption)

No. of animals per sex per dose:

22 - 23 females/dose

Control animals:

yes

yes, plain diet

Details on study design:

- Dose selection rationale: not specified
- Rationale for animal assignment: Mated fermales were assigned to each group in rotation. The sequence was continued from where it ended on the previous day until each group contained the desired number of mated females.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: general condition and behaviour were checked

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: daily from day 0 until day 21 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Ovaries and empty uteri as well as placentas of live fetuses were weighed

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Both soft tissue and skeletal examinations were carried out on the foetuses of the control and high dose group as well as of foetuses of the restricetd feeding group.

Statistics:

For the statistical analyses of differences in degree of ossification between the test and control groups the Student t-test was applied to transformed ossification values (DgOt) expressed in degrees and calculated by the formula:
DgOt = arcsine √DgO°

Statistical analysis of differences in body weight, food consumption, organ weights, litter data, foetus weights and lengths and placenta weights was carried out by applying analysis of co-variance, followed by Dunnett's multiple comparison tes, whereas skeletal and visceral anomalies were evaluated by the Chi-square test.

Indices:

Percentage pre-implantation loss (PRIL) was calculatde for each litter by the formula:
PRIL = number of corpora lutea - number of implantation sites / number of corpora lutea x 100%

Percentage post-implantation loss (POIL) was calculated for each litter by the formula:
POIL = number of implantation sites - number of live young / number of implantation sites x 100%

The degree of ossification of foetus skeletons (DgO) was calculated for each litter by the formula:
DgO = number of bones without ossification (or with incomplete ossification) / numbers of bones examined

Historical control data:

Historical control data on reproduction data and on fetal malformations and variations (external, skeletal and visceral) are provided in the report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Some females in each of the groups showed focal baldness. No signs of ill-health nor any reaction to the treatment were observed.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean maternal body weights and weight gain during pregnancy were similar for both the controls and all test item-fed females. The body weights of pregnant females which had access only to restricted amounts of food stayed significantly behind as compared with the controls. During the full gestation period these animals gained only 60% of the mean weight gain of control animals.

Corrected body weight gains were similar in all groups with diet offered ad libitum. In the restricted feeding group corrected body weight gain was only 16% of that of the control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean food intake was similar in all test item groups and the controls.
Since, the animals of the restricted-feeding group were provided daily with only 80% of the food consumed by the controls during the preceeding day, these animals consumed practically all the food offered. However, since the amounts of food were calculated on the basis of body weights, the actual food intake per animal in this group appeared to be even less than 80% of the food consumed by controls.

Food efficiency:

no effects observed

Description (incidence and severity):

There were no consistent or dose-related differences in food efficiency between the test item groups and the controls.
The grand means of the restricted-feeding group were lower than the control value.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Gravid and empty uterus weights were significantly decreased in the restricted feeding group only.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At autopsy a minor degree of haemorrhagic nasal discharge was noticed in one dam of the 5.0% test item and in two dams of the restricted-feeding group. One female of the 10.0% test item group showed diarrhoea and one control animal exhibited a periocular haemorrhage. Focal baldness was observed in several dams in all groups.
No gross maternal organ changes were seen in in dams of any of the groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The numbers of early and late resorptions were similar in the test item groups and the control. A slight increase of the number of resorptions and through that of post-implantation loss was observed in the restricted­ feeding group.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

The feeding of restricted amounts of stock diet to pregnant females resulted in decreased maternal weight gain and consequently, in lower uterus weights. The effects on foetuses were principally of an embryotoxic nature as expressed by increased numbers of resorptions, smaller foetuses, decreased foetus weights and retardation in bone development. It is therefore concluded that restriction of the diet of pregnant rats during gestation induces embryotoxic effects.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 7 970 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects noted up to and including the highest dose tested

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Foetal weights as well as foetal rump lengths were significantly decreased in the restricted-feeding group only.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Spina bifida was observed in 3 foetuses of one litter in the high dose group. Further, one foetus of the high dose group and one foetus of the restricted-feeding group showed a ringtail. No other externally visible malformations were observed.

