Trehalose

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13. Jan 1997 - Jan. 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 50615
- Expiration date of the lot/batch: 14. Jun. 1998
- Purity test date: not stated

RADIOLABELLING INFORMATION (if applicable)
none

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, in a sealed container protected from
moisture and direct sunlight.
- Stability under test conditions: Stable under the specified storage conditions
- Solubility and stability of the test substance in the solvent/vehicle: The homogeneity and stability of the test article in the diet
were established over at least 3 weeks before the start of
treatment. Intercurrent sampling for content analyses was
performed at monthly intervals (total: 3 times).
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: The homogeneity and stability of the test article in the diet
were established over at least 3 weeks before the start of
treatment. Intercurrent sampling for content analyses was
performed at monthly intervals (total: 3 times).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
TREHALOSE was mixed with microgranulated feed from
which pellets were prepared. Water (approximately 1: 10
volume/weight ratio) was added to aid pelleting. The pellets
were dried with warm air for approximately 48 hours before
storage.

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

Recognized by the international guidelines as a recommended
test system

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd.
CH-4414 Fiillinsdorf I Switzerland
- Females (if applicable) nulliparous and non-pregnant: not stated
- Age at study initiation: 4 weeks
- Weight at study initiation: Males: 19.0-26.7 grams (mean: 23.0 grams), Females: 16.2-23.9 grams (mean: 20.0 grams)
- Fasting period before study: not stated
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
Pelleted standard Kliba 343 mouse maintenance diet (KLIBA
Mtihlen AG, CH-4303 Kaiseraugst/Switzerland) was
available ad libitum (Batch Nos. 82/96, 84/97). The feed
batch was analyzed for contaminants (see Attachment 2, pp.
311-315).
Tap water was available ad libitum in water bottles. The
water was analyzed for chemical contaminants and for
bacteriological contaminants (see Attachment 1, pp. 307-
310).
No contaminants expected to interfere with the study were
present in the feed or water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 3 °C
- Humidity (%): 40 - 70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

Oral, by feeded mixture. Control animals received similarly prepared pellets but without the test article.
rationale: Simulates the route of human exposure.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

- DIET PREPARATION
TREHALOSE was mixed with microgranulated feed from which pellets were prepared. Water (approximately 1:10 volume/weight ratio) was added to aid pelleting. The pellets were dried with warm air for approximately 48 hours before storage.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity and stability of the test article in the diet were established over at least 3 weeks before the start of treatment. Intercurrent sampling for content analyses was performed at monthly intervals (total: 3 times). Analyses were performed in the RCC UMWELTCHEMIE AG Analytical Chemistry Laboratory, according to a method supplied by the Sponsor.

Duration of treatment / exposure:

13 weeks, 1 day: animal numbers 1-10, 21-30, 41-50, 61-70
13 weeks, 2 days: animal numbers 81-90, 101-110, 121-130,
141-150
13 weeks, 3 days: animal numbers 11-20, 31-40, 51-60, 71-80

Frequency of treatment:

Ad libitum

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

stated as 5000 ppm, conversion factor 1 ppm = 0.15 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

2 250 mg/kg bw/day (nominal)

Remarks:

stated as 15000 ppm, conversion factor 1 ppm = 0.15 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

7 500 mg/kg bw/day (nominal)

Remarks:

stated as 50000 ppm, conversion factor 1 ppm = 0.15 mg/kg bw/day

No. of animals per sex per dose:

20

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The selection of dietary concentrations is based upon results
from a 14-day oral toxicity (feeding) study with
TREHALOSE in mice
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random): all control and high dose animals, additionally animals found dead

Positive control:

none

Examinations

Observations and examinations performed and frequency:

