1,4:3,6-dianhydro-2,5-di-O-methyl-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:

23 Apr - 31 Jul 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

No neurobehavioural examination was performed during the course of the study.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,4:3,6-dianhydro-2,5-di-O-methyl-D-glucitol

EC Number:

226-159-8

EC Name:

1,4:3,6-dianhydro-2,5-di-O-methyl-D-glucitol

Cas Number:

5306-85-4

Molecular formula:

C8H14O4

IUPAC Name:

1,4:3,6-dianhydro-2,5-di-O-methyl-D-glucitol

Test material form:

other: liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Canada Inc., St. Constant, Quebec, Canada
- Age at study initiation: 4 weeks
- Weight at study initiation: 117 – 133 g (males), 101 – 116 g (females)
- Housing: Animals were housed in groups of 2 or 3 in the first week of acclimatization period. Thereafter, each animal was housed individually in a stainless-steel cage of conventional design.
- Diet: commercially available certified rodent diet (Purina Laboratory Chow#5002 cubes), ad libitum
- Water: (tap-filtered) water, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 50 ± 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 08 Apr 1986 To: 31 Jul 1986

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
- The test solutions of each treatment group were prepared once weekly by direct dilution of appropriate weights of test article with appropriate quantities of sterile water for injection. All test solutions were kept refrigerated (ca. 3 °C) when not in use. Prior to dosing each day an appropriate amount of each test solution was poured into prelabeled containers.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before the start of treatment, samples of the test material and the test solutions were sent to the Sponsor for analysis to confirm the stability of the test material solutions. The accuracy of formulation was checked during the study by analysis of samples taken from solutions prepared for administration during weeks 1, 6 and 13 of treatment. Analysis revealed that the test material was stable during the study period.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

once daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

375 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20 (main study)
8 (bioavailability study)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels in this study were selected by the Sponsor and were based on the results of previous studies and a multiple of expected human exposure levels.

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined twice daily, before and after treatment, for mortality and signs of reaction to treatment.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded once weekly commencing 1 week prior to the start of treatment and prior to necropsy. Group mean body weights and body weight gains were calculated from these values.

FOOD CONSUMPTION:
- Individual food consumption values were measured once weekly during the last week of the pre-treatment and treatment periods. From these data, group mean and overall food intakes were calculated.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Following the administration of atropine sulfate (0.5% solution), funduscopic and biomicroscopic (slit lamp) examinations were performed on all animals during the pre-treatment period and again during week 13.
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to commencement of treatment, during weeks 6 and 12/13 blood was obtained from 10 animals of each sex per dose group. Selected animals which died before the scheduled investigations were not replaced.
- Anesthetic used for blood collection: Yes (ether anesthesia)
- Animals fasted: Yes
- How many animals: 10 animals of each sex per dose group
- Parameters examined: hematocrit, hemoglobin, red blood cell count, white blood cell count (total and differential), platelet count, cell morphology, Wintrobe´s constants (calculated)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to commencement of treatment, during weeks 6 and 12/13 blood was obtained from 10 animals of each sex per dose group. Selected animals which died before the scheduled investigations were not replaced.
- Animals fasted: Yes
- How many animals: 10 animals of each sex per dose group
- Parameters examined: blood urea nitrogen (BUN), total protein, alkaline phosphatase (AP), glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), total bilirubin. A/G ratio (calculated from albumin and globulin values), cholesterol, glucose, albumin, sodium, potassium, calcium, chloride, phosphorous

URINALYSIS: Yes
- Time schedule for collection of urine: Prior to the start of treatment and again during weeks 6, 12 (males) and 13 (females), urine samples were collected from individual animals placed in metabolism cages, during which time they were deprived of food and water. At week 7, the urine sampling was repeated for 2 females since no urine or insufficient urine sample was obtained at week 6.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: color and appearance, pH, glucose, ketones, urobilinogen, blood, nitrite, volume, specific gravity, protein, bilirubin, microscopy of centrifuged deposit

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: TEST ARTICLE ABSORPTION
- Prior to the start of treatment, 8 animals/sex/group were randomly selected to provide blood samples for plasma test material levels after a single dose. These animals remained untreated until April 28 (males) and 29 (females), 1986. Blood samples were obtained from the abdominal aorta following ether anesthesia approximately 1 hour after a single dose of the appropriate control or test solutions. These animals were not starved overnight prior to blood collection. After 13 weeks of treatment, blood samples were obtained at necropsy, from 8 randomly selected rats/sex/group surviving to termination. These animals were starved overnight prior to blood collection and gross pathological examination. Additional blood samples were also obtained at necropsy from 9 control rats in order to provide analytical standards.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- A complete gross pathological examination was performed on all animals which died during the course of the study and animals sacrificed pre-treatment (health-screen) and at study termination. Animals selected to provide blood samples for bioavailability study after a single dose of the test material were not subjected to gross pathology examinations or tissue preservation. Animals were fasted overnight prior to scheduled sacrifice. Gross pathological examination consisted of an external examination, including identification of all clinically recorded lesions as well as a detailed internal examination.