External variants were seen in several foetuses; subcutaneous haemorrhages or petechia occurred in one or a few foetuses in all groups, while one dismature foetus was observed in the high dose group. None of these findings were considered to be related to the treatment.

Although spina bifida occurred in 3 foetuses of the top-dose group only, the total number of foetuses with skeletal or visceral malformations in this group was similar to the number of affected control foetuses. In addition, the percentage of litters with one or more foetuses showing malformations was even higher in the control (4 out of 17) than in the high dose group (3 out of 19). Therefore, and since all 3 spina bifida foetuses originated from the same litter, no significance is attached to these findings.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were observed in 5 foetuses: in 2 foetuses from the control, in 2 from the high dose, and in 1 foetus from the restricted­ feeding group. Neither the type nor the incidence of the observed malformations indicated any treatment-relationship.

Minor skeletal anomalies and skeletal variants were observed in several foetuses in all groups or occasionally in one or a few foetuses.
The number of foetuses showing dislocation of one or several sternebrae was increased in both the high dose and the restricted-feeding group. Further, the incidences of supernumerary ribs and skull bones was increased in the restricted-feeding group only.

A slightly higher number of incompletely ossified cervical vertebral bodies in the high dose group was considered an incidental finding and reflects the variation in ossification normally found in skeletons of foetuses of the strain of rats used. For the remaining data on ossification degrees no differences were observed between the control and high dose group.
In the restricted-feeding group however decreased degrees of ossification were observed in several bones such as metacarpals, metatarsals and cervical thoracic vertebral bodies.

The somewhat higher number of dislocated sternebrae in the high dose group as compared with the control group, is not considered of significance since, firstly no other indications were noticed that might point to a skeletal effect of the test substance and secondly, the observed control values were rather low in comparison with those of historical control series.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Visceral malformations were observed in 7 foetuses: the control group revealed 2 foetuses with microphthalmia and 1 foetus with anophthalmia. They originated all three from different litters. In the high dose group spina bifida was observed in 2 foetuses from one litter. One of these foetuses also showed microdactyly of a hindlimb. Further, a double kidney was observed in one foetus from a different litter. The restricted-feeding group revealed only one foetus with visceral malformations consisting of right-sided testicular cryptorchism and left-sided testis atrophy.

The other observations listed,were either minor anomalies or visceral variants.

Neither meningial haemorrhages nor dilated brain ventricles were observed in the control group. However, since these anomalies are regularly being observed in our control series, these findings were considered to reflect the variation normally found, rather than to be related to the treatment. The other minor anomalies were about equally spread among the high dose, restricted-feeding and the control animals or occurred occasionally in one or a few foetuses.
No differences were noted in the nature of the observed visceral variants. Although the number of foetuses with unilaterally increased renal pelvic cavitation was slightly higher in the high dose group as compared with controls, the observed incidence is fully comparable with values observed in historical control series. Moreover, there were no other indications of a treatment-related soft tissue effect. Therefore, no significance was attached to the somewhat higher incidence of minor kidney variants.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 7 970 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects noted up to and including the highest dose tested

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Table 2. Analytical results of test substance concentration in diet

Nominal concentration (%)	Actual concentration (%)
2.5	2.5
5.0	5.0
10.0	9.6

Table 3. Achieved test item intake

	Nominal concentration of test item in diet (%)
	0.0	2.5	5.0	10.0
Body weights (g, mean bw at end of organogenesis, day 16)	238.4	241.7	240.1	240.9
Food consumption (g/food/animal/day, mean food consumption at end of organogenesis, day 16)	18.5	19.0	19.8	19.2
Food consumption (g/food/kg bw/day, mean of means)	77.6	78.6	82.5	79.7
Test item intake (g/kg bw/day)	0.0	2.0	4.1	8.0
Test item intake (mg/kg bw/day)	0.0	1965.2	4123.3	7970.1