VIABILITY/MORTALITY
Each mouse was checked for viability twice daily.
CLINICAL SIGNS
Observations for clinical signs of toxicity were performed at least once daily.
FOOD CONSUMPTION
The food consumption was recorded weekly using an on-line electronic recording system
consisting of a Mettler balance connected to the RCC computer.
BODY WEIGHTS
The body weight of each animal was recorded weekly using an on-line electronic recording
system consisting of a Mettler balance connected to the RCC computer.
OPHTHALMOSCOPIC EXAMINATIONS
Ophthalmoscopic examinations were performed at pretest on the 10 mice with the highest
identification numbers per sex and group. During Week 13 an examination was performed on
the same animals from groups 1 and 4. As no treatment-related changes were determined at
the highest dietary concentration, no animals from the mid or low dose groups (2 and 3) were
examined.
About ten minutes after the application of a mydriatic solution (DISPERSA AG, Winterthur I
Switzerland) the cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes
were examined using a Heine Miroflex 2 Ophthalmoscope (Eisenhut Vet AG, Allschwil I
Switzerland).
CLINICAL LABORATORY INVESTIGATIONS
GENERAL
Blood samples for hematology and clinical biochemistry were collected from all animals
under light ether anesthesia. The animals were fasted in metabolism cages for approximately
18 hours before blood sampling. Twenty ml of tap water/kg body weight was administered to
each animal before the fasting period. Blood samples were collected between 0630 and 0800 to
reduce biological variation caused by circadian rhythms. Blood samples were drawn from the
retro-orbital plexus using a micro-hematocrit glass capillary tube.
Blood samples from the 10 mice with the lowest identification numbers per group and sex
were used for hematology and samples from the 10 mice with the highest identification
numbers per group and sex were used for clinical biochemistry.
Urine was collected during the 18-hour fasting period into a specimen vial. Five mice were
placed in each metabolism cage to ensure a sufficient volume of urine was obtained for
analysis. Each sample of pooled urine was identified in the tables of individual values by the
lowest individual animal number of the five animals.
Blood and urine sampling:
Blood samples from the 10 mice with the lowest identification numbers per group and sex
were used for hematology and blood samples from the 10 mice with the highest identification
numbers per group and sex were used for clinical biochemistry.
Blood and urine sampling:
At 5 weeks February 18/19, 1997
At 9 weeks
After 13 weeks
March 18119, 1997
April 22/23/24/25, 1997
The assays of blood and urine parameters were performed, at BRL Biological Research
Laboratories Ltd., under internal laboratory quality control conditions to assure reliable test
results.
The summary and individual tables were generated by a computer. The program used limits
the width of each column to 10 characters. Therefore, the nanies of some parameters have
been abbreviated. Any abbreviation has been defined in this section under "Parameter" in
upper-case letters enclosed by parentheses.
Clinical laboratory data are expressed in general accordance with the International System of
Units (Sn, which in structure comprises base units, derived units and supplementary units. It
also includes a series of prefixes by means of which decimal multiples and submultiples of
these units can be formed. In some cases non-SI units or conventional units may be used.
HEMATOLOGY
The following anticoagulants were used during blood collection:
EDTA-K2 (hematology)
Sodium citrate, 3.8 % (coagulation; 1 part anticoagulant to 9 parts blood)
The following commercial reference controls were used to monitor the performance of the method:
Hematology:
Eightcheck-3WP (normal range) Eightcheck-L-3WP (low abnormal range) Ret-check (reticulocyte
control)
(TOA Medical Electronics Co. Ltd., Kobe/Japan)
The following methods were used to determine the values of the parameters listed:

Hematology Parameter Abbreviation Unit Instrumentation
Erythrocyte count RBC T/L 1
Hemoglobin HB mmol/L 1
Hematocrit HCT L/L 1
Mean corpuscular volume MCV fL 1
Mean corpuscular hemoglobin MCH fmol 1
Mean corpuscular hemoglobin MCHC mmol/L 1
concentration
Platelet count PLATELETS GIL 1
Reticulocyte count RETIC. %(rel.) 2
T/L(abs.)
Reticulocyte fluorescence HFR= high, % 2
ratios MFR = middle,
LFR=low

Nucleated erythrocytes NEN NEN/1OOWBC 3
(normoblasts)
Total leukocyte count WBC G/L 1
Differential leukocyte count Diff. WBC 1(rel.) 3
Count GIL( abs.) 3
Red blood cell morphology normal/abnormal

The following anticoagulant was used during blood collection:
Lithium heparin (15 l.U./ml).
The following commercial reference controls were used to monitor the performance of the
method:
Clinical Biochemistry:
QualitrolR HS-N (normal range) and QualitrolR HS-P (high abnormal range)
(E. Merck, Darmstadt/Germany)

