Trehalose

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

one Guideline study available: a two-generation study in the rat

key value is taken from the two-generation study in the rat, NOAEL > 6.16 g/kg bw/day (gestation)

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16. December 1997 - 27. September 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was used because this species is
considered a suitable species for this type of study.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 4-5 wks; (F1) 3 wks
- Mean Weight at study initiation: (P) Males: 167 - 171 g; Females: 145 - 146 g; (F1) Males: 131 - 142 g; Females: 110 - 115 g
- Fasting period before study: no
- Housing: During the acclimatization and pre-mating periods, the males and females were housed in groups of 4 per sex,
For mating one male and one female were housed together
Mated F0-females were housed individually
After the mating period, non-mated females were housed individually until sacrifice and
males were returned to their group cage.
At or shortly after postnatal (PN) day 21, F1-animals were housed in groups of about 4 per sex,
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 10 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 °C
- Humidity (%): 40-70%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12
IN-LIFE DATES: From: 7. January 1998 To: 2. October 1998

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

The feed was provided as a powder, in stainless steel cans, covered by a perforated
steel plate that serves to reduce spillage. The feed in the feeders was refreshed once
per week and topped up when necessary.
During the quarantine and acclimatization period, the rats were fed a closed formula
diet obtained from SDS (Special Diets Services, Witham, England). Each batch of the
diet was analysed by the supplier for nutrients and contaminants. The analytical
certificates pertaining to the batches used in this study (batch no. 4070, 4177 and 4670)
are presented in Annex 2.
From the start of the study the rats were fed a modified diet. The
modification consisted of the omission of 20% barley from the diet which was
replaced by the test substance and/or pregelatinized potato starch (Paselli WA 4,
AVEBE, Foxhol, the Netherlands). The
various ingredients were homogeneously distributed in the diets by mixing them in a
mechanical blender. The diets were stored in a refrigerator (2-10°C) for maximally 39
days. The diets were prepared ten times.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 3 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after one unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses to determine the homogeneity, content and stability of the test substance in
the diets were conducted using HPLC. The trehalose concentrations measured in the
test diets were corrected for the amount of disaccharides measured in blank RM3 diet.
The stability of the test substance under (simulated) experimental conditions was
demonstrated by analysing samples of diets on the day of diet preparation (day 0),
after storage in an open container at (animal) room temperature for 7 days and after
storage in a closed container in a refrigerator (2-10°C) for 6 weeks.
The homogeneity of the test substance was determined by analysing samples of diets
of the low-, mid- and high-dose groups, taken once at 5 different locations in the feed
containers.
The content of the test substance in the diets was determined in the diets of all
batches prepared. Diet samples were taken immediately after preparation of the diets
and stored at ca. -18°C pending analysis.
All analysis were performed at the Analytical Sciences Division of TNO Nutrition and
Food Research Institute.

Duration of treatment / exposure:

16 weeks

Frequency of treatment:

daily

Details on study schedule:

- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.

Dose / conc.:

0 mg/kg diet

Dose / conc.:

25 000 mg/kg diet

Remarks:

2.5 %

Dose / conc.:

50 000 mg/kg diet

Remarks:

5 %

Dose / conc.:

100 000 mg/kg diet

Remarks:

10 %

No. of animals per sex per dose:

28

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: not stated
- Rationale for animal assignment (if not random): random
- Other:

Positive control:

none

Parental animals: Observations and examinations:

Clinical signs
Each animal was carefully observed daily in the morning hours. On working days, all
cages were checked again in the afternoon. On Saturdays, Sundays and public holidays
only one check per day was performed. All abnormalities, signs of ill health or
reaction were recorded.
Body weight
Body weights of all F0-parents were recorded once during the acclimatization period
at allocation to the various treatment groups. For both generations, body weights of
male animals were recorded weekly until sacrifice. Body weights of female rats were
recorded during the premating and mating periods, on days 0, 7, 14 and 21 of
gestation, and during lactation on PN days 1, 7, 14 and 21. Body weights of mated
females which produced no litter were recorded up to day 21 of the presumed
gestation period.
Non-mated females were weighed weekly after the mating period. All animals were
weighed at sacrifice.
Food consumption
The quantity of food consumed by the animals was measured on a cage basis, by
weighing the feeders. In the report the food consumption is expressed in g/kg body
weight/day and g/animal/day.
Food consumption of the male animals was measured weekly, except during the
mating period when food consumption was not measured.
Food consumption of female animals was measured weekly during the premating
period. Food consumption of females was not recorded during the mating period.
Food consumption of mated females was recorded weekly during pregnancy (days 0-
7, 7-14 and 14-21) and lactation (days 1-7, 7-14 and 14-21). Food consumption of
mated females which produced no litter was recorded up to day 21 of the presumed
gestation period.
Test substance intake
The test substance intake was assessed on the basis of food intake, body weight and
nominal dietary levels of the test substance.

Litter observations:

Parturition and litter evaluation
At the end of the gestation period, females were examined twice daily for signs of
parturition. Any difficulties that occurred during parturition were recorded.
To keep nest disturbance to a minimum, the litters were examined only once daily for
dead pups.
The total litter size, number of each sex, the number of stillbirths, livebirths, grossly
malformed pups, and pups showing abnormalities were recorded on PN day 1.
Furthermore, the number of live and dead pups, pups showing malformations or
abnormalities were recorded on PN days 4, 7, 14, and 21.
Pup weight
The litters were weighed on PN days 1, 4 (before and after culling), 7 and 14. At
weaning (PN day 21), all pups were weighed individually.

Postmortem examinations (parental animals):

Gross necropsy and histology of parental animals
All surviving male and female parents of the F0- and F1-generation were killed by
decapitation after ether anaesthesia, after weaning of their litters, and/or if they were
no longer necessary for assessment of reproductive effects. A complete gross
examination of each animal and collection of tissue samples for microscopic
observations was performed.
At necropsy the spleen was weighed.
Samples of the following tissues and organs of all parents of the F0- and F1-
generation were preserved in a neutral, aqueous phosphate buffered solution
containing 4% formaldehyde, except for the testes which was preserved in Bouin's
fixative:
- ovaries
- uterus
- vagina
- testes
- epididymides
- seminal vesicles (with coagulating glands and their fluids)
- prostate
- pituitary
- spleen
- organs or tissues showing macroscopic abnormalities
Tissues for microscopic examination were processed, embedded in paraffin wax,
sectioned and stained with haematoxylin and eosin, except for the sections of the
testes which were stained with PAS haematoxylin. Microscopic examination was done
on the collected organs of all rats of the high-dose group and of the control group
and on the macroscopic abnormalities of all groups.
In addition, the reproductive organs of the males that fail to sire and non-pregnant
females of the low- and mid-dose group were examined.

Postmortem examinations (offspring):

Gross necropsy of pups and weanlings
All stillborn pups, pups found dead and pups that were killed because they were
moribund during the study were examined macroscopically for structural
abnormalities or pathological changes.

Statistics:

Statistical procedures used in the evaluation of data were as follows:
- for (pup) body weights and food consumption: one-way analysis of variance
(ANOVA) followed by Dunnett's multiple comparison tests
- for clinical signs and developmental markers: Fisher's exact probability test
- for pre-coital time and duration of gestation: Kruskal-Wallis followed by
Mann-Whitney U-tests
- for number of females pregnant, females with liveborn, females surviving
delivery, females with (all) stillborn pups, number of live- and stillborn pups,
number of pups/litters lost, number of male pups and number of implantation
sites: Fisher's exact probability test
- for mean number of pups delivered, mean number of pups alive, mean number of
implantations and post-implantation loss: Kruskal-Wallis followed by
Mann-Whitney U-tests
- for pathological changes: Fisher's exact probability test
All tests were two-sided and a value of P<0.05 was considered statistically significant
(significant).
Statistical evaluations on variables associated with the pups were considered on a litter
basis in accordance with standard procedures. Additional evaluations on a per pup
basis were performed to attempt to identify any specific dose-related effect that may
have occurred.

