D-Glucitol, 1-deoxy-1-(methylamino)-, N-[C8-16 (even numbered) and C18 unsaturated acyl] derivs.

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented guideline study according to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

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:

no

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)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Glucamide 24

IUPAC Name:

Glucamide 24

Constituent 2

Reference substance name:

287735-50-6

Cas Number:

287735-50-6

IUPAC Name:

287735-50-6

Test material form:

other: solid

Details on test material:

- Name of test material: Glucamide 24
- TSIN: E4086.01
- Substance type: Other
- Physical state: White opaque solid gel
- Stability under test conditions: Not reported
- Storage condition of test material: Room temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

bi-distilled

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test solution was prepared daily prior to each application. The test substance was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using homogenizer. Homogeneity of the test substance in the vehicle was maintained during treatment using a magnetic stirrer.
Dose volume: 10 mL/kg bw
VEHICLE
- Amount of vehicle: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSIS OF DOSE PREPARATIONS: Concentrations, homogeneity and stability of the dose formulations were determined prior to pretest start. Further samples for analysis were taken during weeks 2, 7 and 11 of the test and subsequently analyzed. The analyses were performed in the Analytical Laboratories of RCC umweltchemie AG, according to a method which was supplied by the sponsor.

Duration of treatment / exposure:

91 days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 10, 50, 200 and 500 mg/kg bw/day
Basis:

No. of animals per sex per dose:

10 rats/sex/dose (groups 1 to 5)

Details on study design:

- Dose selection rationale: The dose levels end route of application used in this study were based on the data from a 14-day (range-finding) feeding toxicity study in rats (RCC 287458) and from a 14-day (range-finding) oral toxicity study (RCC 292195).
- Rationale for animal assignment: Animals were randomized by computer generated random algorithm.
- Rationale for selection of animal: Recognized by the international guidelines as the recommended test system.

TREATMENT GROUPS:
Group 1: Control (Bidistilled water)
Group 2: 10 mg/kg bw/day active test substance (equivalent to 22.2 mg/kg bw of test solution)
Group 3: 50 mg/kg bw/day active test substance (equivalent to 111.1 mg/kg bw of test solution)
Group 4: 200 mg/kg bw/day active test substance (equivalent to 444.4 mg/kg bw of test solution)
Group 5: 500 mg/kg bw/day active test substance (equivalent to 1111 mg/kg bw of test solution)
Recovery groups of 10 rats/sex/dose group were also utilized in this study. The recovery group rats were sacrificed after a 4-week recovery period following 13 week treatment with the test substance.

Examinations

Observations and examinations performed and frequency:

MORTALITY: Yes
- Time schedule: Animals were observed twice daily for viability and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Signs of toxicity were assessed once daily. Descriptions of all abnormalities were recorded and the subsequent progress was monitored.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded on the same days as the food consumption using the same recording system. Additionally, terminal body weights were recorded at necropsy.

FOOD CONSUMPTION :
- The food consumption was recorded once during the acclimatization period and weekly thereafter using an on-line electronic recording system consisting of a Mettler PM 4800 balance connected to the RCC computer.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmoscopic examinations were performed on all animals. A description of any abnormality was recorded. Examinations were performed at treatment week 4 and treatment week 13 (shortly before the termination or treatment) and a third time on the recovery animals of group 1 and 5 at recovery week 4 (shortly before the termination of the recovery period). 10 to 90 minutes after the application of a mydriatic solution (Dispersa AG, CH-8442 Hettlingen) the cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes were examined under dimmed light using a Heine Ophthalmoscope BETA 200 (Eisenhut Vet. AG, CH-4123 Allschwil).
- Dose groups that were examined: On all surviving animals at Week 4 and 13. At week 17, on control and high dose (500 mg/kg bw) group animals.

