1,4:3,6-dianhydro-D-glucitol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2003 to May 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Sufficiently compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions. The few deviations have no impact on the reliability and completeness of the conclusions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

No microscopic examination was performed on the Harderian glands of one male; the microscopic examinations of the skin of one male of group 4 and the ovaries of one female of group 2 were not performed (technical error)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,4:3,6-dianhydro-D-glucitol

EC Number:

211-492-3

EC Name:

1,4:3,6-dianhydro-D-glucitol

Cas Number:

652-67-5

Molecular formula:

C6H10O4

IUPAC Name:

1,4:3,6-dianhydro-D-glucitol

Details on test material:

- Name of test material (as cited in study report): Isosorbide
- Physical state: whitish crystalline powder
- Analytical purity: 99.8%
- Purity test date: 01 October 2002
- Lot/batch No.: E0031
- Expiration date of the lot/batch: not specified
- Storage condition of test material: at room temperature and protected from humidity

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: laboratory breeder
- Age at study initiation: 7 week old
- Weight at study initiation: 219 g to 287 g for the males and 160 g to 199 g for the females.
- Fasting period before study: no
- Housing: suspended wire-mesh cages
- Diet (e.g. ad libitum): A04 C P2.5 powdered maintenance diet
- Water (e.g. ad libitum): tap water (filtered with a 0.22 μm filter)
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°
- Humidity (%): 50 +/-20
- Air changes (per hr): 12 cycles per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 27 March 2003 to 11 July 2003

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): the dietary admixtures were prepared once a week for groups 1 to 3 and twice a week for group 4 from week 6
- Mixing appropriate amounts with (Type of food): A04 C P2.5 powdered maintenance diet, batch Nos. 21118, 30130, 30227 and 30423
- Storage temperature of food: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of duplicate samples taken from each dietary admixture (including the control) prepared for use in weeks 1, 4, 7 and 13 was determined.
There was a good correspondence between the nominal and the measured concentrations of the test item in the diet: a satisfactory agreement was observed between the nominal and actual concentrations of the test item in the dietary admixtures administered since the deviations from nominal concentrations were in an acceptable range of ± 10%.
Method: Gas Liquid Chromatography (GC) with Flame Ionisation Detection
Limit of quantification: 7500 ppm

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Continuous administration was achieved by dietary admixture.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
12500 ppm
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
25000 ppm
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
50000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

10 animals per sex and per dose

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The dose-levels were determined on the basis of the results of a preliminary 2-week dietary toxicity study performed in the same species in which neither clinical signs nor perturbation of the body weight evolution or food consumption were noted at 12500, 25000 or 50000 ppm. In addition, there were no treatment-related macroscopic findings or changes in organ weights.

- Rationale for animal assignment (if not random): according to a computerized stratification procedure

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day
- Cage side observations .

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the beginning of the treatment period and then once a week until the end of the study.

BODY WEIGHT: Yes
- Time schedule for examinations: the body weight of each animal was recorded at least once before allocation of the animals to
groups, on the first day of treatment, and then once a week until the end of the study

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight data: Yes
The quantity of food consumed by the animals of each cage was recorded once a week (over a 7-day period) for groups 1 to 3. Food consumption of group 4 animals was recorded at weekly intervals until week 4 inclusive, and twice a week from week 5.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: recorded on one occasion before the beginning of the treatment period, and then at weekly intervals during the treatment period

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: performed on all animals before the beginning of the treatment period and on one occasion at the end of the treatment period (control and high-dose groups).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on week 13
- Anaesthetic used for blood collection: Yes (isoflurane anesthesia)
- Animals fasted: Yes
- How many animals: all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on week 13
- Animals fasted: Yes
- How many animals: all animals

URINALYSIS: Yes
- Time schedule for collection of urine: on week 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION (Functional Observation Battery (FOB)): Yes
- Time schedule for examinations: on week 11
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity / detailed clinical examination

OTHER: URINE SUBSTANCE LEVELS
- Time schedule for collection of urine: on week 13 (during periods of 0-8 and 8-24 hours)
- Metabolism cages used for collection: yes
- Animal fasted: yes
- How many animals: animals of group 4

