Cyclooctapentylose

Repeated dose toxicity: other routes

Administrative data

Endpoint:

sub-chronic toxicity: other route

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The rats received single daily doses of .gamma.-cyclodextrin on 90/91 consecutive days. The compound was dissolved in saline (9 mg/ml) and administered intravenously into a lateral tail vein (injection speed 2 ml/min).

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Cyclooctapentylose

EC Number:

241-482-4

EC Name:

Cyclooctapentylose

Cas Number:

17465-86-0

Molecular formula:

C48H80O40

IUPAC Name:

cyclooctapentylose

Details on test material:

Test material: .gamma.-cyclodextrin
Batch No.: V991
Compound was freshly dissolved in saline (9 mg/mL)

Test animals

Species:

rat

Strain:

other: Wistar: Hoe:WISKf(SPF71)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Immature Wistar rats, strain Hoe:WISKf(SPF71), from the company's own breeding colony, with a mean body weight of 102 g (M) or 113 9 (F) and an age of about 5 weeks at the start of the study were used.

Maintenance and feeding:
During the study, the rats were kept conventionally in groups of two male and four female animals (maximum) per cage of the same sex on wood shavings in transparent plastic cages and were distinguished by consecutively numbered ear tags. They received Altromin(R)-1324 feed from Altromin GmbH, Lage/Lippe (for composition see pages 321 - 322) and tap water ad libitum. The study was carried out from Sept. 17, 1991 to Jan. 22, 1992, in air-conditioned rooms at a temperature of 22 to 23 °C and a relative humidity of 29 to 70 %.

Administration / exposure

Route of administration:

intravenous

Vehicle:

physiological saline

Remarks:

9 mg/ml

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

3 months

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
dosages: 0/ 60/ 120/ 600 mg/kg bw
concentrations: 0 / 24 / 48 / 120 mg/ml

No. of animals per sex per dose:

15 per sex per group

Control animals:

yes, concurrent vehicle

Details on study design:

.Gamma.-Cyclodextrin was used from batch No. V 991. The compound was freshly dissolved in saline (9 mg/ml) every day prior to admininistration.

Examinations

Observations and examinations performed and frequency:

Clinical examinations:
Body weight: weekly (twice in the first week)
Feed consumption: continuously, with measurement weekly (twice in the first week)
Survival check: twice daily
Behaviour and general condition: daily
Eyes: weekly
Teeth: weekly
Visible mucous membranes: weekly
Neurological condition: weekly

Haematology:
Blood was examined at the times given after the individuall parameters from the number of animals specified:
after 1 month: 10 M / 10 F per group
end of dosing : 10 M / 10 F per group
after 37 (M) or 36 (F)
days recovery: 5 M / 5 F per group
Examinations were carried out in accordance with the methods listed in the List of Methods dated April 5, 1990. They comprised:
Erythrocytes
Hemoglobin
Hematocrit
MCV
MCH
MCHC
Reticulocytes
Thrombocytes
Coagulation time
Leukocytes
Differential blood count
Heinz bodies
Methemoglobin
The blood samples were taken from a sublingual vein of non-fasting animals according to the method of E. SCHÜTZ, Arneim. Forsch. 12, 444-445 (1962)

Clinical chemistry:
Serum analysis were carried out in the following numbers of animals at the times indicated:
end of dosing: 10 M / 10 F per group
after 37 (M) or 36 (F)days recovery (R): 5 M / 5 F per group.
The examinations included:
Sodium
Potassium
Calcium
Chloride
Inorg. phosphorum
Bilirubin
*Glucose
Uric acid
Creatinine
Urea
*ASAT (GOT)
*ALAT (GPT)
*Alk. phosphatase
For the parameters marked with a "*", blood samples were taken from a sublingual vein. For the other parameters, blood was obtained from the vena cava when killing the animals by exsanguination. In all cases, the animals were non-fasted.

Urinalysis:
The urinalysis were carried out in the following numbers of animals at the times indicated:
after 1 month : 10 M/10 per group
end of dosing : 10 M/10F per group
after 37 (M) or 36 (F) days recovery ( R) : 5 M / 5 F per group
The urine was obtained overnight in diuresis cages after withdrawal of the feed (this may explain deviations in body weights or feed consumption values at these particular times). The urinalysis included the follwing parameters:
Appearance
Colour
pH
Glucose
Protein
Bilirubin
Hemoglobin
Sediment

Organ weights: The weights of the following organs were determined and calculated per 100 g body weight (= relative organ weights):
heart, lungs, liver, kidneys, spleen, adrenals, testes or ovaries, thyroid, pituitary and brain.