HAEMATOLOGY: Yes
- Collection of blood samples: Blood samples were drawn from retro-orbital plexus.
- Time schedule for collection of blood: After 13 weeks of treatment (between hours of 06:30 and 09.00). Blood was collected from recovery group at week 17.
- Anaesthetic used for blood collection: Yes; light ether anesthesia.
- Animals fasted: Yes; animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- How many animals: All animals
- Parameters checked: Erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count, nucleated erythrocytes, Heinz bodies, methemoglobin, total leukocyte count, differential leukocyte count, red cell morphology, coagulation: thromboplastin time, activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Collection of blood samples: Blood samples were drawn from retro-orbital plexus.
- Time schedule for collection of blood: After 13 weeks of treatment (between hours of 06:30 and 09.00). Blood was collected from recovery group at week 17.
- Animals fasted: Yes; animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- How many animals: All animals
- Parameters checked: Glucose, urea, creatinine, uric acid, total bilirubin, total cholesterol, triglycerides, phospholipids, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, creatine kinase, alkaline phosphatase, gamma-glutamyl-transferase, calcium, phosphorus, sodium, potassium, chloride, total protein, albumin, globulin, albumin/globulin ratio.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected during the 18-h fasting period into a specimen vial.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Volume (18 hour), specific gravity, color, pH, protein, glucose, ketones, bilirubin, urobilinogen, erythrocytes, urine sediment

Statistics:

The following statistical methods were used to analyze the body weights, food consumption, organ weights and all ratios as well as clinical laboratory data:
- Univariate one-way analysis of variance was used to assess the significance or intergroup differences.
- If the variables could 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 between the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher test was not applied to the overall spontaneous mortality data nor to the overall ophthalmoscopy data, as there was a considerable mortality rate in group 5 in the absence of mortality in the control group and as only one rat showed an ophthalmoscopic abnormality.
- Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
- Individual values, means, standard deviations and statistics were rounded-off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded-off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

MORTALITY
- Four male and two female rats of group 5 (500 mg a.i./kg bw) died or were killed in extremis between treatment days 27 and 91. The mortality of three of these males and of one of these females was considered to be treatment related.
- However, the causes of death of the other male and female of group 5 and of one male of each of groups 2 (10 mg a.i./kg bw) and 4 (200 mg a.i./kg bw) were found to be intubation errors rather than the test substance itself. No other death occurred.

CLINICAL SIGNS
- There was a dose related increase in incidence and severity of respiratory findings from group 3 (males only) to group 5. The main finding was breathing rales, which was accompanied by dyspnea and labored respiration in some rats of group 5.
- Slight to moderate sedation and emaciation was found in several rats and ruffled fur in all rats of group 5 and considered to reflect some general discomfort.
- All other findings were considered to be toxicologically irrelevant and/or treatment unrelated.
Further details on clinical signs are provided in the study report.

BODY WEIGHT AND WEIGHT GAIN
- No treatment-related effect on the body weight gain of any female rats treated at 10, 50, 200 or 500 mg/kg bw, nor on that of male rats treated at the 10 or 50 mg/kg bw dose levels.
- There was an apparently dose related retardation of body weight gain in male rats of groups 4 (200 mg/kg bw) and of group 5 (500 mg/kg bw). Body weights were statistically significantly lower than controls in males of group 4 at treatment weeks 9, 12 and 13 and in males of group 5 from treatment week 2 until termination of treatment in week 14. This effect lasted on at a statistically significant level in recovery males of group 5 until week 1 of the subsequent recovery period. Afterwards, the male rats of groups 4 and 5 gained weight at a rate similar to that of controls.

FOOD CONSUMPTION
- No effect on food consumption was observed at doses of 10, 50 and 200 mg a.i./kg bw.
- Food consumption of male rats of the 500 mg a.i./kg bw group was statistically significantly lower than that of controls during weeks 1-2 and 5 to 10 and that of females during weeks 1-2, 7-8 and 12-13. Male and occasionally female rats of the 500 mg a.i./kg bw group showed a similar, statistically non-significant trend during the remaining treatment period.
- During the treatment-free recovery period food consumption of the animals of the treatment groups was comparable to that of controls.

