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Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14. January 1998 - 9. July 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Trehalose
EC Number:
202-739-6
EC Name:
Trehalose
Cas Number:
99-20-7
Molecular formula:
C12H22O11
IUPAC Name:
trehalose
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: 7L111
- Expiration date of the lot/batch: 10 December 1999
- Purity test date: not stated

RADIOLABELLING INFORMATION (if applicable)
not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at ambient temperature
- Stability under test conditions: assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: assumed stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
none

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga, Germany
- Age at study initiation: 11 weeks (male) / 12 weeks (female)
- Weight at study initiation: ca. 210 g
- Fasting period before study: no
- Housing:
During the quarantine and acclimatization period, the males and females were housed
in groups of 4 per sex
For mating one male and two females were housed
together
Mated females were housed individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/ 12

IN-LIFE DATES: From: 14. January 1998 To: 24. February 1998

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
During the quarantine and acclimatization period, the rats were fed a closed formula
diet obtained from SDS.
From day 0 of gestation, the female rats were fed a somewhat modified diet (batch no.
4070) supplemented with different concentrations of the test substance. The
modification consisted of the omission of 20% barley from the diet. The barley was replaced by the test substance and/or pregelatinized potato starch.
Diets were prepared by mixing the various ingredients in a mechanical blender and
stored in a refrigerator.

VEHICLE
none
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses to determine the content, homogeneity and stability of the test substance in
the carrier were conducted in all diets using HPLC method.
Before analysis of samples from the study, the method was validated to conform with
the following criteria:
- linearity: the correlation coefficient of the calibration curves should be greater than
or equal to 0.996;
- selectivity: no peak should be found in the blank carrier with a retention time of 95-
105% of that of the test substance;
- repeatability: the relative standard deviation in the percentage recovery and the
retention time when the recovery test is performed 3 times at each concentration
used in the study should be less than 10 and 2% respectively;
- recovery: the recovery of the test substance from the carrier should be between 80
and 110% at all concentrations used in the study.
The stability of the test substance under (simulated) experimental conditions was
demonstrated by analyzing samples with (low-, mid- and high dose) and without
(control) the test substance on the day of diet preparation, after storage at room
temperature in an open container (for 7 days) and after storage in a refrigerator in a
closed container (for about 5 weeks).
To determine the homogeneity of the test substance in the diet, 5 samples per dose
level (low-, mid- and high-dose) taken once at different locations in the feed container
and 1 control sample were analyzed.
Diet samples will be taken immediately after preparation of the diets and stored at
< -18 °C pending analysis.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2
- Length of cohabitation: until pregnancy occurs
- not applicable: After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: not applicable
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol:
Duration of treatment / exposure:
The test substance was administered in the diet from fertilization (gestation day 0) up to
Cesarian section (gestation day 21).
Frequency of treatment:
once dayly
Duration of test:
from fertilization (gestation day 0) up to
Cesarian section (gestation day 21)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg diet
Remarks:
control group
Dose / conc.:
25 000 mg/kg diet
Dose / conc.:
50 000 mg/kg diet
Dose / conc.:
100 000 mg/kg diet
No. of animals per sex per dose:
28
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: not stated
- Rationale for animal assignment (if not random):
After mating the mated females were distributed over the 4 experimental groups in
such a way that the animals from the same day of pregnancy were equally distributed
over all groups.
In the mid-dose group 2 females (C151 and C167) were mated by the same male (48);
all other females mated by the same male were placed in different groups
- Other:

Examinations

Maternal examinations:
Each female was observed daily from the start of the study and, if necessary, handled
to appraise its physical condition. Signs of ill health and reaction to treatment as well
as mortality were recorded. On working days, all cages were checked again late in the
afternoon for dead or moribund animals to minimize loss of animals from the study.
During weekends and holidays only one check per day was carried out.

Body weights of the rats were recorded on days 0, 7, 14 and 21 of gestation.

The quantity of food consumed by each animal was measured over the periods days 0-
7, 7-14 and 14-21 of gestation by weighing the feeders.