HISTOPATHOLOGY: Yes
- The following tissues were prepared for histological examination by embedding in paraffin wax, sectioning and staining with hematoxylin and eosin. Histopathological examination were performed on the tissues from all animals in group 1 (control) and 4 (high-dose): adrenals, aorta (thoracic), brain, cecum, colon, duodenum, epididymides, esophagus, eyes, heart, ileum, jejunum, kidneys, liver (sample of 2 lobes), lungs (sample of 2 lobes), lymph nodes (mandibular and mesenteric), mammary gland (inguinal), optic nerves, ovaries, pancreas, pituitary, prostate, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical), spleen, stomach, testes, thymus, thyroid (and parathyroids), tongue, trachea, urinary bladder, uterus.

Other examinations:

ORGAN WEIGHTS
- For all animals killed at termination, the following organs from each animal were dissected free of fat and weighed: adrenals, brain, heart, kidneys, liver, ovaries, pituitary, spleen, testes, thyroid and parathyroids (lobes weighed together).

Statistics:

Group mean values and standard deviations were calculated for numerical data. The data were analyzed for homogeneity using Bartlett´s test. The statistical significance of the data was assessed using analysis of variance and the significance of any intergroup differences was determined using Dunnett´s t-test for homogenous data or the Kruskal-Wallis test followed by Dunn´s test for heterogeneous data.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

One control and one high-dose male were found dead during the course of the study. These deaths were not considered to be related to treatment with the test material.

Mortality:

mortality observed, treatment-related

Description (incidence):

One control and one high-dose male were found dead during the course of the study. These deaths were not considered to be related to treatment with the test material.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

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
Two males died during the 13-week treatment period. One control male died shortly after withdrawal of a blood sample during week 12. The gross pathology examination of this rat revealed red staining of the muzzle, red fluid in the oral cavity and frothy red fluid in the trachea, mottled uncollapsed lungs, red discoloration of the gastric mucosa and multiple dark areas on the thymus. During week 13 of treatment, a high-dose male was also found dead with no significant prior clinical history. The gross pathology examination of this rat revealed multiple dark areas on the thymus. No clinical signs considered to be related to the administration of the test material were noted during the 13-week treatment period. The clinical signs observed were those commonly seen in rats of this age and strain.

BODY WEIGHT AND WEIGHT GAIN
Group mean body weights of treated rats were comparable with those of the controls and showed no evidence of a treatment-related disturbance. Likewise, treated rats showed group body weights comparable to the controls and showed no indication of a treatment-related effect.

FOOD CONSUMPTION
Group mean food consumption values and total food consumption values of treated animals were comparable to those of the controls and showed no evidence of any treatment-related effect.

OPHTHALMOSCOPIC EXAMINATION
Ophthalmoscopic examination of all animals performed during week 13 of treatment revealed no eye abnormalities.

HAEMATOLOGY
Hematological results obtained from control and test material-treated rats showed no indication of any adverse treatment-related change. Although some small differences between values obtained for treated rats, on occasion, attained a level of statistical significance when compared to controls, these values are well within the normally expected range for rats of this age and strain and were not considered to be of toxicological significance.

CLINICAL CHEMISTRY
Isolated increases in SGPT and SGOT levels were obtained at week 6 from one high-dose and one mid-dose female, and at week 12 from one high-dose male and SGPT levels for two mid-dose females when compared to controls. However, in view of the small magnitude of these differences and the inherent variability of these parameters, such differences were considered of doubtful toxicological significance. At week 6 the majority of treated males in all groups and females receiving 375 mg/kg bw/day, a large proportion of females receiving 100 mg/kg bw/day and an occasional females receiving 30 mg/kg bw/day showed chloride levels which were marginally higher than the concurrent controls at this time. Similarly at week 12, marginally higher chloride levels were recorded for all females receiving 375 mg/kg bw/day, a large proportion of females and one male receiving 100 mg/kg bw/day, and occasional males and females receiving 30 mg/kg bw/day when compared to the concurrent controls at this time. These differences between chloride levels for treated and control animals were of a small magnitude and individual values showed variability between sampling occasions, particularly for males receiving 100 or 375 mg/kg bw/day. In view of this and the lack of an obvious disturbance of other electrolyte levels, the significance of the marginally higher chloride levels is uncertain. Other small differences noted in the biochemical parameters, which in some instances attained a level of statistical significance since individual values were within the normally expected range for rats of the age and strain used.