Histology:
The following organs or parts thereof were submitted to microscopic examination:
heart, lungs, liver,kidneys, spleen, brain, testes, epididymides, prostate, seminal vesicle or ovaries, uterus and vagina, adrenals, pituitary, thyroid, thymus, salivary glands (parotid and submandibular), trachea, esophagus, stomach, sections of the intestines (duodenum, jejunum, ileum, cecum, colon, rectum), urinary bladder, pancreas, abdominal aorta, diaphragm, eyes, tongue, skeletal muscle(psoas), bone marrow (femoral), femur, lymph nodes (cervical and iliac), sternum, spinal cord, medulla oblongata, sciatic nerve, and skin with mammary gland and injection site.

Sacrifice and pathology:

Killing and post-mortem examinations:
Dissection: 10 M / 10 F rats from each group were killed on the day after the last dose, and the remaining animals were killed 37(M) or 36 (F) days after the last dose. For this purpose, the animals were first anaesthetised by intraperitoneal injection of 50 mg/kg pentobarbital sodium and then killed by opening the thorax and severing the cranial vena cava. All organs were assessed macroscopically.

Statistics:

For statistical evaluation of the study, the following parameters were tested at a significance level of p=0.05 using the methods given in the summary tables:
body weight, haemoglobin, erythrocytes, hematocrit, MCV, MCH, MCHC, reticulocytes, thrombocytes, blood coagulation time, leukocytes, all clinical chemistry parameters, and all relative (per 100 g body weight) organ weights.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

slightly retarded (males)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

dose related decrease (both sexes)

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

interim values for erythrocyte, haemoglobin and hematocrit were slightly lower than those of control animals (females) etc.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

bilirubin marginally decreased (both sexes), alkaline phosphatase increased (males)

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

traces of haemoglobin and mean protein values were noted in the urine (males); traces of glucose were noted in the urine (females); generally unobstrusive sediment (both sexes) etc.

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

increased weights of lungs, liver and spleen (males); increased weights of heart, lungs, kidneys, spleen and adrenals (females)

Gross pathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

minor local, reactive changes at the application site (tail) and in the regional lymphnodes; reactive and reversible hyperplasia of the mucosae of the urinary bladder (both sexes) etc.

Histopathological findings: neoplastic:

not examined

Details on results:

Body weight development:
The body weight development was slightly retarded in the high dose group males (600 mg/kg b.w.) over the whole study period, however, only being significant at study days 7 and 10. In the remaining animals, the body weight development did not show any substance-related differences.

Feed consumption:
Only during the first few days of the study a dose-related decrease in food consumption was observed in the treated male and female animals when compared with the control. Subsequent to this initial period, the food consumption was comparable in all groups.

Intercurrent deaths:
There were no intercurrent deaths.

Behavior and general condition were normal in all animals.
Local reactions at the injecton sites were not observed.

Eyes, teeth, visible mucous membranes and neurological condition showed no indication of compound-induced changes.

Haematology:
In the high dose group females (600 mg/kg) the interim values for erythrocyte, haemoglobin and hematocrit were slightly lower than those of the control animals and statistically significant after 3 months. These parameters were not altered in the high dose group males after 3 months. Therefore, the significantly decreased values observed after one month only in the males of the mid dose group (partly concerned also the males of the low dose group) were not considered as compound induced effects.
The reticulocyte counts were significantly increased after 3 months in the female rats from the 600 mg/kg group. A significantly increased interim value was detected for the males of the low and high dose group, but not in the mid dose (120 mg/kg). Thus, only the increase in the high dose group animals was considered as a compound induced effect. At the end of the four-week recovery period, the reticulocyte counts of all group animals were again within the range of the historical control data.
In addition, the trombocyte counts were reduced at 600 mg/kg, however, being significant only in the female rats after one month. The decreased leucocyte counts observed in the treated females after one month, only, were considered as incidental variations since they were not dose related and were not observed after three months.
At the end of the four-week recovery period, the above changes were no longer observed.
No Heinz bodies or methemoglobin (hemoglobin) were detected.

Clinical chemistry:
Bilirubin was marginally decreased in the high dose group animals (600 mg/kg). Alkaline phosphatase was only increased in the males of the high dose group, when compared with the control animals.
These findings might be considered as compound induced effects although they were well within the historical range of variation for this animal strain.
There were several further small deviations from the control group, which, in individual parameters, were calculated to be significant. However, they were not regarded as having compound-induced, but as being coincidental since, for example, their occurrence was not dose-dependent (urea for males, GPT for females) or since the values for the control group animals were incidentally at the upper limit of the historical range of variation for this animal strain (chloride in females).
The above encountered period mentioned changes were no longer encountered at the end of the four-week recovery period.

Urinanalysis:
At the end of the dosing period, traces of haemoglobin and mean protein values at about 0.30 g/l were noted in the urine for the male rats of all groups including the control. These findings were considered as non-compound induced, incidental and normal findings.
Traces of glucose (up to 5.5 mmol/l) were noted in the urine of 6/10 high dose group females (600 mg/kg) and were related to the test compound.
A generally unobstrusive sediment was found in the urine of the animals. However, a slightly to moderately increased number of irregular forms of epithelial cells was noted in the urinary sediment of the males and females from the high dose group(600 mg/kg) at the end of the dosing period as well as at the end of recovery (males, only) when compared with the control animals.

