Trehalose

Acute Toxicity: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Type of information:

other: discussion of available information

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: adapting information from repeated dose toxicity studies

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

8. March -22. July 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study, conducted for Quadrant Holdings, was designed to investigate the toxicity
associated with the daily administration of oral, intravenous, or subcutaneous doses of
trehalose in beagle dogs for 14 days.

GLP compliance:

yes

Limit test:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: lot no. 22128, Pfanstiehl Laboratories, Inc. (Waukegan, IL)
- Expiration date of the lot/batch: not stated
- Purity test date: not stated
RADIOLABELLING INFORMATION (if applicable)
not applicable
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not stated
- Stability under test conditions: not stated but assumed uncritical
- Solubility and stability of the test substance in the solvent/vehicle: not stated but assumed uncritical
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not stated but
assumed uncritical
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
none

Species:

dog

Strain:

Beagle

Details on species / strain selection:

no further details given

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., (Madison, WI).
- Females (if applicable) nulliparous and non-pregnant: not stated
- Age at study initiation: 4-6 month
- Weight at study initiation: 6.4 - 11.7 kg (male), 5.5 - 7.8 kg (female)
- Fasting period before study: none
- Housing: single
- Diet (e.g. ad libitum): 400 g once daily for 2 h
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 17 days
DETAILS OF FOOD AND WATER QUALITY:
Certified Harlan Teklad diet 8727 (approximately 400 grams) was offered for approximately
2 hours once daily, at midday, and municipal tap water filtered by reverse osmosis was
available ad libitum to the animals. Results of analytical certification of batches of feed
provided by the manufacturer and water analysis performed periodically by the municipal
authority and FRC are retained on file at FRC. There were no known contaminants in the feed
or the water which could have influenced the outcome of the study.
ENVIRONMENTAL CONDITIONS
- Temperature (°F): 68° ± 4° F
- Humidity (%): 30%-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 19 Apr. 1994 To: 26 May 1994

Type of coverage:

other: subcutaneous

Vehicle:

physiological saline

Details on exposure:

- PREPARATION OF DOSING SOLUTIONS:
For the intravenous and subcutaneous preparations, the appropriate amount of trehalose was
weighed and added in a stepwise manner with normal saline, sterile solution, to a sterile,
calibrated container. Normal saline, sterile solution, was added to the final volume and the
liquid was stirred until the test article was dissolved. Formulated test article for the
intravenous and subcutaneous administration was dispensed into sterile vials. The formulated
test articles were prepared weekly and were stored refrigerated.
For the oral dose, 300 capsules were prepared in advance of the study. Ten grams of bulk test
article (equivalent to a 2-kg dose) was weighed into each capsule. On the day of body weight
collection, an additional capsule was prepared for each dog. The weight of trehalose in each
additional capsule was calculated for individual dogs based on the difference of the total
weight of compound in the prepared capsules and the total required dose (10 g trehalose/2 kg
body weight). A sufficient number of capsules were prepared for one week's dosing. The control
animals received the number of empty capsules equivalent to their individual body weight
(i.e., one capsule per 2 kg body weight).
- VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0.5 g/mL (intravenous), 0.5 g/mL (subcutaneous)
- Amount of vehicle (if gavage): 0.5 mL/kg bw (subcutaneous), 2 mL/kg bw (intravenous)
- The vehicle control article, normal saline, sterile solution (Am Vet Pharmaceuticals, Ft.
Collins, CO, lot no. 3000), a clear liquid, was received on April 25 and May 10, 1994 (1000 mL
and 250 mL, respectively) from the storage area of Frederick Research Center. The vehicle
control article was stored at room temperature.
All animals were shaved in the jugular region for blood collection; dogs treated intravenously
were shaved in the cephalic regions; dogs treated subcutaneously were shaved in the dorsal
scapular region. Gelatin capsules packed with bulk trehalose or empty gelatin capsules were
administered orally; trehalose solutions were administered intravenously (cephalic vein) with
a 25-gauge butterfly catheter or subcutaneously via a 25-gauge sterile needle and syringe.
Animals were dosed once daily for 14 days. All solutions were administered at room
temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

analytical verification was done by sponsor, no further documentation available

Duration of treatment / exposure:

14 d

Frequency of treatment:

1 per day

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

5 000 mg/kg bw/day (nominal)