URINALYSIS
Qualitative evaluation of the urinalysis performed during weeks 6 and 12/13 revealed no clear changes in the quantity or quality of urine voided by treated rats when compared with controls which could be attributed to treatment with the test material.

ORGAN WEIGHTS
Organ weight analysis performed on rats killed at termination revealed a small (statistically significant) increase in absolute and relative liver weights among males and females receiving 375 mg/kg bw/day when compared to controls. Absolute and relative liver weights for other treated rats were comparable to those of the controls. A marginal, but statistically significant, increase in absolute kidney weights was also noted for males receiving 375 mg/kg bw/day, with an associated increase in relative weights when compared with controls. Females receiving this dose level and all other treated rats showed absolute and relative kidney weights comparable with those of the controls. Other small differences observed between values obtained for treated rats when compared with controls, although attaining a level of statistical significance in some instances, were not considered to be of toxicological significance.

GROSS PATHOLOGY
Gross pathology examination of rats found dead during the course of the study revealed no consistent changes which could be associated with the treatment of the test material. Similarly, examinations of animals surviving to termination revealed a low incidence of commonly occurring pathology changes. The distribution and type of changes recorded showed no indication of any disturbance attributable to test material treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological examination of vehicle controls and rats receiving the test material at a dose of 375 mg/kg bw/day revealed a low incidence of commonly occurring changes which showed no evidence of any treatment-related disturbance.

OTHER FINDINGS (TEST ARTICLE ABSORPTION)
On each occasion, test material plasma levels showed a linear proportionality with dose. Higher test material plasma levels were noted at all dose levels after 13 weeks of treatment when compared to the day 1 plasma levels. These differences were statistically significantly different for all treated males and for females receiving 100 or 375 mg/kg bw/day. Females receiving 100 mg/kg bw/day also showed significantly higher plasma levels than males receiving this dose level on day 1 and females receiving 375 mg/kg bw/day showed significantly higher levels than males on day 1 and after 13 weeks of treatment.

Effect levels

Dose descriptor:

NOAEL

Effect level:

375 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reactions to treatment were limited to small increases in absolute and relative liver and kidney weights for males and females receiving 375 mg/kg bw/day which were not, however, associated with any morphological changes (non-adverse).

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Group Mean Haematological Values (Males and Females, 6-week treatment)

Dose Group [mg/kg bw/day]		MCHC [g/dL]		HCT [%]		MCV [µm³]
		M	F	M	F	M	F
0	Mean S.D.	37.0 0.40	36.9 0.56	46.1 1.77	43.7 1.75	55.6 1.24	54.5 2.27
30	Mean S.D.	36.3* 0.56	36.7* 0.57	45.9 1.16	43.5* 1.01	56.6 2.39	53.9* 1.54
100	Mean S.D.	35.9* 0.49	35.9* 0.63	46.9 1.53	46.2* 1.86	55.8 1.30	56.0* 2.09
375	Mean S.D.	35.5* 0.65	35.4* 0.50	46.8 1.35	46.3* 2.30	56.7 1.42	55.9* 1.46

* p<0.05

S.D.: standard deviation

M: Males

F: Females

 Table 2: Group Mean Haematological Values (Males and Females, 12-week treatment)

Dose Group [mg/kg bw/day]		MCHC [g/dL]
		M	F
0	Mean S.D.	36.8 1.04	34.9 0.85
30	Mean S.D.	36.8 0.86	35.6 0.60
100	Mean S.D.	36.9 0.65	36.1* 0.91
375	Mean S.D.	36.5 0.98	35.9* 0.64

* p<0.05

S.D.: standard deviation

M: Males

F: Females

Table 3: Group Mean Clinical Biochemistry Values (Males and Females, 6-week treatment)