Post-mortem examinations
Macroscopic findings: The macroscopic examination at the autopsy revealed an increased incidence of enlarged iliacal lymphnodes in both the high dose group males and females (600 mg/kg).
Renal pelvic dilatations observed independent of the dose in a few animals occur spontaneously from time to time in the rat strain used and are probably of genetic origin.

Organ weights:
Statistical evaluation of relative organ weights revealed several significant differences between control and .gamma.-cyclodextrin treated animals. The increased weights of lungs, liver and spleen in male rats (600 mg/kg) and heart, lungs, kidneys, spleen and adrenals in females (600 mg/kg) are considered compound- or treatment related.
Other significant differences are considered to be incidental findings since there was no dose-dependency and the values were within the normal range known for rats of this strain and age used in the study.
All changes were reversible after the four-week recovery period.

Histology:
Beside minor local, reactive changes at the application site (tail) and in the regional lymphnodes, the daily intravenous administration of 60 and 120 mg/kg body weight over a period of 90 days did not cause any compound induced changes in the examined organs.
Beside these local findings, .gamma.-cyclodextrin caused a reactive and reversible hyperplasia of the mucosae of the urinary bladder in the high dose group males and females as well as pulmomonary histiocytosis (foam-cell macrophage aggregates similar to a phospholipidosis), which also showed a clear tendency toreversibility after the recovery period.
Slight reversible vacuolizations were found in the epithelia of the proximal tubules in the kidneys of individual animals.

Effect levels

Dose descriptor:

NOAEL

Effect level:

120 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effect

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In conclusion, the intravenous application of .gamma.-cyclodextrin in daily doses of 60 and 120 mg/kg body weight over a period of 90 days was tolerated by the male and female rats without any biologically significant changes. At 600 mg/kg, compound induced alveolar toxicity (phospholipidosis-type) could be detected as well as a slight alteration of epithelial kidney tubules and urinary bladder mucosae, which could be confirmed by the urinary sediment. The marginal and reversible signs of a toxic haemolytic anemia observed only in some animals of the high dose group did not show any clinico-chemical or histopathological correlate.

Executive summary:

.gamma.-cyclodextrin, dissolved in saline (9 mg/ml), was administered intravenously in single daily doses of 60 / 120 or 600 mg per kg body weight to groups of 15 male and 15 female rats on 90 or 91 consecutive days. A further group of the same composition serving as control received only the vehicle. Five male and five female rats of each group were used as recovery animals.

 

There were no intercurrent deaths.

 

The body weight development was slightly retarded in the high dose group males (600 mg/kg), while - except of a dose-related decrement observed only in the first few days of the study – the feed consumption was comparable in all study groups.

 

After 3 months the erythrocytes, hemoglobin and hematocrit values were slightly lowered in the females of the high dose group (600 mg/kg) and accompanied with slightly increased reticulocyte counts. No clear changes of these parameters could be detected for the males. In addition, the thrombocyte counts were slightly reduced in the males and females at 600 mg/kg.

 

Except of slightly increased alkaline phosphatase values observed in the males at 600 mg/kg no changes of toxicological significance could be found in the clinical chemistry parameters. Traces of glucose were noted in the urine of the females at 600 mg/kg and were related to the test compound. In addition, the urinanalysis revealed an increase in the number of irregular forms of epithelial cells in the urinary sediment of the high dose group animals (600 mg/kg).

 

Postmortem examination at autopsy revealed various treatment related changes in the tail region, however also implying a local compound induced effect since resulting in an enlargement of the regional lymph nodes especially in the high dose group animals (600 mg/kg). In the organ weights, increased weights of lungs, liver and spleen in male rats at 600 mg/kg and heart, lungs, kidneys, spleen and adrenals in females (600 mg/kg) are considered compound- or treatment related.

 

Histopathological examination revealed no discernable damage in the organs of the low and mid dose group animals (60 and 120 mg/kg). At 600 mg/kg, besides various local changes in the tail region, a reactive hyperplasia of the mucosa of the urinary bladder and foam cell macrophage aggregates in the lungs (similar to a phospholipidosis) were observed. In addition, slight vacuolizations were found in the epithelia of the proximal tubules in the kidneys of individual animals.

 

In the four week recovery period, all of the above changes were either completely reversible or showed a tendency to reverse.

In conclusion, the intravenous application of .gamma.-cyclodextrin in daily doses of 60 and 120 mg/kg body weight over a period of 90 days was tolerated by the male and female rats without any biologically significant changes. At 600 mg/kg, compound induced alveolar toxicity (phospholipidosis-type) could be detected as well as a slight alteration of epithelial kidney tubules and urinary bladder mucosae, which could be confirmed by the urinary sediment. The marginal and reversible signs of a toxic haemolytic anemia observed only in some animals of the high dose group did not show any clinico-chemical or histopathological correlate.