Reproductive indices:

- number of females placed with males
- number of males mated with females
- number of successful copulations (= number of females mated)
- number of males that became sires
- number of pregnant females
- number of females surviving delivery
- number of females with liveborn and (all) stillborn pups
- number of pups delivered (live- and stillborn)
- number of live pups at day n
- number of pups lost (= number of pups dying after live birth)
- number of pups culled and alive after culling
- number of litters lost entirely (= number of litters in which all pups died or were
stillborn)
- number of male pups at day n
- number of implantations
- number of lost implantations (= number of implantations that did not result in
live or stillborn births)
The following factors were calculated:
- pre-coital time = time between the start of mating and successful copulation
- duration of gestation = time between gestation day 0 and day of delivery
-mating index= (number of females mated/number of females placed with
males) x 100
-male fertility index = (number of males that became sire/number of males
placed with females) x 100
- female fertility index = (number of pregnant females/number of females
placed with males) x 100
- female fecundity index = (number of pregnant females/number of females mated)x100
- gestation index = (number of females with live pups/number of females
pregnant) x 100
- live birth index = (number of pups born alive/number of pups born) x 100
- viability index (days 4-21) = (number of live weanlings/number of pups alive on day
4 post partum) x 100
- pup mortality day n = (number of dead pups on day n/total number of pups
on day n) x 100
- sex ratio day n = (number of live male pups on day n/ number of live pups on day
n) x 100
- number of lost implantations = number of implantations sites - number of
pups born alive
- post-implantation loss = [(number of implantation sites - number of pups born
alive)/number of implantation sites] x 100

Offspring viability indices:

- number of pups delivered (live- and stillborn)
- number of live pups at day n
- number of pups lost (= number of pups dying after live birth)
- number of pups culled and alive after culling
- number of litters lost entirely (= number of litters in which all pups died or were
stillborn)
- number of male pups at day n
- gestation index = (number of females with live pups/number of females
pregnant) x 100
- live birth index = (number of pups born alive/number of pups born) x 100
- viability index (days 4-21) = (number of live weanlings/number of pups alive on day
4 post partum) x 100
- pup mortality day n = (number of dead pups on day n/total number of pups
on day n) x 100
- sex ratio day n = (number of live male pups on day n/ number of live pups on day
n) x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The number of F1-females with a sparsely haired skin was statistically significantly
increased in all dose groups during the premating period, in the mid- and high-dose
groups during the gestation period and in the high-dose group during the lactation
period. This finding was considered not to be related to treatment since sparsely
haired skin is a normal finding in this strain of rats.
Daily clinical observations of the F0- and F1-animals during the premating, gestation
and lactation period did not reveal other remarkable findings in the animals'
appearance, general condition or behaviour amongst the dosing and control groups.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weight changes of the F0-males of the mid- and high-dose groups were
significantly increased in week 8-9 of the premating period. On day 21 of the
gestation period of the F0-generation, body weights of the females of the mid-dose
group were significantly increased. Mean body weight changes of the F1-females of
the low-dose group were significantly decreased in weeks 7-8 and 8-9 of the
premating period. Mean body weight changes of the F1-females of the high-dose
group were significantly decreased between days 14-21 of the lactation period.
The significant differences in body weights and body weight changes between the
control and treatment groups were not dose dependent and inconsistent over time
and reproductive period and generation. Therefore, trehalose was considered to have
no effect on maternal body weights and body weight changes.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption (g/animal/day) of the F0-males of the mid-dose group was
significantly decreased in week 1-2 of the premating period. Food consumption
(g/animal/day) of the F0-females of the mid-dose group was significantly increased in
week 5-6 of the premating period and between days 7-14 of the lactation period. Food
consumption (g/animal/day) of the F0-females of the high-dose group was
significantly increased between days 7-14 and 14-21 of the lactation period.
Furthermore, food consumption (g/kg/day) of the F0-females of the mid- and highdose
groups was significantly increased in week 5-6 of the premating period.
In the F1-generation, food consumption (g/animal/day) of the males of the mid-dose
group was significantly increased in week 8-9 of the premating period. In the
premating period of the F1-generation, food consumption (g/kg/day) of the males of
the high-dose group was significantly increased in weeks 0-1, 1-2, 3-4 and 8-9 and of
the females of the high-dose group in week 9-10.
In conclusion, the significant differences in food consumption between the control
and treatment groups were not dose dependent and inconsistent over time,
reproduction period and generation. Therefore, trehalose was considered to have no
effect on food consumption.