Protein Electrophoresis: Seronorm™ Protein
(Nycomed Pharma AS, Oslo/Norway)
The following methods were used to determine the values of the parameters listed:

Clinical Biochemistry Abbreviation Unit Instrumentation
Parameter
Glucose mmol/L 1
Urea mmol/L 1
Bilirubin, total BILI. T µmol/L 1
Cholesterol, total CHOLEST. T mmol/L 1
Triglycerides TRIGL. mmol/L Aspartate 1
Aspartate aminotransferase ASAT/GOT µkat/L (37°C) 1
Alanine aminotransferase ALAT/GPT µkat/L (37°C) 1
Lactate dehydrogenase LDH µkat/L (37°C) 1
Alkaline phosphatase ALP µkat/L (37°C) 1
Calcium mmol/L 1
Phosphorus mmol/L 1
Sodium mmol/L 1
Potassium mmol/L 1
Chloride mmol/L 1
Protein, total PROTEIN T. g/L 1
Protein electrophoresis PROT. 1(rel.) 2
ELECTROPH. g/L (abs.)


URINALYSIS
The following commercial reference control was used to monitor the performance of the
method:
Lyphochek Quantitative Urine Control - Normal I and Abnormal II - (for the assay control of
Osmolality, pH, Protein, Glucose, Ketone, Bilirubin, Blood and Urobilinogen).
(Bio-Rad, ECS Division, Anaheim, California/USA)
Liquichek™ Routine Urine Chemistry Control Levels 1 and 2 （for the assay control of pH,
Protein, Glucose, Ketone, Bilirubin, Blood and Urobilinogen)
(Bio-Rad, ECS Division, Anaheim, California/USA)
The following methods were used to determine the values of the parameters listed:
Urinalysis Parameter
Volume (18 hour)
Specific gravity
Osmolality
Color
Appearance
pH
Protein
Glucose
Ketone
Bilirubin
Blood
Urobilinogen
Urine sediment

Sacrifice and pathology:

PATHOLOGY
NECROPSY
After 13 weeks April 22/23/24/25, 1997
All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were
recorded. Necropsies were performed by experienced prosectors supervised by a veterinary
pathologist. All animals surviving to the end of the observation period were anesthetized by
intraperitoneal injection of pentobarbitone and killed by exsanguination.
Samples of the following tissues and organs were collected from all animals at necropsy and
fixed in neutral phosphate-buffered 4% formaldehyde solution:
Adrenal glands
Aorta
(Auricles)
Brain - including sections of medulla/pons, cerebral and cerebellar cortex
Cecum
Colon
Duodenum
Epididymides
Esophagus
(Extra-orbital lacrimal gland)
Eyes with optic nerve and Harderian gland
Femur - including articular surface
Heart
Ileum
(\ Jejunum
Kidneys
Liver with gall bladder
Lungs, infused with formalin
Lymph nodes - mesenteric, mandibular
Mammary gland area
Nasal Turbinates
Ovaries
Pancreas
Pituitary gland
Prostate gland
Rectum
Salivary glands - mandibular, sublingual
Sciatic nerve
Seminal vesicles
Skeletal muscle
(\ Skin
Spinal cord - cervical, midthoracic, lumbar
Spleen
Sternum with bone marrow
Stomach
Testes
Thymus
Thyroid gland I parathyroid gland
Trachea
Urinary bladder, infused with formalin
Uterus
Vagina
All gross lesions
ORGAN WEIGHTS
The following organ weights were recorded on the scheduled dates of necropsy:
Adrenals
Brain
Heart
Kidneys
Liver
Ovaries
Pituitary gland
Prostate
Spleen
Testes
Thyroid
Thymus
HISTOPATHOLOGY
Representative tissue samples from all mice of Groups 1 and 4, as well as from any
unscheduled deaths from Groups 2 and 3, were examined. Numbers of sections made are
given in parentheses.
Adrenal glands (2), aorta ( 1 ), bone - sternum ( 1) and femur ( 1 ), bone marrow - sternal ( 1 ),
brain (4) - cerebrum, cerebellum and medulla/pons, cecum (1), colon (1), duodenum (1),
epididymides (2), esophagus (1), eyes (2), Harderian glands (2); heart (1), ileum (1), jejunum
(1), kidneys (2), [lacrimal glands exorbital (2)], liver (2), lungs (2), lymph nodes - mandibular
(2) and mesenteric (1), mammary gland area (1), nasal cavity (3), optic nerves (2), ovaries (2),
pancreas (1 ), pituitary gland (1 ), prostate gland (1 ), rectum (1 ), salivary glands - mandibular
(2) and sublingual (2), sciatic nerve (1), seminal vesicles (2), skeletal muscle (1), skin (1),
spinal cord (3) - cervical, midthoracic and lumbar, spleen (1), stomach (1), testes (2), thymus
(1), thyroid with parathyroid glands (2), trachea (1), urinary bladder (1), uterus (3), vagina (1)
and all organs or tissues with macroscopic abnormalities.
All organs or tissues with macroscopic abnormalities were also prepared and examined from
mice of Groups 2 and 3