Remarks:

oral capsule

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

subcutaneous

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

intravenous

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: not stated
- Rationale for animal assignment (if not random): Animals were assigned randomly to groups by a
computerized body weight randomization
program that ensured homogeneity of group means and reduced variances for body weights
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random):

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Animals were examined twice daily (in the morning and the afternoon) for mortality and signs
of reaction to treatment. Each animal received individual weekly physical examinations,
including observation of external surfaces, all orifices, injection sites (as applicable), palpation
for masses, and measurement of heart rate, body temperature, and respiration. Observed
clinical signs and physical measurements were recorded for individual animals.
DETAILED CLINICAL OBSERVATIONS: Yes
Animals were examined twice daily (in the morning and the afternoon) for mortality and signs
of reaction to treatment. Each animal received individual weekly physical examinations,
including observation of external surfaces, all orifices, injection sites (as applicable), palpation
for masses, and measurement of heart rate, body temperature, and respiration. Observed
clinical signs and physical measurements were recorded for individual animals.
BODY WEIGHT: Yes
Animals were weighed individually for randomization, on day 1 before dosing, and weekly
thereafter.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was measured for 2 days (3 days for the females) 2 weeks before dosing
commenced and then 4 days each week beginning the week before dosing and continuing
throughout the study.
FOOD EFFICIENCY:
- Body weight gain calculated
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:
OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
HAEMATOLOGY: Yes
and
CLINICAL CHEMISTRY: Yes
Blood was collected for glucose analyses on day 14 of dosing at selected intervals after dosing.
Blood was collected from the jugular vein of each animal into serum separator tubes and was
centrifuged and sent to Ani Lytics, Inc., for analysis. Blood from the oral groups was collected
before dosing and at approximately 30 minutes and 1, 4, and 24 hours after dose administration;
blood from the intravenous group was collected before dosing and at approximately 15 and 30
minutes, 1, 4, and 24 hours; and blood from the subcutaneous group was collected before dosing
and at approximately 30 minutes, 1, 4, and 24 hours after dosing.
Before treatment and before necropsy, blood was collected from the jugular vein of each animal
for routine hematology and clinical chemistry evaluation.
A list of parameters examined and the abbreviations and units used in the laboratory
investigations precedes the appropriate appendix. Clinical pathology services were provided
by Ani Lytics, Inc. Methods used in laboratory investigations are described in Standard
Operating Procedures retained in the clinical pathology laboratory of Ani Lytics, Inc.
URINALYSIS: No
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.
NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
IMMUNOLOGY: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.
OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
On day 15, all animals were humanely killed by sodium pentobarbital anesthesia, followed by
exsanguination from the femoral or carotid arteries. Macroscopic examination of all animals,
performed by qualified necropsy technicians in the presence of a pathologist, included the
external surface of the carcass and a detailed internal examination of the major body cavities,
organs, and viscera.
HISTOPATHOLOGY: Yes (see table)
The tissues listed were saved from each animal. Tissues were preserved in 10% NBF. Lungs
were infused with 10% NBF. Bone marrow slides were prepared but not evaluated.
animal identification eyes mammary gland spleen
adrenals gallbladder optic nerves stomach
aorta (thoracic) heart ovaries testes
bone/marrow (sternum) ileum pancreas thymus
brain (three levels) injection site pituitary thyroid lobes (and
cecun1 jejtmurn prost:lte parathyroids)
colon kidneys salivary glands tongue
duodenum liver sciatic nerve trachea
epididymides lungs skeletal muscle urinary bladder
esophagus lymph nodes (mandibular skin uterus
and mesenteric) spinal cord vagina
Tissues listed above were trimmed, embedded in paraffin wax, sectioned, and stained with
hematoxylin and eosin. Histopathologic examination was performed on tissues listed above for
the control and oral trehalose-treated dose groups. Histopathologic examination was
performed on the kidneys, liver, pancreas, urinary bladder, and injection sites for the
intravenous and subcutaneous groups. Pathology services were performed by Pathology
Associates, Inc. 005R

Statistics:

Means and standard deviations were calculated for numerical data. Subsequently, data in the
study groups were analyzed for homogeneity using ANOV A and then subjected to Dunnett's ttest.
Calculations for body weight and weight change were performed with the commercially
available Labcat® statistical program. Calculations for food consumption and clinical
pathology data were performed with the commercially available StatView s12+ statistical
program. Most statistical comparisons for the intravenous, oral, and subcutaneous groups were
made against the oral control group (group 1 ). Serum glucose values were compared with the
predose value for each group.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs of toxicity were
apparent in the animals treated by the intravenous route. Two dogs showed some evidence that
a small amount of test material was injected outside the vein, however, no subsequent effects
were noted. Diarrhea was seen for all males and females treated by the oral route. The
frequency of occurrence increased, especially for the females, as the study progressed. One male
vomited before dosing. Regurgitation of a capsule, accompanied by red or yellowish vomitus,
was observed on two occasions for the females; one capsule was redosed. Capsule regurgitation,
accompanied by yellowish vomitus was seen on one occasion for a control female. In an effort to
decrease the incidence of capsule regurgitation, animals were not fed for at least 1 hour after
oral dosing. One male in the subcutaneous group had a swollen nictitating membrane that was
apparent prior to dose initiation and was not attributed to the test material. At week 2 a slight
abrasion on the top of the head was noted for two males in the oral control group.
For the males treated by the subcutaneous route, diarrhea and vomitus (yellow) were each seen
on one occasion. No sign of toxicity was apparent for females treated by the subcutaneous route.

Dermal irritation:

no effects observed

Description (incidence and severity):

no effects reported.
Although no direct application on the skin of the animals was performed,
the subcutaneous route may serve as an indicator for systemic toxicity after dermal exposure.
No treatment related effects for this route are observed

Mortality:

no mortality observed

Description (incidence):

There were no deaths on the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Group mean body weight and body weight change were essentially comparable between the
vehicle and treated groups for each route of administration over the 14-day study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Treatment with trehalose by any route had no effect on food consumption throughout the study.
Group mean food consumption was variable among all groups. Statistically significant
differences from control values were observed pretest for females treated by the oral or
subcutaneous route.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A summary of the group mean hematology values is presented in Table 5 and individual
hematology values are presented in Appendix I.
Before dose administration, all values were within normal limits, although slight but
statistically significant decreases from the control group were noted for erythrocyte count for
males in the intravenous group and for MCV and MCH for females in the intravenous group.
After 14 days of treatment, no toxicologically meaningful changes were observed in hematologic
status of the trehalose-treated animals. The calculated index of MCHC was slightly
decreased from the control values for orally treated males and intravenously treated females.
MCH was also decreased from control for the intravenously treated females. The differences
were attributed to normal biologic variation and were not test article related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Serum Glucose
With the exception of a slight decrease in serum glucose at the 4-hour time point for males, the
orally dosed controls were comparable to predose values over the sampling interval. Further,
the predose baseline values were comparable across all groups.
On the 14th day of oral administration, a slight increase was noted at the 30-minute (male
only) and/or 1-hour interval (males and females). Intravenous administration resulted in
slight increase through the 1-hour period for males and the 30-minute interval for females.
Glucose levels decreased over the remainder of the 24-hour sampling interval.
Repeated subcutaneous administration of trehalose did not affect glucose levels for males or
females.
Clinical Chemistry
Slight, but statistically significant, differences from control for sodium and/ or albumin were
noted for male intravenous and oral groups before initiation of treatment. After 14 days of
treatment, blood urea nitrogen was slightly elevated for females dosed orally with trehalose
and the albumin/globulin ratio was slightly decreased due to an increase in globulin for males
treated subcutaneously with trehalose. The lack of a consistent response between sexes makes
the biologic significance of these findings questionable.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic lesions
noted at necropsy were few in number and were not test article related. One dog given trehalose
intravenously (no. 251) and one dog in the subcutaneous group (no. 453) had red discoloration at
the injection site which corresponded to perivenous hemorrhage attributed to trauma from
repeated injections.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No lesion directly related to the test
article was noted. The lesions noted were considered incidental. As noted above, perivenous
hemorrhage was observed with or without inflammation in several dogs, but this was
considered a result of repeated trauma from daily injections rather than a test-article-related
problem.
Minimal-to-mild acinar degeneration of the exocrine pancreas was observed in dogs in all
groups including controls. Lesions as observed in this study have previously been reported in the
literature and are commonly described in historic control data.
One male in the subcutaneous group had an area of marked necrosis and hemorrhage that was
consistent with an infarct. The cause is unknown, but it is a common finding and should not be
considered a test-article-related phenomenon.
Overall, it can be stated that intravenous, oral, or subcutaneous administration of trehalose for
14 days did not produce abnormal morphology.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Heart Rate, Body Temperature, and Respiration
These parameters were normal and comparable between the control and treated groups for each
route of administration over the 14-day study.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

dermal irritation

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

Conclusions:

Trehalose, given by oral, intravenous, or subcutaneous route of administration (at 5, 0.25 and 1 g/kg
bw/day resp.), was not associated with macro- or microscopic changes.