RELATIVE FOOD CONSUMPTION
- There was no toxicologically significant effect on the relative food consumption (i.e. food consumption related to body weight) of both sexes of groups 2 (10 mg a.i./kg bw) and 3 (50 mg a.i./kg bw) and of female rats or groups 4 (200 mg a.i./kg bw) and 5 (500 mg a.i./kg bw).
- The relative food consumption of group-4 males was statistically significantly higher than control during treatment week 13, and that of group-5 males during treatment weeks 10-11, 12-13 and 14-15 after having been significantly lower than control during treatment week 1-2. The relative food consumption of group-4 recovery males was statistically significantly higher than control during recovery week 2-3 that of group-5 recovery males throughout the recovery period.
- The findings in group-4 and group-5 males may partly reflect the treatment related retardation of body weight gain described below, and during the recovery period probably also some compensational response.

OPHTHALMOSCOPIC EXAMINATION
- There were no treatment-related effects on any ophthalmoscopy parameters. In the right eye of one female rat in 10 mg a.i./kg bw group, blood vessels were slightly visible at the 4- and 13-week observation points. This finding was considered to be spontaneous and therefore treatment unrelated.
- On recovery day 4, the left eye of one female rat in 50 mg a.i./kg bw group was found to be injured probably from blood collection.

HAEMATOLOGY
- The assessment of hematological data indicated no changes of toxicological significance at termination of the treatment; however, the following effects were noted for the treated rats when compared to the controls:
i) slightly increased erythrocyte count (RBC) by 5% on average for both sexes of group 5 (500 mg a.i./kg bw)
ii) slightly increased hemoglobin concentration (HB) by 4 % on average for males of group 5
iii) slightly increased hematocrit (HCT) value by 6 to 7 % on average for both sexes of group 5 and by 3 % for males of group 4 (200 mg a.i./kg bw)
iv) slightly decreased mean corpuscular hemoglobin concentration (MCHC) by 2 to 4 % on average for both sexes of group 5 and by 2 % for females of group 4
v) slightly increased methemoglobin (MET-HB) concentration by 32 % on average for females of group 5.
- The changes noted reflect slight hemoconcentration and suggest changes in basal fluidity. From a functional point of view, these findings have no hematological implications. At termination of the treatment-free recovery period these findings were found to be reversed and comparable to those of the controls.
- All other statistical differences in the results of the hematology parameters were considered to be incidental and of normal biological variation for
rats of this strain and age.

CLINICAL CHEMISTRY:
- For clinical biochemical data the following effects were noted for rats of the treated groups at termination of the treatment when compared to the controls:
i) slightly increased uric acid and triglyceride concentration for females of group 5;
ii) slightly increased alanine aminotransferase and alkaline phosphatase activity for males or group 5;
iii) slightly decreased calcium concentration for males of all treated groups;
iv) slightly decreased chloride concentration for both sexes of group 5;
v) slightly increased total protein and globulin concentration, and slightly decreased A/G ratio for females or groups 3, 4 and 5.
- The findings noted were not considered to be of toxicological significance but more likely a reflection of metabolic adaptation due to an increased functional load on the liver. At termination of the treatment-free recovery period these findings were, with the exception of the chloride concentration and the A/G ratio for females of group 5, found to be reversed and comparable to those of the controls.
Further details on clinical chemistry are provided in the study report.
URINALYSIS
- Urinalysis data indicated no changes of toxicological significance at termination of the treatment. The only change noted was a slight increase in the overnight urinary output (Volume/18 hour) by 43 to 63 % on average for both sexes of group 5 (500 mg a.i./kg bw). The finding reflects an increased fluid intake. At termination of the treatment-free recovery period this finding was found to be reversed and comparable to that of the controls.
- All other differences in the results of the urinalysis parameters were considered to be incidental and of normal biological variation for rats of this
strain and age.

ORGAN WEIGHTS AND ORGAN WEIGHT RATIO
- After 13 weeks of treatment, the liver weights and their ratios to body weight and brain weight of female rats of group 5 (500 mg a.i./kg bw) as well as the liver to body weight ratio of males of this group were statistically significantly higher than the corresponding controls. These findings were considered to be treatment related, but no longer significant in recovery rats at termination of the 4-week recovery period following treatment.
- Additionally, the kidney to body weight ratios of group-5 females was slightly higher than the respective controls at termination of treatment. This may have been a reflection or their terminal body weights being slightly (statistically non-significantly) lower than that of controls or related to the concomitant slight increase in overnight urinary output.
- All other statistically significant differences noted at termination of the treatment period were considered to be either a reflection of the concomitant low terminal body weights in male rats of group-5 (statistically significantly below controls) or incidental and therefore treatment unrelated. The latter applied also to any statistically significant differences in absolute or relative kidney weights found at termination of the recovery period.
Further details on organ weights and organ weight ratio are provided in the study report.