Statistics:

The following statistical methods were used to analyze the body weight, food consumption,
organ weights and clinical laboratory data:
If the variables can be assumed to follow a normal distribution, the Dunnett-test (many to one
t-test) based on a pooled variance estimate was applied for the comparison of the treated
groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied instead of the Dunnett-test if the data can not
be assumed to follow a normal distribution.
The Fisher's Exact Test was applied to ophthalmoscopy data and macroscopic findings.
References: - C.W. Dunnett: A Multiple Comparison Procedure for Comparing Several
Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
- R.G. Miller: Simultaneous Statistical Inference, Springer Verlag, New York
(1981).
- R.A. Fisher: Statistical Methods for Research Workers, Oliver and Boyd,
Edinburgh (1950).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

The few clinical signs noted did not distinguish treated groups from controls and were those
commonly seen in mice of this strain and age.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were four deaths during the treatment period.
Male No. 78 (50000 ppm) was killed in extremis on Day 78 (Week 12) of treatment. A
progressive reduction in food consumption and a loss of body weight of 7 .9 g had been
observed during the previous weeks. This animal was in poor condition two days before
sacrifice when it appeared emaciated, with ruffled fur and uncoordinated movements. A
deterioration in condition necessitated the early sacrifice of this animal.
Gravel was detected in the urinary bladder and pelvis of the right kidney at necropsy. Isolated
gray/white foci were also observed on the surface of both kidneys.
Histopathologic evaluation of the tissues revealed severe pyelonephritis which was considered
not to be related to treatment.
Male No. 12 (Control) was found dead on Day 64 (Week 10). A severe reduction in food
consumption had been recorded over the previous week while a loss in body weight of 11.4 g
had been recorded between Weeks 8 and 10. This mouse had appeared emaciated on the day
before death. No findings were observed at necropsy with the exception of a thickened
thymus.
Histopathologic examination indicated an extensive thoracic inflammation, which contributed
to the animal's death.
Male No. 16 (Control) died during anaesthesia and male No. 57 (15000 ppm) died after blood
sampling during the investigations in Week 5.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight gain. of male mice treated at 50'000 ppm or 15'000 ppm TREHALOSE was
slightly retarded in comparison with the control animals. On only one week, however, were
the differences shown to be statistically significant (Week 12, 50'000 ppm, p < 0.05).
Body weight gain of the females was unaffected by the administration of TREHALOSE.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Throughout the treatment period food consumption was slightly reduced for males treated at
50000 ppm and to a lesser extent at 15000 ppm in comparison with the control group.
Differences often achieved statistical significance (p < 0.05 or 0.01).
Food consumption amongst female mice was generally similar for all groups although, on
occasions, mice treated at 50000 ppm or 15000 ppm consumed slightly less than those of the
control group. These intergroup differences occasionally attained statistical significance
(p < 0.05).

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related ophthalmoscopic changes.