The females were killed by decapitation after ether anaesthesia on gestation day 21 and
examined for gross abnormalities. Maternal tissue showing severe macroscopic
abnormalities was removed and fixed in a neutral, aqueous phosphate buffered 4%
solution of formaldehyde.
Ovaries and uterine content:
The uteri (including the fetuses), ovaries and placentas of all females killed on day 21
were examined for the following parameters:
- number of corpora lutea
- number of implantation sites
- number of early and late resorptions
- number of live and dead fetuses
- sex of the fetuses
- number of grossly visible malformed fetuses and fetuses with external
abnormalities
- weight of ovaries
- weight of uterus, containing placentas and fetuses
- weight of uterus, empty
- weight of fetuses
- weight of the placentas
- gross evaluation of the placentas
Fetal examinations:
First all fetuses of each litter were examined carefully for anomalies. The sex of the
fetuses was determined. Half of the number of fetuses in each litter was fixed in
Bouin's fixative and subsequently examined for soft tissue anomalies according to a
method modified after Barrow and Taylor (1969) and then discarded.
The other half of the foetuses were fixed in 70% ethanol, subsequently partly
eviscerated and then cleared in potassium hydroxide and stained with Alizarin Red S
modified after Dawson (1926). These foetuses were examined for skeletal
abnormalities and then retained.
The fetopathological examinations were initially restricted to all foetuses of the animals
of the control group and the high dose group. Only when alterations were observed in
the high-dose group, the examinations were, after consultation with the sponsor,
extended to the intermediate-dose groups.
Statistics:
The resulting data were analyzed using the methods mentioned below. P < 0.05 was
considered as a level of significance.
Clinical findings were evaluated by Fisher's exact probability test.
Body weight, body weight gain, organ weights and food consumption data were
subjected to one-way analysis of variance (ANOVA) followed by Dunnett's multiple
comparison tests.
Fisher's exact probability test was used to evaluate the number of pregnant females
and females with live fetuses. Number of corpora lutea, implantations, live and dead
fetuses and early and late resorptions were evaluated by Kruskal-Wallis nonparametric
analysis of variance followed by the Mann-Whitney U-test .
Indices:
For each group the following data were recorded:
- female fecundity index = (number of pregnant females/number of females
mated) x 100
- pre-implantation loss = [(number of corpora lutea - number of implantation
sites)/ number of corpora lutea] x 100
- post-implantation loss = [(number of implantation sites- number of live
fetuses)/number of implantation sites] x 100
- gestation index = (number of females with live fetuses/number of females
pregnant) x 100
- sex ratio = (number of live male fetuses/number of live fetuses) x 100
Historical control data:
not stated