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Kolmogorov-Lilliefors test
Bartlett test
Fisher test
Dunn test
Mann-Whitney/Wilcoxon test
Dunnett test

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

effects observed, treatment-related

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality occurred during the treatment period.
Hair loss (sometimes associated with the presence of scabs) on the right and/or left forelimbs, the head, the neck and/or the back and thorax was observed in a few control and test-treated animals at all dose-levels. As this was seen with a similar incidence in both control and treated animals
and in view of its non-specific nature, it was considered to be of no toxicological relevance

BODY WEIGHT AND WEIGHT GAIN
When compared to control values, a slightly lower mean body weight gain was observed at 50000 ppm from days 29 to 57, statistically significant in females only.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
During the study, the daily food consumption was generally similar for all groups.

WATER CONSUMPTION
When compared with the mean control values, water consumption of animals treated at 50000 ppm was statistically higher during the first three weeks of the treatment period. A tendency to higher values (not significant) was also recorded in the female group treated at 25000 ppm over the same dosing period. These changes in water consumption were not accompanied by relevant modifications of blood biochemistry parameters.

OPHTHALMOSCOPIC EXAMINATION
Pallor of the fundus (bilateral), increase in corneal thickness (bilateral) and chorio-retinitis (unilateral) were noted with a very low incidence in both males and females pre-study and at the end of the study period (top dose-level) in a few individuals.
As these ophthalmological findings are commonly recorded spontaneously in the untreated laboratory rat of this strain and age and were also noted in control animals, they were thus considered to be of no toxicological importance.

HAEMATOLOGY
There were no treatment-related differences in the hematological parameters investigated.

CLINICAL CHEMISTRY
As the above mentioned differences were slight, sometimes not dose-related and/or of opposing trend on the different groups and sexes and the individual values were within the range of our historical background data, they were considered to be of no toxicological importance, although
they were sometimes statistically significant.

URINALYSIS
Increase in urinary specific gravity was noted for all test-treated males at all dose-levels and for females given 50000 ppm. Taking into account that this increase was minimal, that no dose-relationship was demonstrated in males, and that no statistical significance was reached in the high dose female group, a relationship to treatment with test item could be excluded.

NEUROBEHAVIOUR
There were no treatment-related findings during the detailed clinical observations.
No perturbation of autonomic or physiological functions was noted at the end of the study period.

ORGAN WEIGHTS
No treatment-related effect on organ weights.

GROSS PATHOLOGY
No treatment-related necropsy findings were noted.

HISTOPATHOLOGY:
None of the changes seen were considered to be related to treatment.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

50 000 ppm

Sex:

male/female

Basis for effect level:

other: Corresponding to 3347 mg/kg/day in males and 3970 mg/kg/day in females (achieved dosages of the substance)

2

Dose descriptor:

NOEL

Effect level:

12 500 ppm

Sex:

male/female

Basis for effect level:

other: Corresponding to 748 mg/kg/day in males and 937 mg/kg/day in females (achieved dosages of substance)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

URINE SUBSTANCE LEVELS:

Sex	Total excreted 0-24h (mg)	Body weight Day 92 (g)	Achieved dosage Week 13 (mg/kg/day)	Dose-level (mg)	% of the dose 0-24h
Males	356	505	2511	1269	28
Females	146	311	2803	872	17

Applicant's summary and conclusion

Conclusions:

The ISOSORBIDE (batch No. E0031), was given ad libitum by dietary admixture to Sprague-Dawley rats at concentrations of 12500, 25000 and 50000 ppm (corresponding to daily dose-levels of 784, 1577 or 3347 mg/kg/day for the males and 937, 2060 or 3970 mg/kg/day for the females) for 13 weeks.
The test item was clinically well tolerated at all dose-levels. No mortality or clinical signs were observed. In addition, no relevant differences related to the test item were noted in the body weight gain or food consumption of treated groups. Treatment with the test item at 25000 or 50000 ppm caused transient increase of water consumption.
No treatment-related hematological, blood biochemistry or urinary changes were recorded.
No histopathological findings were noted at necropsy.