Food efficiency:

not examined

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

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Microscopic examination did not reveal treatment-related histopathologic changes in
either generation. The histopathologic changes observed are common findings in rats
of this age and strain. Furthermore, they were about equally distributed amongst the
groups or they occurred in only one or a few animals.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Microscopic examination did not reveal treatment-related histopathologic changes in
either generation. The histopathologic changes observed are common findings in rats
of this age and strain. Furthermore, they were about equally distributed amongst the
groups or they occurred in only one or a few animals.

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

All females of all groups were mated; all mating indices were 100%.
Pre-coital time was comparable amongst all groups for both generations.
The number of pregnant females was 24, 26, 24, 27 and 23, 25, 25, 25 for the control,
low-, mid- and high-dose group of the F0- and F1-generation, respectively. The
female fecundity index and male- and female fertility index ranged from 86-96% in
the F0-generation and from 82-89% in the F1-generation. Duration of gestation was
comparable in all groups for both generations.
The number of females with liveborn pups was 24, 26, 24, 27 and 23, 25, 25, 25 for
the control, low-, mid- and high-dose group of the F0- and F1-generation,
respectively. In both generations, there were no females which delivered only dead
pups. Stillborn pups were observed in 7, 6, 4, 4 and 3, 3, 3, 2 litters of the control,
low-, mid- and high-dose group of the F0- and F1-generation, respectively. The
gestation index was 100% for all groups of both generations. Post-implantation loss
was 16.05, 14.84, 12.87, 13.91% and 13.81, 15.78, 11.32, 11.69% for the control, low-,
mid- and high-dose group of the F0- and F1-generation, respectively.

Key result

Dose descriptor:

NOAEL

Effect level:

> 100 000 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no toxicological relevant effects reported in all observed endpoints

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The number of F1-females with a sparsely haired skin was statistically significantly
increased in all dose groups during the premating period, in the mid- and high-dose
groups during the gestation period and in the high-dose group during the lactation
period. This finding was considered not to be related to treatment since sparsely
haired skin is a normal finding in this strain of rats.
Daily clinical observations of the F0- and F1-animals during the premating, gestation
and lactation period did not reveal other remarkable findings in the animals'
appearance, general condition or behaviour amongst the dosing and control groups.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weight changes of the F0-males of the mid- and high-dose groups were
significantly increased in week 8-9 of the premating period. On day 21 of the
gestation period of the F0-generation, body weights of the females of the mid-dose
group were significantly increased. Mean body weight changes of the F1-females of
the low-dose group were significantly decreased in weeks 7-8 and 8-9 of the
premating period. Mean body weight changes of the F1-females of the high-dose
group were significantly decreased between days 14-21 of the lactation period.
The significant differences in body weights and body weight changes between the
control and treatment groups were not dose dependent and inconsistent over time
and reproductive period and generation. Therefore, trehalose was considered to have
no effect on maternal body weights and body weight changes.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption (g/animal/day) of the F0-males of the mid-dose group was
significantly decreased in week 1-2 of the premating period. Food consumption
(g/animal/day) of the F0-females of the mid-dose group was significantly increased in
week 5-6 of the premating period and between days 7-14 of the lactation period. Food
consumption (g/animal/day) of the F0-females of the high-dose group was
significantly increased between days 7-14 and 14-21 of the lactation period.
Furthermore, food consumption (g/kg/day) of the F0-females of the mid- and highdose
groups was significantly increased in week 5-6 of the premating period.
In the F1-generation, food consumption (g/animal/day) of the males of the mid-dose
group was significantly increased in week 8-9 of the premating period. In the
premating period of the F1-generation, food consumption (g/kg/day) of the males of
the high-dose group was significantly increased in weeks 0-1, 1-2, 3-4 and 8-9 and of
the females of the high-dose group in week 9-10.
In conclusion, the significant differences in food consumption between the control
and treatment groups were not dose dependent and inconsistent over time,
reproduction period and generation. Therefore, trehalose was considered to have no
effect on food consumption.