Executive summary:

This study, conducted for Quadrant Holdings, was designed to investigate the toxicity

associated with the daily administration of oral, intravenous, or subcutaneous doses of

trehalose in beagle dogs for 14 days. Three male and three female dogs per group received

treatments of trehalose at doses of 1 g/kg/ d by intravenous injection in the cephalic vein,

5 g/kg/d by oral capsule, or 0.25 g/kg/d by subcutaneous injection for 14 consecutive days. Three

male and three female dogs per group that served as vehicle controls received empty capsules

at the same number of capsules/kg body weight as those treated with the test article for 14

consecutive days.

Observations for mortality and signs of reaction to treatment were performed twice daily.

Detailed individual physical examinations were performed weekly. Body weight was

recorded before dosing and weekly thereafter. Weekly food consumption was calculated

beginning during acclimation. Blood was collected for determination of serum glucose at

specified intervals on the last day of dosing (up to 24 hours after dosing) for each route of

administration. Rlood was collected for hematology and clinical chemistry evaluations before

dose initiation and before necropsy. On day 15, animals were humanely killed and necropsied,

and tissues were preserved. Histopathologic examination of all protocol-required tissues was

performed for all animals in the control and oral trehalose-treated groups and for selected

tissues of animals in the intravenous and subcutaneous groups.

Oral, intravenous, or subcutaneous administration of trehalose at a limit dose for 14 days did

not have an effect on mean body weight or food consumption nor did it produce acute lethality.

All animals treated by the oral route experienced diarrhea. Occasional vomiting was observed

for a few animals treated orally or subcutaneously.

Fourteen days after dosing, slight, transient elevations occurred in glucose for the oral and

intravenous groups through the 30-minute or I-hour post dose interval. Subcutaneous

parameters. None of the differences was directly attributed to administration of the test

material.

No macro- or microscopic lesion was noted that could be attributed to administration of the test

article.

Under the conditions of this study, oral, intravenous, or subcutaneous administration of

trehalose at a limit dose did not produce systemic toxicity. After 14 days of dose

administration, baseline serum glucose levels were not dramatically affected which suggested

that trehalose did not accumulate after repeated dosing. Further, only transient elevations

were noted after oral or intravenous dose administration; the small differences were considered

not toxicologically significant.

administration of trehalose did not affect glucose levels.

After 14 days of treatment, no toxicologically meaningful changes in hematologic or clinical

chemistry status was observed for any route of administration. Slight, but statistically

significant, differences from control were noted in a few hematologic and clinical chemistry

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

8. March -3 October 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study, conducted for Quadrant Holdings, was designed to investigate the toxicity
associated with the daily administration of oral, intravenous, or subcutaneous doses of
trehalose in CD®-1 mice for 14 days.

GLP compliance:

yes

Limit test:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: lot no. 22128, Pfanstiehl Laboratories, Inc. (Waukegan, IL)
- Expiration date of the lot/batch: not stated
- Purity test date: not stated
RADIOLABELLING INFORMATION (if applicable)
not applicable
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not stated
- Stability under test conditions: not stated but assumed uncritical
- Solubility and stability of the test substance in the solvent/vehicle: not stated but assumed uncritical
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not stated but
assumed uncritical
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
none

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

no further details given

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Raleigh, NC)
- Females (if applicable) nulliparous and non-pregnant: not stated
- Age at study initiation: 6 weeks
- Weight at study initiation: 25.2 -33.2 g (male), 19.6 - 25.4 g (female)
- Fasting period before study: none
- Housing: single
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days
DETAILS OF FOOD AND WATER QUALITY:
Powdered Purina Certified Rodent Chow® 5002 and municipal tap water filtered by reverse
osmosis were available ad libitum to the animals. Results of analytical certification of
batches of feed provided by the manufacturer and water analysis performed periodically by
the municipal authority and FRC are retained on file at FRC. There were no known
contaminants in the feed or the water which could have influenced the outcome of the study.
ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72° ± 4° F
- Humidity (%): 30%-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 19 Apr. 1994 To: 11 May 1994