Consequently, following administration of ISOSORBIDE to rats for 13 weeks by dietary admixture, the No Observed Effect Level (NOEL) was established at 12500 ppm (corresponding to 748 mg/kg/day in males and 937 mg/kg/day in females) and the No Observed Adverse Effect Level (NOAEL) was set at 50000 ppm (corresponding to 3347 mg/kg/day in males and 3970 mg/kg/day in females).

Executive summary:

A total of 80 (40 males and 40 females) Sprague-Dawley rats was assigned to one control and three test-treated groups, each of ten males and ten females. The animals were treated with the test item, ISOSORBIDE, by dietary administration ad libitum, for 13 weeks as follows:

. group 1 (controls): untreated diet,

. group 2 (low dose-level): diet with test item at 12500 ppm,

. group 3 (mid dose-level): diet with test item at 25000 ppm,

. group 4 (high dose-level): diet with test item at 50000 ppm.

The animals were checked daily for mortality and clinical signs. In addition, detailed clinical observations were carried out weekly on all animals as well as a functional observation battery at the end of the treatment period. Body weight and water consumption were recorded once pre-study and then weekly during the study. Food consumption was recorded every week throughout the study. Urine for toxicokinetic investigations was collected from the animals of group 4 at the end of the study at designated intervals.

Ophthalmological examinations were performed on all animals once pre-study and on those of the control and high-dose groups at the end of the treatment period. Hematology and blood biochemical investigations and urinalysis were carried out at the end of the study.

On completion of the treatment period, the animals were killed and submitted to a full macroscopic post-mortem examination. Designated organs were weighed and selected tissues were preserved. A microscopic examination was performed on selected tissues from all animals of the control and high-dose groups and macroscopic lesions of the low- and intermediate-dose groups.

Results:

Achieved dosages

The concentrations of 12500, 25000 or 50000 ppm resulted in achieved dosages (as a.i.: active ingredient) of 784, 1577 or 3347 mg a.i./kg/day for the males and 937, 2060 or 3970 mg a.i./kg/day for the females, respectively.

Morbidity and mortality

No unscheduled deaths occurred during the study.

General clinical signs

No test item-related clinical signs were observed during the study.

Detailed clinical observation and functional observation battery

There were no treatment-related findings during the detailed clinical observations. No perturbation of autonomic or physiological functions was noted at the end of the study period in the test-treated animals in the Functional Observation Battery test.

Body weight and food consumption

Compared to controls, there were no changes in the mean body weight or mean food consumption attributed to treatment in the test item.

Water consumption

Transient increase of water consumption was recorded in females treated at 25000 ppm and in males and females treated at 50000 ppm in the first three weeks of the study.

Ophthalmological examinations

No relevant ophthalmological findings were observed at the end of the treatment period in the test-treated animals.

Hematology and blood biochemistry

No changes of toxicological importance were noted in the hematological and blood biochemical parameters, when compared to controls.

Urinalysis

No treatment-related effects were noted in urinary parameters.

Organ weights

No treatment-related effects on organ weights were observed.

Macroscopic post-mortem examination

No changes were seen in the test-treated animals that were considered to be related to the test item.

Microscopic examination

No treatment-related abnormalities were observed.

Urine analysis for test item levels

The amount of ISOSORBIDE excreted via the urine over 24 hours on day 92 represented approximately 28% (males) and 17% (females) of the administered dose in group 4.

The test item was clinically well tolerated at all dose-levels. No mortality or clinical signs were observed. In addition, no relevant differences related to the test item were noted in the body weight gain or food consumption of treated groups. Treatment with the test item at 25000 or 50000 ppm caused transient increase of water consumption.

No treatment-related hematological, blood biochemistry and urinary parameter changes were recorded.

No histopathological findings were noted at necropsy.

Consequently, following administration of ISOSORBIDE to rats for 13 weeks by dietary admixture, the No Observed Effect Level (NOEL) was established at 12500 ppm (corresponding to 748 mg a.i./kg/day in males and 937 mg a.i./kg/day in females) and the No Observed Adverse Effect Level (NOAEL) was set at 50000 ppm (corresponding to 3347 mg a.i./kg/day in males

and 3970 mg a.i./kg/day in females).