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Toxicological information

Toxicity to reproduction

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction 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 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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

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

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

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.

Effect levels (P1)

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

Target system / organ toxicity (P1)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

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

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

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

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

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

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

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.