Type of coverage:

other: subcutaneous

Vehicle:

physiological saline

Details on exposure:

A trehalose solution or the vehicle was administered orally via an 18-gauge stainless steel
gavage needle; trehalose solutions were administered intravenously in a lateral tail vein or
subcutaneously in the dorsal surface via a 25-gauge sterile needle and syringe. Animals were
dosed once daily for 14 days. All solutions were administered at room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

analytical verification was done by sponsor, no further documentation available

Duration of treatment / exposure:

14 d

Frequency of treatment:

1 per day

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

5 000 mg/kg bw/day (nominal)

Remarks:

oral gavage

Dose / conc.:

2 500 mg/kg bw/day (nominal)

Remarks:

subcutaneous

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

intravenous

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: not stated
- Rationale for animal assignment (if not random): Animals were assigned randomly to groups by a
computerized body weight randomization
program that ensured homogeneity of group means and reduced variances for body weights
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random):

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Animals were examined twice daily (in the morning and the afternoon) for mortality and signs
of reaction to treatment. Each animal was examined individually, including observation of
external surfaces, all orifices, injection sites (as applicable), and palpated for masses, weekly.
Observed clinical signs were recorded for individual animals.
DETAILED CLINICAL OBSERVATIONS: Yes
Animals were examined twice daily (in the morning and the afternoon) for mortality and signs
of reaction to treatment. Each animal was examined individually, including observation of
external surfaces, all orifices, injection sites (as applicable), and palpated for masses, weekly.
Observed clinical signs were recorded for individual animals.
BODY WEIGHT: Yes
Animals were weighed individually for randomization, on day 1 before dosing, and weekly
thereafter.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was measured weekly, beginning one week before dosing commenced.
FOOD EFFICIENCY:
- Body weight gain calculated
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:
OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
HAEMATOLOGY: Yes
and
CLINICAL CHEMISTRY: Yes
Blood was collected for glucose analyses on day 14 of dosing at selected intervals after dosing.
Blood was collected into Microtainer® serum separator tubes from the orbital sinus from the
first five animals/sex/group and was centrifuged and sent to Ani Lytics for analysis. Blood
from the oral groups was collected at approximately 30 minutes and 1, 4, and 24 hours after dose
administration, blood from the intravenous group was collected at approximately 5 and 30
minutes, 2 and 24 hours, and blood from the subcutaneous group was collected at approximately
15 minutes, 1, 2, and 24 hours after dosing. In addition, blood was collected from these animals
at 24 hours for routine hematology evaluation.
Before necropsy, blood was collected from the remaining five animals/sex/group for routine
clinical chemistry evaluation. Blood was collected for glucose, hematology, and clinical
pathology from only four males in the intravenous dose group (group 2) due to the removal of
two animals from the study.
Clinical pathology services were provided by Ani Lytics, Inc. Methods used in laboratory
investigations are described in Standard Operating Procedures retained in the clinical
pathology laboratory of Ani Lytics, Inc.
URINALYSIS: No
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.
NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
IMMUNOLOGY: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.
OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
On day 15, all surviving animals were humanely killed by carbon dioxide asphyxiation,
followed by .exsanguination from the abdominal aorta. Macroscopic examination of all
surviving animals, performed by qualified necropsy technicians in the presence of a
pathologist, included the external surface of the carcass and a detailed internal examination of
the major body cavities, organs, and viscera. Complete macroscopic examination of the animal
found dead on day 6 of the study was performed by a qualified necropsy technician.
HISTOPATHOLOGY: Yes (see table)
Tissues listed were trimmed, embedded in paraffin wax, sectioned, and stained with
hematoxylin and eosin. Histopathologic examination was performed on tissues listed above in
the control and oral dose groups. Histopathologic examination was performed on the kidney,
liver, pancreas, urinary bladder, and injection sites for the intravenous and subcutaneous groups.
Pathology services were performed by Pathology Associates, Inc. (Frederick, MD).
The tissues listed were saved (if applicable) from all animals. Tissues were preserved in 10 % NBF. L
ungs were infused with 10% NBF.
animal identification
adrenals
aorta (thoracic)
bone/ marrow (sternum)
brain (three levels)
cecum
colon
duodernun
epididymides
esophagus
eyes
gallbladder
heart
ileum
injection site
jejunum
kidneys
liver
lungs
lymph nodes (mandibular and mesenteric)
mammary gland
optic nerves
ovaries
pancreas
pituitary
prostate
salivary glands
sciatic nerve
seminal vesicles
skeletal muscle
skin
spinal cord
spleen
stomach
testes
thymus
thyroid lobes (and
parathyroids)
tongue
trachea
urinary bladder
uterus
vagina