Food efficiency:

not examined

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):

No intergroup differences were observed on absolute and relative spleen weights of
the males and females of the F0- and F1-generation.

Gross pathological findings:

no effects observed

Description (incidence and severity):

At necropsy of both generations, no treatment-related macroscopic changes were
observed.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Microscopic examination did not reveal treatment-related histopathologic changes in
either generation. The histopathologic changes observed are common findings in rats
of this age and strain. Furthermore, they were about equally distributed amongst the
groups or they occurred in only one or a few animals.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Microscopic examination did not reveal treatment-related histopathologic changes in
either generation. The histopathologic changes observed are common findings in rats
of this age and strain. Furthermore, they were about equally distributed amongst the
groups or they occurred in only one or a few animals.

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

All females of all groups were mated; all mating indices were 100%.
Pre-coital time was comparable amongst all groups for both generations.
The number of pregnant females was 24, 26, 24, 27 and 23, 25, 25, 25 for the control,
low-, mid- and high-dose group of the F0- and F1-generation, respectively. The
female fecundity index and male- and female fertility index ranged from 86-96% in
the F0-generation and from 82-89% in the F1-generation. Duration of gestation was
comparable in all groups for both generations.
The number of females with liveborn pups was 24, 26, 24, 27 and 23, 25, 25, 25 for
the control, low-, mid- and high-dose group of the F0- and F1-generation,
respectively. In both generations, there were no females which delivered only dead
pups. Stillborn pups were observed in 7, 6, 4, 4 and 3, 3, 3, 2 litters of the control,
low-, mid- and high-dose group of the F0- and F1-generation, respectively. The
gestation index was 100% for all groups of both generations. Post-implantation loss
was 16.05, 14.84, 12.87, 13.91% and 13.81, 15.78, 11.32, 11.69% for the control, low-,
mid- and high-dose group of the F0- and F1-generation, respectively.

Key result

Dose descriptor:

NOAEL

Effect level:

> 100 000 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no toxicological relevant effects reported in all observed endpoints

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No significant findings in any variables were
obtained when evaluated using the standard litter basis.
When a per pup basis was used statistical significances were obtained.
On PN day 1 of the F0-generation, the number of pale pups, small pups and pups
having no milk in the stomach in the low- and high-dose groups was significantly
decreased. In the mid-dose group the number of cold pups was significantly increased
compared to the control group.
On PN day 4 of the F0-generation, the number of small pups was significantly lower
in the low- and high-dose groups than in the control group. Furthermore, on PN day
7 of the F0-generation, the number of large pups was significantly higher in the midand
high-dose groups than in the control group.
On PN day 1 of the F1-generation, the number of small pups in the low-dose group
was significantly increased and the number of large pups was significantly decreased.
On PN day 4 the number of small pups was significantly increased in the low-dose
group and significantly decreased in the high-dose group. In the low-dose group, the
number of small pups was significantly increased on PN day 7 and the number of
sparsely haired pups was significantly increased on PN day 14. On PN day 21, the
number of large pups was significantly increased in the high-dose group.
All these findings were only significant on a pup basis and not on a litter basis.
Furthermore, no dose or generational (F0, F1) relationships were observed. The
findings were normal for pups of this age. For these reasons the findings are
considered to be not related to treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The pup mortality (days 1-4) was significantly decreased in the low-and high-dose
groups of the F0-generation.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean pup weight and pup body weight changes as calculated from the litter
weights on PN days 1, 4, 7, 14 and the mean pup weight on PN day 21 by weighing
the individual male and female pups are presented in Tables 41 and 42 for the F0- and
F1-generation, respectively.
Mean pup weights of all groups were comparable on PN days 1, 4, 7, 14 and 21 for
both generations except for the pup body weights of the mid-dose group of the F0-
generation measured on PN day 7 and 21 which was significantly increased (Table
41).
Body weight changes of the pups of the mid-dose group of the F0-generation were
significantly increased between PN days 4 and 7 compared to the control group
(Table 41). In the F1-generation, pup body weight changes of the low-dose group was
statistically significantly decreased between PN days 1-4 (Table 42).
Since the statistically significant differences in body weights and body weight changes
between the control and treatment groups were not consistent for treatment group,
time period or generation, the differences were considered incidental. Therefore,
trehalose was considered to have no effect on body weights and body weight changes
of the pups of the F0- and F1-generation.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