GROSS PATHOLOGY:
- No treatment related macroscopic findings were identified.

HISTOPATHOLOGY: NON-NEOPLASTIC
- The premature decedents from groups 2 (10 mg a.i./kg bw) and 4 (200 mg a.i./kg bw) showed lung changes clearly indicative of an accident at dosing, as did one male and one female from group 5 (500 mg a.i./kg bw).
- The remaining group-5 premature decedents gave no microscopic indication of the mechanism of toxicity, but the deaths are considered to be related to treatment. The group-5 survivors were affected by treatment but gave no microscopic evidence of toxicity.
- Several group-5 animals, particularly the premature decedents, showed exudation in the nasal cavity but this was thought to be due to the general poor condition of the animal and was certainly not of sufficient severity to cause death or morbidity. The term exudate was used to represent a range from simple mucous to frankly purulent which may be apparent in the same section. Vegetable matter was sometimes present in the exudate.
- Thymic atrophy which was largely cortical, and lung lesions were seen in some group-5 premature decedents but the changes are thought to be related to the poor general condition of the animals and not specifically treatment-related. Similarly, the small thymic hemorrhages identified are thought to be agonal and of no toxicological significance.
- Adrenocortical rest was seen in a number of animals. This lesion was congenital, consisted of normal cortex and may also be named 'Accessory cortical nodule'. The purpose of reporting this clearly incidental lesion was to ensure that there was no danger of confusion with the potentially significant 'Extruded hyperplastic nodule'. The distinction was easily made as the hyperplastic nodule was not completely surrounded by a fibrous capsule whereas the accessory nodule or rest has a complete capsule.

Effect levels

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Based on mortality, food consumption, body weight and clinical signs observed at 500 mg a.i./kg bw dose group.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Glucamide 24 when administered by oral gavage daily for 13 weeks to male and female Wistar rats at dose levels of 0, 10, 50, 200 and 500 mg a.i./kg bw/day, revealed a no observed adverse effect level (NOAEL) at 200 mg a.i./kg bw/day for male and female rats.

Executive summary:

The repeated dose oral toxicity study ofGlucamide 24was performed by following method similar to the OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents).

Male and female Wistar (HanIba, SPF bred) rats obtained from SRL, Biological Research Laboratories Ltd., Wolferstrasse 4, CH-4414 Fuellinsdorf were used in this study. The body weight range of animals was 187- 225 g (males) and 151-184 g (females). Animals were individually housed in Makrolon type-3 cages and maintained under controlled environmental conditions (Temperature: 22±3°C, Relative humidity: 40-70%, 10-15 air changes/ hour and 12 hours fluorescent light /12 hours dark). The study consisted of 4 test treatment groups and 1 control group containing 10 rats/sex/group. Test substance formulations were prepared in bi-distilled water. The animals of different groups were treated as follows: 

Group 1: Control (Bi-distilled water)

Group 2: 10 mg/kg bw/day active test substance (equivalent to 22.2 mg/kg bw of test solution)

Group 3: 50 mg/kg bw/day active test substance (equivalent to 111.1 mg/kg bw of test solution)

Group 4: 200 mg/kg bw/day active test substance (equivalent to 444.4 mg/kg bw of test solution)

Group 5: 500 mg/kg bw/day active test substance (equivalent to 1111 mg/kg bw of test solution)

Recovery groups of 10 rats/sex/dose group were also utilized in this study. The recovery group rats were sacrificed after a 4-week recovery period following 13 week treatment with the test substance.

The rats were observed for mortality and clinical signs throughout the study period. Animals were observed for ophthalmoscopic observation at pretest and after 4 and 13 weeks of treatment. Detailed observations, body weights and food consumption were recorded weekly. Urinalysis, hematology and clinical chemistry were performed at study termination prior to sacrifice.