Statistics:

Means and standard deviations were calculated for numerical data. Subsequently, data were
analyzed for homogeneity using ANOV A and then subjected to Dunnett's t-test. Calculations
for body weight, food consumption, and weight change were performed with the commercially
available Labcat® statistical program. Calculations for clinical pathology data were
performed with the commercially available StatView s12+ statistical program. All
statistical comparisons for the intravenous, oral, and subcutaneous groups were made against
the oral control group (group I ).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs of toxicity were apparent in the animals treated by the oral or subcutaneous routes. One male treated
subcutaneously showed clipper burn, which was not related to treatment, from days 8 to 15.
In the intravenously treated animals, mild necrosis of the tail was seen in one male on day 2 and
a second male on day 3. The necrosis increased in severity and by day 7, both animals were
removed from the study due to severe necrosis of the tail (and eventual tail loss in both
animals) which prevented further dosing. Three of the remaining eight males in the
intravenous group showed lesions on the tail which included redness, crust, swelling, sore,
necrosis, and/or loss of portions of the tail. These signs began to appear on day 7 for one animal
and on day 8 for the two remaining animals. One female dosed by the intravenous route had
crust and a sore on the tail beginning on day 8 and another female in this group had black
discoloration on the tail at day 14. No abnormal gross post mortem findings were noted that
corroborated the tail discoloration.
The tail findings, which progressed to necrosis in several cases, were most likely attributed to
the hypertonic solution administered. This could have occurred as a result of the
administration of some test article outside the vein or through intravascular irritation, which
may have allowed infiltration of the test substance into the perivascular space. Due to the
limited tissue space, normal osmotic forces could not dilute the hypertonic solution. Extended
contact of the concentrated trehalose solution resulted in cell death.
Other clinical observations noted included ocular opacity for two males in the intravenous

Dermal irritation:

no effects observed

Description (incidence and severity):

upon subcutaneous dosing, no effects observed.
upon intraveneous dosing, crust, necrosis, and/ or missing portions of the tail were seen in five males
and one female which showed similar clinical signs.
Although no direct application on the skin of the animals was performed,
the subcutaneous route may serve as an indicator for systemic toxicity after dermal exposure.
No treatment related effects for this route are observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female (no. 2504) treated by
the intravenous route died after dosing on day 6. No clinical signs were observed for this
animal prior to the day of death. Necropsy findings were unremarkable therefore, a cause of
death was not determined. No other deaths occurred during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Group mean body weight and body weight change was essentially comparable between the
vehicle control and treated groups for each route of administration. A slightly lower gain in
the group 4 males during week 2 was attributed to weight loss in one animal. The difference
noted was not attributed to administration of the test article.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Treatment of trehalose by any route had no effect on food consumption throughout the study.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Decreased white blood cell counts for males were apparent 14 days after oral or subcutaneous
dosing of trehalose. The decreased total white blood cell count was attributed to a decrease in
total lymphocytes as well as slight (not statistically significant) decreases in monocytes and
eosinophils noted in the same group. Total lymphocytes and monocytes tended to be lower than
control values for the intravenously dosed males, although the differences were not
statistically significant. A similar trend for lymphopenia was not evident in the orally or
subcutaneously treated female groups. Further, no histopathologic evidence of bone marrow
changes were observed that could corroborate the clinical pathology findings. Other
hematologic parameters affected were mean corpuscular volume (MCV) and mean corpuscular
hemoglobin (MCH) for the group 3 males. In the absence of effects on erythrocyte count or
hemoglobin, the statistically significant decreases for MCV and MCH were considered not test
article related.
No differences were noted in hematologic parameters for females. This suggests that there may
be a sex difference with respect to hematologic effects.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Serum Glucose
A moderate degree of intra-animal and intra-group
variability, expected with small group sizes, was observed.
The orally treated animals (group 3) showed a slight elevation over control glucose values at
30 minutes (males and females) and 1 hour (females only) after dosing on day 1~, which then
declined through the 24-hour interval.
After intravenous administration of trehalose, males had higher serum glucose levels at 5 and
30 minutes compared with the 30-minute post dosing control value. Females treated
intravenously had a slightly higher glucose level at the 5-minute post dosing interval. Over
the remaining sample intervals, glucose levels were comparable to historic values for mice.
Fifteen minutes after subcutaneous administration, the serum glucose level was slightly
increased for males which was comparable to the level seen after the intravenous dose. In the
corresponding group of females, no change over time was observed for serum glucose and the
mean values were comparable to the oral control values at corresponding time points.
The changes in glucose observed for each route of administration were considered transient.
Therefore, it can be stated that oral, intravenous, or subcutaneous administration of trehalose in
repeated doses did not produce accumulation of glucose.