Sexual maturation:

no effects observed

Description (incidence and severity):

At macroscopic examinations no findings were observed which indicated an abnormal
development of the pups for either generation.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

No grossly malformed pups were observed.

Histopathological findings:

not examined

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 100 000 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no toxicological relevant effects reported in all observed endpoints

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No significant findings in any variables were
obtained when evaluated using the standard litter basis.
When a per pup basis was used statistical significances were obtained.
On PN day 1 of the F0-generation, the number of pale pups, small pups and pups
having no milk in the stomach in the low- and high-dose groups was significantly
decreased. In the mid-dose group the number of cold pups was significantly increased
compared to the control group.
On PN day 4 of the F0-generation, the number of small pups was significantly lower
in the low- and high-dose groups than in the control group. Furthermore, on PN day
7 of the F0-generation, the number of large pups was significantly higher in the midand
high-dose groups than in the control group.
On PN day 1 of the F1-generation, the number of small pups in the low-dose group
was significantly increased and the number of large pups was significantly decreased.
On PN day 4 the number of small pups was significantly increased in the low-dose
group and significantly decreased in the high-dose group. In the low-dose group, the
number of small pups was significantly increased on PN day 7 and the number of
sparsely haired pups was significantly increased on PN day 14. On PN day 21, the
number of large pups was significantly increased in the high-dose group.
All these findings were only significant on a pup basis and not on a litter basis.
Furthermore, no dose or generational (F0, F1) relationships were observed. The
findings were normal for pups of this age. For these reasons the findings are
considered to be not related to treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean pup weight and pup body weight changes as calculated from the litter
weights on PN days 1, 4, 7, 14 and the mean pup weight on PN day 21 by weighing
the individual male and female pups are presented in Tables 41 and 42 for the F0- and
F1-generation, respectively.
Mean pup weights of all groups were comparable on PN days 1, 4, 7, 14 and 21 for
both generations except for the pup body weights of the mid-dose group of the F0-
generation measured on PN day 7 and 21 which was significantly increased (Table
41).
Body weight changes of the pups of the mid-dose group of the F0-generation were
significantly increased between PN days 4 and 7 compared to the control group
(Table 41). In the F1-generation, pup body weight changes of the low-dose group was
statistically significantly decreased between PN days 1-4 (Table 42).
Since the statistically significant differences in body weights and body weight changes
between the control and treatment groups were not consistent for treatment group,
time period or generation, the differences were considered incidental. Therefore,
trehalose was considered to have no effect on body weights and body weight changes
of the pups of the F0- and F1-generation.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

Sexual maturation:

no effects observed

Description (incidence and severity):

At macroscopic examinations no findings were observed which indicated an abnormal
development of the pups for either generation.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No grossly malformed pups were observed.

Histopathological findings:

not examined

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

> 100 000 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no toxicological relevant effects reported in all observed endpoints

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

From the data of this study it can be concluded that dietary administration of
trehalose to rats at concentrations up to 10% (w/w) for two consecutive generations
had no maternal toxic effects and had no effects on reproduction of the parental F0-
and F1-generation or the development of the pups of the F0- and F1-generation. The
no observed adverse effect level for reproductive effects after dietary administration
of trehalose to rats is at least 7.09 (males premating), 7.61 (females premating), 6.16
(gestation) and 14.09 (lactation) g/kg body weight/day.