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
Animal A53 and D 219 of the control and high-dose group showed haemorrhagic
discharge on gestation day 21 and 14, respectively.
Further, daily clinical observations during the gestation period did not reveal any
remarkable findings in the animals' appearance, general condition or behaviour
amongst the dosing and control groups.
Mortality:
no mortality observed
Description (incidence):
No mortalities were observed.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No statistically significant differences in mean body weights and body weight changes
were observed amongst the control and the groups fed trehalose in the diet.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No statistically significant difference in food consumption was observed amongst the
control and the groups fed trehalose in the diet.
Food efficiency:
not specified
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):
No remarkable differences in gravid and empty uterus
weight, ovary weight, carcass weight and net weight change (body weight gain from
day 0 to day 21 of gestation minus gravid uterine weight) were observed between the
control group and the groups fed trehalose in the diet.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross examination did not reveal any significant differences of the maternal organs
and tissues amongst the groups. The few macroscopic findings observed are common
in rats.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
For all dose groups 28 females were mated; early delivery was observed in 1 female
(A51) of the control group and 2 females (B93 and B111) of the low-dose group.
At Cesarian section, 21, 23, 24 and 24 females in the control group, low-dose, middose
and high-dose group were pregnant.
There were no statistically significant differences between the control group and the
groups fed trehalose in the diet in the number of corpora lutea, implantations, live and
dead fetuses and early and late resorptions nor in the pre- and post implantation loss
or in the sex ratio of the fetuses.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
For all dose groups 28 females were mated; early delivery was observed in 1 female
(A51) of the control group and 2 females (B93 and B111) of the low-dose group.
At Cesarian section, 21, 23, 24 and 24 females in the control group, low-dose, middose
and high-dose group were pregnant.
There were no statistically significant differences between the control group and the
groups fed trehalose in the diet in the number of corpora lutea, implantations, live and
dead fetuses and early and late resorptions nor in the pre- and post implantation loss
or in the sex ratio of the fetuses.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
For all dose groups 28 females were mated; early delivery was observed in 1 female
(A51) of the control group and 2 females (B93 and B111) of the low-dose group.
At Cesarian section, 21, 23, 24 and 24 females in the control group, low-dose, middose
and high-dose group were pregnant.
There were no statistically significant differences between the control group and the
groups fed trehalose in the diet in the number of corpora lutea, implantations, live and
dead fetuses and early and late resorptions nor in the pre- and post implantation loss
or in the sex ratio of the fetuses.
Early or late resorptions:
no effects observed
Description (incidence and severity):
For all dose groups 28 females were mated; early delivery was observed in 1 female
(A51) of the control group and 2 females (B93 and B111) of the low-dose group.
At Cesarian section, 21, 23, 24 and 24 females in the control group, low-dose, middose
and high-dose group were pregnant.
There were no statistically significant differences between the control group and the
groups fed trehalose in the diet in the number of corpora lutea, implantations, live and
dead fetuses and early and late resorptions nor in the pre- and post implantation loss
or in the sex ratio of the fetuses.
Dead fetuses:
no effects observed
Description (incidence and severity):
For all dose groups 28 females were mated; early delivery was observed in 1 female
(A51) of the control group and 2 females (B93 and B111) of the low-dose group.
At Cesarian section, 21, 23, 24 and 24 females in the control group, low-dose, middose
and high-dose group were pregnant.
There were no statistically significant differences between the control group and the
groups fed trehalose in the diet in the number of corpora lutea, implantations, live and
dead fetuses and early and late resorptions nor in the pre- and post implantation loss
or in the sex ratio of the fetuses.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
For all dose groups 28 females were mated; early delivery was observed in 1 female
(A51) of the control group and 2 females (B93 and B111) of the low-dose group.
At Cesarian section, 21, 23, 24 and 24 females in the control group, low-dose, middose
and high-dose group were pregnant.
There were no statistically significant differences between the control group and the
groups fed trehalose in the diet in the number of corpora lutea, implantations, live and
dead fetuses and early and late resorptions nor in the pre- and post implantation loss
or in the sex ratio of the fetuses.
Changes in number of pregnant:
not specified
Description (incidence and severity):
For all dose groups 28 females were mated; early delivery was observed in 1 female
(A51) of the control group and 2 females (B93 and B111) of the low-dose group.
At Cesarian section, 21, 23, 24 and 24 females in the control group, low-dose, middose
and high-dose group were pregnant.
There were no statistically significant differences between the control group and the
groups fed trehalose in the diet in the number of corpora lutea, implantations, live and
dead fetuses and early and late resorptions nor in the pre- and post implantation loss
or in the sex ratio of the fetuses.
Other effects:
no effects observed
Details on maternal toxic effects:
no maternal toxic effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
> 100 000 mg/kg diet
Based on:
test mat.
Basis for effect level:
body weight and weight gain
changes in number of pregnant
changes in pregnancy duration
clinical signs
dead fetuses
early or late resorptions
effects on pregnancy duration
food consumption and compound intake
food efficiency
gross pathology
maternal abnormalities
mortality
necropsy findings
number of abortions
organ weights and organ / body weight ratios
pre and post implantation loss
total litter losses by resorption