Clinical Chemistry
Sodium and chloride levels were slightly elevated over control values for males treated by the
intravenous, oral, or subcutaneous route. Phosphorus was also elevated in the males treated
subcutaneously with trehalose. In comparison, electrolyte levels for females given trehalose by
any route were comparable to control values with the exception of chloride in the subcutaneous
group which was slightly elevated.
In the males given trehalose orally, blood urea nitrogen (BUN) was decreased from the control
value and albumin and, as a result, total protein were slightly elevated over the control values.
Total protein also was elevated slightly in the subcutaneously treated males, whereas globulin
was statistically significantly elevated in the intravenously treated males without a
corresponding increase in total protein. Decreases in BUN are not usually associated with
clinically relevant changes and, therefore, the decrease in this study is not considered
toxicologically meaningful. The increases in total protein, albumin, or globulin were slight,
variable, and considered to be normal biologic variability.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related findings were observed in the animals treated orally or subcutaneously. In animals treated by
the intravenous route, crust, necrosis, and/ or missing portions of the tail were seen in five males
and one female which showed similar clinical signs. The tail lesions were attributed to the
manipulations performed during the intravenous procedure which resulted in compromised
circulation. Microscopic examination revealed that a control male had an esophageal mass
with plant material and bacteria at the center of the lesion, which suggested t.hat the mass
was the result of a gavage injury. One female in the intravenous group had an enlarged spleen
and kidney mass that was determined to be a chronic abscess; neither lesion was related to
administration of the test article.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Various individual gross lesions occurred but were considered not test article related.
No microscopic test-article-related lesions were observed. Incidental lesions were observed
among mice in all dose groups but none could not be associated with the test article or route of
administration. No evidence of decreased lymphocytes was observed in the clinical pathology
data specific to the bone marrow.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

dermal irritation

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

Conclusions:

Trehalose, given by oral, intravenous, or subcutaneous route of administration (at 5, 2.5 and 1 g/kg
bw/day resp.), was not associated with macro- or microscopic changes.

Executive summary:

This study, conducted for Quadrant Holdings, was designed to investigate the toxicity

associated with the daily administration of oral, intravenous, or subcutaneous doses of

trehalose in CD-1 mice for 14 days. Ten male and 10 female mice per group received

treatments of trehalose at doses of 1 g/kg/ d by intravenous injection into the tail vein, 5 g/kg/ d

by oral gavage, or 2.5 g/kg/d by subcutaneous injection for 14 consecutive days. Ten male and

10 female mice received oral doses of sterile saline at the same dose volume as those treated

orally with test article for 14 consecutive days and served as vehicle controls.

Observations for mortality and signs of reaction to treatment were performed twice daily.

Detailed individual examinations were performed weekly. Body weight and food consumption

were measured before dosing and weekly thereafter. Blood was collected at specified intervals

on the last day of dosing (up to 24 hours after dosing) for each route of administration for

determination of serum glucose. Blood was collected for hematology and clinical chemistry

evaluations before necropsy. On day 15, surviving animals were humanely killed and

necropsied, and tissues were preserved. Histopathologic examination was carried out for all

tissues of animals in the control and oral dose groups and for selected tissues of animals in the

intravenous and subcutaneous groups.

Repeated intravenous, subcutaneous, or oral administration of trehalose for 14 days did not

affect body weight or food consumption of the mice. Further, no adverse clinical signs were

evident in the subcutaneously or orally treated animals throughout the 14-day treatment. No

treatment-related macro- or microscopic effects were noted.

One female treated intravenously died on day 6; a cause of death could not be determined based

on clinical signs or necropsy findings.