Executive summary:

1. Trehalose was given for two successive generations to male and female Wistar

rats in the diet at concentrations of 0, 2.5, 5 and 10%. In each generation one

litter was raised.

2. From the data of this study it can be concluded that dietary administration of

trehalose to rats at concentrations up to 10% (w/w) for two consecutive

generations had no maternal toxic effects and had no effects on reproduction of

the parental F0- and F1-generation or the development of the pups of the F0-

and F1-generation. The no observed adverse effect level (NOAEL) for

reproductive effects after dietary administration of trehalose to rats is at least 7.09

(males premating), 7.61 (females premating), 6.16 (gestation) and 14.09 (lactation)

g/kg body weight/day.

3. The test substance was homogeneously distributed in the diets and was stable

when stored for 7 days at room temperature or stored for 6 weeks in the

refrigerator (2-10°C). The concentrations of the test substance measured in each

batch of the diets prepared were close to the intended concentration (2.5, 5 and

10%).

4. Daily clinical observations of the F0- and F1-animals during the premating,

gestation and lactation period did not reveal remarkable findings in the animals'

appearance, general condition or behaviour which could be related to trehalose

treatment.

5. Statistically significant intergroup differences in maternal body weights and body

weight changes were not-consistent by reproductive period, dose or generation

and were considered not related to treatment.

6. The statistically significant differences in food consumption between the control

and treatment groups of the F0- and F1-generation were not considered to be

treatment related, because of the inconsistent nature of the affected groups.

7. The test substance intake for the F0- and F1 males during the premating period

ranged from 1.24-2.90 (mean ± standard error = 1.73±0.10), 2.38-5.65

(3.45±0.20) and 4.89-12.43 (7.09±0.45) g/kg body weight/day for the low-, midand

high-dose group, respectively. The test substance intake for the F0- and F1

females during the premating period ranged from 1.47-2.85 (1.86±0.09), 2.88-5.48

(3.74±0.16) and 5.94-11.73 (7.61±0.34)g/kg body weight/day for the low-, midand

high-dose group, respectively. During the gestation period the test substance

ranged from 1.06-1.74 (1.49±0.13), 2.22-3.51 (3.06±0.23) and 4.40-

7.21(6.16±0.48) g/kg body weight/day for the low-, mid- and high-dose group,

respectively. During the lactation period the test substance ranged from 2.30-4.34

(3.33±0.54), 4.98-8.94 (6.88±1.09), 10.10-17.94 (14.09±2.19) g/kg body

weight/day for the low-, mid- and high-dose group, respectively.

8. From the results of this study it was concluded that in both generations no

effects of trehalose treatment were observed on any reproduction variables

determined: precoital time, mating index, male and female fertility, female

fecundity index, gestation index, duration of gestation, and number of females

with (all) stillborn pups and post-implantation loss.

9. For both generations, no adverse effects of trehalose were observed on the

number of pups delivered, the number of liveborn and stillborn pups, pup

mortality, sex ratio, pup observations, pup body weights and pup body weight

changes.

10. No effect of the test substance was observed on absolute and relative spleen

weights.

11. At autopsy of both generations, no treatment-related macroscopic changes were

observed.

Microscopical examination did not reveal treatment-related histopathological

changes in either generation. The histopathological changes observed are

common findings in rats of this age and strain. Furthermore, they were about

equally distributed amongst the groups or they occurred in only one or a few

animals.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

6 160 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

one well documented Guideline study present

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

one Guideline study available: a developmental toxicity study in the rat

key value is taken from the developmental toxicity study in the rat, NOAEL > 7.8 g/kg bw/day (gestation)

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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

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

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)

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:

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

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

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

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

Key result

Abnormalities:

no effects observed

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.

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

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

7 800 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

one well documented Guideline study present

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

No mode of action identified.

Justification for classification or non-classification

The presented information is conclusive but not sufficient for classification.