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
No significant differences in the mean fetal body weight and placenta weights were observed
between the control group and the groups fed trehalose in the diet.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no statistically significant differences between the control group and the
groups fed trehalose in the diet in the number of corpora lutea, implantations, live and
dead fetuses and early and late resorptions nor in the pre- and post implantation loss
or in the sex ratio of the fetuses.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no statistically significant differences between the control group and the
groups fed trehalose in the diet in the number of corpora lutea, implantations, live and
dead fetuses and early and late resorptions nor in the pre- and post implantation loss
or in the sex ratio of the fetuses.
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
In the low- and high-dose group a statistically significantly decreased number of large
fetuses (i.e. fetus weight > 125% of the mean fetal body weight) were observed.
Furthermore, in the low-dose group a statistically significantly decreased number of
small fetuses (i.e. fetus weight < 75% of the mean fetal body weight) was observed.
The differences in the number of large and small fetuses were not considered to be an
effect of the test substance for reason of their incidental nature and lack of dose
response.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In the low-dose group a fetus with a flexed hindlimb and in the control and high-dose
groups a fetus with a filiformed tail were observed. No other external findings were
observed.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):

No skeletal malformations were observed in the fetuses of the control and high-dose
group
Visceral malformations:
no effects observed
Description (incidence and severity):
No visceral malformations were observed in the fetuses of the control and high-dose
group
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
no embryotoxic / teratogenic effects observed.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
> 100 000 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
On the basis of the results obtained in this study it was concluded that:
trehalose, when administered in the diet did not induce maternal nor developmental
toxicity at concentration of 2.5, 5 and 10% (1.4-2.0, 2.8-3.9 and 5.5-7.8 g trehalose/kg
body weight/day) for the low-, mid and high-dose group, respectively.
Executive summary:

1. Trehalose was fed in the diet to mated female Wistar rats (28 animals per dose

group) from days 0-21 of gestation. The concentrations in the diet were 0, 2.5, 5

and 10%. On gestation day 21 the dams were killed and macroscopically

examined. Reproductive organs were weighed and fetuses were examined after

Cesarian section. The viscera and skeletons of the fetuses of the control and highdose

group were examined.

2. The test substance was homogeneously distributed in the diets. The diets were

stable at room temperature and for 42 days in the refrigerator (2-10°C). The

content of the test substance measured in the batch diets prepared for this study

was close to intended at all dose levels.

3. No mortalities were observed.

One animal of the control and high-dose group showed haemorrhagic discharge

on gestation day 14 and 21, respectively.

Further, daily clinical observations during the gestation period did not reveal any

remarkable findings in the animals' appearance, general condition or behaviour

amongst the dosing and control groups.

4. No statistically significant differences in mean body weights, body weight changes

and food consumption were observed amongst the control and the groups fed

trehalose in the diet. The test substance intake during the gestation period ranged

from 1.4-2.0, 2.8-3.9 and 5.5-7.8 g trehalose/kg body weight/day for the low-,

mid and high-dose group, respectively.

5. For all dose groups 28 females were mated; early delivery was observed in 1

female (A51) of the control group and 2 females (B93 and B111) of the low-dose

group. At Cesarian section, 21, 23, 24 and 24 females in the control group, lowdose,

mid-dose and high-dose group were pregnant.

There were no statistically significant differences between the control group and

the groups fed trehalose in the diet in the number of corpora lutea, implantations,

live and dead fetuses and early and late resorptions nor in the pre- and post

implantation loss or in the sex ratio of the fetuses.

No remarkable differences in gravid and empty uterus weight, ovary weight,

carcass weight and net weight change (body weight gain from day 0 to day 21 of

gestation minus gravid uterine weight) were observed between the control group

and the groups fed trehalose in the diet.

6. Fetal external observations of the fetuses and placentas at Cesarian section did not

reveal any remarkable findings which could be related to treatment.

Furthermore, no significant differences in the mean fetal body weight and

placenta weights were observed between the control group and the groups fed

trehalose in the diet.

7. Upon fetal examination there were no treatment-related changes in fetal soft

tissues or fetal skeletal examination.

8. On the basis of the results obtained in this study it was concluded that:

trehalose, when administered in the diet did not induce maternal nor

developmental toxicity at concentration of 2.5, 5 and 10% (1.4-2.0, 2.8-3.9 and

5.5-7.8 g trehalose/kg body weight/day) for the low-, mid and high-dose group,

respectively.