After completion of treatment, all rats were sacrificed by exsanguination under sodium pentobarbitone anesthesia and necropsied. All animals were observed for gross pathological changes during necropsy and tissues were collected to perform histological examination. Absolute and relative organ weights were also determined.

There was no effect on food consumption at 10, 50 and 200 mg a.i./kg bw dose levels. In male and female rats of 500 mg a.i./kg bw, a statistically significant decrease in food consumption was observed when compared with the respective controls. There was no such effect in recovery rats during the treatment-free recovery period following treatment.

There was no toxicologically significant effect on the relative food consumption of either sex of groups 2 and 3, and of female rats of groups 4 and 5. The relative food consumption of group-4 males was statistically significantly higher than control during treatment week 13. Similarly, a statistically significant increase in relative food consumption (when compared to the control group) was observed in group-5 males during treatment weeks 10-11, 12-13 and 14-15 after having been significantly lower than control during treatment week 1-2. The relative food consumption of group-4 recovery males was statistically significantly higher than control during recovery week 2-3 than that of group-5 recovery males throughout the recovery period.

There were no effects on the body weight of any treated female rats or on that of males treated at the 10 or 50 mg a.i./kg bw dose levels. However, the body weight gain appeared to be retarded in a dose- related manner in male rats of groups 4 and 5. The body weights of group-4 males were significantly lower than the respective controls at treatment weeks 9, 12 and 13 and those of group-5 males from treatment week 2 until treatment week 14. This effect lasted in recovery males of group 5until week 1 of the subsequent treatment-free recovery period.

There was a dose related increase in incidence and severity of respiratory findings from group 3 (males only) to group 5 (males and females). The main finding was breathing rales, which was accompanied by dyspnea and labored respiration in some rats of group 5. Slight to moderate sedation and emaciation was found in several rats and ruffled fur in all rats of group 5and considered to reflect some general discomfort. All other findings were considered to be toxicologically irrelevant and/or not related to treatment.

Four male and two female rats of group 5 died or were euthanized in extremis between treatment days 27 and 91. The mortality of three of these males and of one of these females was considered to be treatment related. However, the causes of death of the other male and female of group 5 and of one male of each of groups 2 and 4 were found to be intubation errors rather than the test substance itself. No other death occurred.

There were no treatment related findings on ophthalmoscopy.

The hematological data reflected slight hemoconcentration and changes in basal fluidity. The findings were not considered to be of toxicological significance. At termination of the treatment-free recovery period these findings were found to be reversed and comparable to those of the controls.

The clinical biochemical data noted were not considered to be of toxicological significance but more likely a reflection of metabolic adaptation due to an increased functional load on the liver. At termination of the treatment-free recovery period these findings were, with the exception of the chloride concentration and the A/G ratio for females of group 5, found to be reversed and comparable to those of the controls.

For urinalysis, no changes of toxicological significance were noted at termination of the treatment or at the end of the treatment-free recovery period. The only change noted was a slight increase in the overnight urinary output (Volume/18 hour) for both sexes of group 5 after 13 weeks. This finding was found to be reversed at termination of the treatment-free recovery period. The finding was considered to be related to an increased fluid intake.

After 13 weeks of treatment, the liver weights and their ratios to body weight and brain weight of female rats of group 5as well as the liver to body weight ratio of males of this group were significantly higher than the corresponding controls. These findings were considered to be treatment related, but no longer significant in recovery rats at termination of the 4-week recovery period following treatment.

There were no treatment related necropsy findings identified.

The group-2 and group-4 males which died showed clear microscopic evidence of death being due to an accident at dosing, as did some of the group-5 animals. The premature deaths of the remaining group-5 animals were considered to be treatment related, but there were no histological changes identified to indicate the mechanism of toxicity.

There was no treatment related death or morbidity in group 4 and so the oral administration of the test substance to rats for 13 weeks at the dose level of up to 200 mg a.i./kg bw/day was considered to produce no pathological evidence of toxicity.

Based on above,Glucamide 24when administered by oral gavage daily for 13 weeks to male and female Wistar rats at dose levels of 0, 10, 50, 200 and 500 mg a.i./kg bw/day, revealed a no observed adverse effect level (NOAEL) at 200 mg a.i./kg bw/day for male and female rats.