Mild necrosis of the tail, which was observed early in the study for two males treated

intravenously with trehalose, progressed to the point where dosing was not possible and these

animals were removed from the study. Three additional males and two females in the

intravenous group showed tail lesions that ranged from redness, crust, swelling, and sores to

black discoloration and necrosis and loss of a portion of the tail. The tail lesions were

attributed to manipulations performed during the intravenous procedure which resulted in

compromised circulation. Other clinical observations noted were considered not related to

treatment.

Blood glucose was elevated at various times after dosing on day 14. Serum glucose from the

intravenously treated animals was slightly elevated from 5 minutes (females) to 30 minutes

(males) after dosing and then declined over the remaining intervals. A minimal increase for

glucose was noted for the orally treated animals from 30 minutes to 1 hour after dosing, after

which the values returned to control levels. After subcutaneous dosing, glucose was slightly

elevated at 15 minutes, in males only, and then declined to values considered to be within

normal limits. Based on transient changes in the glucose levels, no significant accumulation of

glucose was evident after 14 days of dosing.

Total white blood cell count (WBC) was decreased from control value for males after 14 days of

oral or subcutaneous dosing. The decrease in WBC was attributed to a decrease in lymphocytes

and a tendency for decreases in monocytes and eosinophils. A similar trend for lymphopenia

was not evident in the corresponding groups of females nor was a histopathologic change

observed in the bone marrow that would confirm the clinical pathology results. Other

differences in hematologic parameters were considered not treatment related.

Sodium and chloride were slightly elevated from control values for males treated by the

intravenous, oral, or subcutaneous route. Phosphorus was also elevated for males treated

subcutaneously with trehalose. With the exception of chloride in females treated

subcutaneously, ·au electrolyte levels for females were comparable to those of the orally

treated controls. Blood urea nitrogen was decreased from the control value for males treated

orally and albumin, globulin, and total protein were slightly elevated over the control values

for each group. These differences were considered not toxicologically meaningful.

Under the conditions of this study, administration of trehalose, either oral or subcutaneous, at a

limit dose was not associated with any discernible clinical sign of toxicity. Intravenous dosing

was limited by effects leading to necrosis on the tail. Transient increases in serum glucose were

noted for all routes of administration during a 24-hour period after 14 days of dosing. Further,

the data suggest that no accumulation of glucose occurred after repeated dosing with trehalose

in mice. Possible treatment-related changes in clinical pathology included decreased white

blood cell count accompanied by a decrease in lymphocytes and slight elevations in sodium,

chloride, and phosphorus. The decease in lyphocytes for animals in the oral and subcutaneous

groups could not be confirmed histopathologically. Trehalose, given by oral, intravenous, or

subcutaneous route of administration, was not associated with macro- or microscopic changes.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

information from referenced studies is assessed and used to adapt data requirement.

GLP compliance:

no

Test type:

other: details given in referenced study summaries

Test material

Test material information

Constituent 1

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:

details given in referenced study summaries

Administration / exposure

Type of coverage:

other: details given in referenced study summaries

Vehicle:

other: details given in referenced study summaries

Details on dermal exposure:

details given in referenced study summaries

Duration of exposure:

details given in referenced study summaries

Doses:

details given in referenced study summaries

No. of animals per sex per dose:

details given in referenced study summaries

Details on study design:

details given in referenced study summaries

Statistics:

details given in referenced study summaries

Results and discussion

Effect levels

Effect levels 1

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 500 mg/kg bw

Based on:

test mat.

Remarks on result:

other: species mouse

Effect levels 2

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 250 mg/kg bw

Based on:

test mat.

Remarks on result:

other: species dog

Mortality:

details given in referenced study summaries

Clinical signs:

other: details given in referenced study summaries

Gross pathology:

details given in referenced study summaries

Other findings:

details given in referenced study summaries

Any other information on results incl. tables

No signs of toxicity were noted by subcutaneous route in mouse and dog.

This piece of evidence shows that systemic toxicity does not occur, when the substance is administered under the skin. Therefore the results represent absence of effects under the assumption of 100 % dermal absorption.

Applicant's summary and conclusion

Interpretation of results:

other:

Conclusions:

No signs of toxicity were noted by subcutaneous route in mouse and dog.
the results represent absence of effects under the assumption of 100 % dermal absorption.
The stated LD50 is valid for the species mouse.