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed. Occasional intergroup differences were shown
to be statistically significant, but in the absence of any dose-related trend these are considered
fortuitous.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

An increase in plasma glucose concentration was recorded at each investigation (Weeks 5, 9
and 13) for both males and females treated at 50000 ppm. The difference from the controls
was shown to be statistically significant for the females in Weeks 5 and 9 (p < 0.05).
Plasma glucose concentrations were also shown to be slightly higher during Weeks 5 and 9 for
males and females treated at 5'000 ppm or 15000 ppm. Similar differences were recorded for
females only during Week 13. No consistent relationship to dose was apparent at the lower
dietary concentrations and the only difference to achieve statistical significance was the
increase in females at 15000 ppm recorded in Week 9 (p < 0.05).
A statistically significant reduction in total plasma bilirubin concentration was recorded for
both males and females at 50000 ppm in Week 5 and for males only in Week 9,
p < 0.05 (females) or 0.01 (males).
Extremely high values for aspartase aminotransferase, alanine aminotransferase and lactate
dehydrogenase were recorded in Week 5 for male No. 72 (50000 ppm).
A statistically significant decrease in plasma calcium concentration was recorded for males at
15000 ppm (p < 0.01) or 50000 ppm (p < 0.05) in Week 13. As similar differences were not
recorded for males during Weeks 5 or 9, or for females on any occasion, an association with
treatment is considered unlikely.
A slight, but statistically significant, increase in plasma phosphorous concentration was
recorded for males (p < 0.05) and females (p < 0.01) at 50000 ppm in Week 5. Similar
increases were seen during Week 9 in females at 15000 or 50000 ppm (p < 0.05) but not in
males where the mean values for animals at 50000 ppm was lower than the concurrent
control.
Although a slight trend for increased plasma phosphorous concentration was recorded for both
sexes in Week 13 statistical significance was not achieved.
In view of the small magnitude of the intergroup differences and the temporal variations seen
in the control plasma phosphorous concentrations, an effect of treatment is considered
unlikely.
On the single occasion that sufficient plasma was available for the determination of potassium
concentration (Week 13), a dosage-related reduction was recorded for both sexes. Statistical
significance was achieved at 15000 and 50000 ppm for males (p < 0.01) and at 50000 ppm
for females (p < 0.05). Although the values recorded for both male and female control groups
were slightly higher than expected, an association with treatment cannot be excluded.
All remaining statistically significant intergroup differences were considered to be fortuitous
and not related to the administration of TREHALOSE.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no treatment-related effects. All minor differences in the results of the urinalysis
parameters are considered to be incidental and reflect the normal biological variation for mice
of this strain and age.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The assessment of organ weights was based on absolute weights as well as on organ/body
weight and organ/brain weight ratios.
Organ weights were unaffected by the administration of TREHALOSE.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no findings which distinguished treated animals from the controls.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no microscopic alterations recorded which were considered related to test article
treatment.
Animal No. 12 had an extensive thoracic inflammation which had contributed to the animal's
death. The cause of morbidity in mouse No. 78 was a severe pyelonephritis which was not
considered related to treatment. Two instances of alveolar/bronchiolar adenomas were
recorded - male No. 58 at 15000 ppm and female No. 153 at 50000 ppm. These benign
pulmonary neoplasms are amongst the commonest spontaneous tumors in this strain of mouse
and these early, isolated occurrences were considered not related to treatment.
The remainder of microscopic findings recorded were also within the range of background
pathology encountered in NMRI mice of this age and strain and occurred at similar incidences
and severity in both control and treated mice.

Histopathological findings: neoplastic:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

> 7 500 mg/kg bw/day (nominal)

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

Key result

Critical effects observed:

no

Any other information on results incl. tables

The daily administration of TREHALOSE to mice for 13 weeks at dietary concentrations of

up to 50000 ppm was generally well tolerated with no evidence of toxicity.

Minor changes were recorded principally in mice at 50000 ppm. These comprised slight

reductions in food consumption and body weight gain in the males, slightly increased plasma

glucose concentration and slightly reduced plasma bilirubin and potassium concentrations.

As the reduction in food consumption was apparent from the onset of dosing and as there was

no clinical or pathological evidence of toxicity, this was probably attributable to palatability.

The slight reduction in food consumption, in comparison with the control group, was reflected

in a slight reduction in body weight gain.