Dose Group [mg/kg bw/day]		Total bilirubin [mg/dL]		K+  [mMol/L]		Cl- [mMol/L]		Na+  [mMol/L]		Albumin [g/dL]		Total protein [g/dL]
		M	F	M	F	M	F	M	F	M	F	M	F
0	Mean S.D.	0.4 0.07	0.4 0.04	4.2 0.31	4.1 0.34	106.6 1.35	107.0 1.49	148.4 3.53	141.4 1.65	4.4 0.15	4.3 0.14	6.9 0.24	6.7 0.24
30	Mean S.D.	0.3* 0.07	0.4 0.06	4.5 0.41	4.3 0.29	110.1* 1.92	108.3 1.34	145.9 3.11	143.8 2.86	4.2* 0.13	4.4 0.17	6.7 0.25	6.7 0.20
100	Mean S.D.	0.4 0.06	0.5 0.05	4.6* 0.26	4.6 0.80	111.8* 1.03	110.1* 2.42	145.7 0.67	148.1* 3.11	4.3 0.12	4.5 0.15	6.8 0.20	6.9 0.30
375	Mean S.D.	0.4 0.05	0.4 0.05	4.7* 0.40	4.3 0.46	110.9* 1.60	112.3* 2.83	149.3 1.06	151.2* 2.90	4.3 0.17	4.5* 0.09	6.9 0.37	7.1* 0.26

* p<0.05

S.D.: standard deviation

M: males

F: females

Table 4: Group Mean Clinical Biochemistry Values (Males and Females, 12-week treatment)

Dose Group [mg/kg bw/day]		Total bilirubin [mg/dL]		Cl- [mMol/L]		Na+  [mMol/L]		Cholesterol [mg/dL]		Ca++ [mg/dL]		Total protein [g/dL]
		M	F	M	F	M	F	M	F	M	F	M	F
0	Mean S.D.	0.5 0.07	0.3 0.07	105.6 1.08	105.7 1.64	146.7 1.89	147.9 2.23	86.5 17.78	87.2 15.61	9.7 0.29	9.7 0.21	7.1 0.28	7.1 0.24
30	Mean S.D.	0.4 0.05	0.3 0.05	107.2* 1.32	107.7* 1.16	149.4* 2.01	147.1 2.08	81.8 18.39	79.9 12.54	9.6 0.40	9.9 0.38	7.1 0.33	7.3 0.35
100	Mean S.D.	0.3* 0.07	0.4* 0.05	105.8 1.55	108.7* 1.70	146.8 2.20	149.2 2.20	80.5 14.05	84.7 11.51	9.9 0.40	10.0 0.50	7.0 0.28	7.2 0.46
375	Mean S.D.	0.4* 0.09	0.4* 0.05	105.0 1.25	111.1* 1.37	146.1 0.99	151.9* 1.79	107.8* 23.18	92.7 16.46	10.3* 0.35	10.0 0.39	7.2 0.48	7.6* 0.44

* p<0.05

S.D.: standard deviation

M: males

F: females

Table 5: Group Mean Absolute Organ Weight [g]

Dose Group [mg/kg bw/day]		Liver		Kidneys
		M	F	M	F
0	Mean S.D.	14.241 1.5197	6.625 0.7397	3.104 0.2648	1.690 0.1283
100	Mean S.D.	14.618 2.0438	6.530 0.4005	3.237 0.2383	1.674 0.1736
300	Mean S.D.	14.604 1.8387	7.104 0.7753	3.180 0.3475	1.770 0.1577
375	Mean S.D.	16.495* 1.9260	7.324** 0.6719	3.436* 0.3076	1.766 0.1652

* p<0.05

**p<0.01

S.D.: standard deviation

M: males

F: females

Table 6: Group Mean Relative Organ Weight [%]

Dose Group [mg/kg bw/day]		Liver		Kidneys
		M	F	M	F
0	Mean S.D.	2.741 0.2093	2.579 0.1053	0.598 0.0320	0.661 0.0508
100	Mean S.D.	2.832 0.2714	2.613 0.1536	0.629 0.0374	0.669 0.0526
300	Mean S.D.	2.851 0.1428	2.636 0.1839	0.625 0.0648	0.658 0.0496
375	Mean S.D.	3.174* 0.2138	2.862* 0.1794	0.664** 0.0579	0.691 0.0572

* p<0.05

**p<0.01

S.D.: standard deviation

M: males

F: females

Applicant's summary and conclusion

Conclusions:

Oral administration of the test material to rats for a period of 13 consecutive weeks, at dose levels of 30, 100 and 375 mg/kg bw/day did not produce any convincing treatment-related adverse changes and on this basis under the conditions of this study the NOAEL was considered to be 375 mg/kg bw/day.