The increase in plasma glucose concentration observed at each investigation in both sexes was

considered to be related to the administration of TREHALOSE. This finding may be attributed

to the metabolism of the test article.

The high values for aspartate aminotransferase, alanine aminotransferase and lactate

dehydrogenase activities recorded on one occasion for a single animal at 50000 ppm were

considered to be spontaneous and of no toxicological significance as similar findings were not

recorded in any other animal.

No treatment-related pathological changes were detected.

From the results obtained in this study the No-Toxic-Effect-Level (NOTEL) is considered to

be 50000 ppm.

Applicant's summary and conclusion

Conclusions:

From the results obtained in this study the No-Observed-Adverse-Effect-Level (NOAEL) is considered to be 50'000 ppm, laying down the conversion factor of 1 ppm = 0.15 mg/kg bw/day, the NOAEL is determined to be 7500 mg/kg bw/day.

Executive summary:

This study was conducted at RCC ltingen / Switzerland according to internationally

recognized Good Laboratory Practice and Conditions. The purpose of the study was to assess

the cumulative toxicity of TREHALOSE when administered in the diet to mice for a period of

13 weeks.

Three groups of 20 male and 20 female mice received TREHALOSE at dietary concentrations

of 5000, 15000 or 50000 ppm for 13 weeks. A fourth group of 20 male and 20 female mice

received untreated diet for the same time period and served as a control.

There were four deaths during the treatment period, two control males, one male at

15000 ppm and one male at 50000 ppm. None of the deaths were attributed to the

administration of TREHALOSE.

Food consumption was slightly reduced for males treated at 50000 ppm and to a lesser extent

at 15000 ppm. Food consumption amongst female mice was generally similar for all groups

although, occasionally, the groups treated at 50'000 ppm or 15'000 ppm consumed slightly less

food than the control group.

Body weight gain of male mice treated at 50000 or 15000 ppm TREHALOSE was slightly

retarded in comparison with the control animals. Body weight gain of the females was

unaffected by the administration of TREHALOSE.

Clinical signs and ophthalmoscopy were unaffected by the administration of TREHALOSE.

Hematology parameters were not affected by treatment with TREHALOSE.

An increase in plasma glucose concentration was recorded at each investigation for both males

and females treated at 50000 ppm. Slight increases were also recorded, on occasions, for

animals at 5000 ppm or 15000 ppm.

Extremely high aspartate aminotransferase, alanine aminotransferase and lactate

dehydrogenase activities were recorded in Week 5 for a single male at 50'000 ppm.

A statistically significant reduction in total bilirubin concentration was recorded for both

males and females at 50000 ppm in Week 5 and for males only in Week 9.

On the single occasion that sufficient plasma was available for the determination of potassium

concentration (Week 13), a dosage-related reduction was recorded for both sexes. Although

the values recorded for both male and female control groups were slightly higher than

expected, an association with treatment cannot be excluded.

Macroscopic and microscopic examination of the tissues did not indicate any evidence of

toxicity to the test article.

The daily administration of TREHALOSE to mice for 13 weeks at dietary concentrations of

up to 50000 ppm was generally well tolerated with no evidence of toxicity.

Minor changes were recorded principally in mice at 50000 ppm. These comprised slight

reductions in food consumption and body weight gain in the males, slightly increased plasma

glucose concentration and slightly reduced plasma bilirubin and potassium concentrations.

As the reduction in food consumption was apparent from the onset of dosing and as there was

no clinical or pathological evidence of toxicity, this was probably attributable to palatability.

The slight reduction in food consumption, in comparison with the control group, was reflected

in a slight reduction in body weight gain.

The increase in plasma glucose concentration observed at each investigation in both sexes was

considered to be related to the administration of TREHALOSE. This finding may be attributed

to the metabolism of the test article.

The high values for aspartate aminotransferase, alanine aminotransferase and lactate

dehydrogenase activities recorded on one occasion for a single animal at 50000 ppm were

considered to be spontaneous and of no toxicological significance as similar findings were not

recorded in any other animal.

No treatment-related pathological changes were detected.

From the results obtained in this study the No-Toxic-Effect-Level (NOTEL) is considered to

be 50000 ppm