Cycloheptapentylose

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1986-11-07 to 1987-05-31

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Similar to OECD Guideline study report, just one dose level

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Principles of method if other than guideline:

Thirty male and 30 female weanling rats, Wistar derived (Bor: WISW (SPF;Cpb)) were obtained from F. Winkelmann, Institute for the Breeding of Laboratory Animals GmbH & Co. KG, Borchen, F.R.G. Each rat had been vaccinated at the premises of the breeder with 0.2 ml of a Sendai vaccine, at an age of 25 or 26 days. The vaccine had been obtained from "European Veterinary Laboratories", Amsterdam, the Netherlands, and was produced by "Statens Veterinarmedicinaska Anstalt", Upsala, Sweden. The animals arrived on October 28, 1986, when they were about 4.5 weeks old. Upon arrival, the rats were checked for overt signs of ill health and anomalies, and housed in a quarantine room. After 9 days, the rats received a second similar vaccination, and on the same day they were transferred to a normal rat room, and were allocated to 5 groups of 5 males and 5 females each, by a computer randomization program. The remaining rats were kept in reserve. The next day, the rats were weighed. A few rats were reallocated or exchanged with reserve rats in order to equalize the mean initial body weight in the various groups. Then the feeding study was started. None of the reserve rats was used after the start of the experimental period. During the experimental period (as well as during the quarantine period), the rats were housed 5 per cage, separated by sex, in suspended, stainless steel cages, fitted with wire-screen bottom and front. The cages were randomized over the cage racks. Housing conditions were conventional. The rats were maintained at 22+/- 2°C, a relative humidity of at least 40% and a ventilation rate of c. 10/hour. Artificial light was provided for 12 hours/day continuously, from 7.30 a.m. till 7.30 p.m. Following the arrival of the rats till the initiation of the experimental period, the Institute's basal diet for rats and mice, and tap water were provided ad libitum.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Cycloheptapentylose

EC Number:

231-493-2

EC Name:

Cycloheptapentylose

Cas Number:

7585-39-9

Molecular formula:

C42H70O35

IUPAC Name:

5,10,15,20,25,30,35-heptakis(hydroxymethyl)-2,4,7,9,12,14,17,19,22,24,27,29,32,34-tetradecaoxaoctacyclo[31.2.2.2~3,6~.2~8,11~.2~13,16~.2~18,21~.2~23,26~.2~28,31~]nonatetracontane-36,37,38,39,40,41,42,43,44,45,46,47,48,49-tetradecol (non-preferred name)

Details on test material:

- Name of test material (as cited in study report): beta-Cyclodextrin
- Substance type: chemical
- Physical state: white powder
- Impurities (identity and concentrations): water, alpha- and gamma-Cyclodextrin
- Composition of test material, percentage of components:
- Stability under test conditions: stable

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Thirty male and 30 female weanling rats, Wistar derived (Bor: WISW (SPF;Cpb)) were obtained from F. Winkelmann, Institute for the Breeding of Laboratory Animals GmbH & Co. KG, Borchen, F.R.G. Each rat had been vaccinated at the premises of the breeder with 0.2 ml of a Sendai vaccine, at an age of 25 or 26 days. The vaccine had been obtained from "European Veterinary Laboratories", Amsterdam, the Netherlands, and was produced by "Statens Veterinarmedicinaska Anstalt", Upsala, Sweden. The animals arrived on October 28, 1986, when they were about 4.5 weeks old. Upon arrival, the rats were checked for overt signs of ill health and anomalies, and housed in a quarantine room. After 9 days, the rats received a second similar vaccination, and on the same day they were transferred to a normal rat room, and were allocated to 5 groups of 5 males and 5 females each, by a computer randomization program. The remaining rats were kept in reserve. The next day, the rats were weighed. A few rats were reallocated or exchanged with reserve rats in order to equalize the mean initial body weight in the various groups. Then the feeding study was started. None of the reserve rats was used after the start of the experimental period. During the experimental period (as well as during the quarantine period), the rats were housed 5 per cage, separated by sex, in suspended, stainless steel cages, fitted with wire-screen bottom and front. The cages were randomized over the cage racks. Housing conditions were conventional. The rats were maintained at 22+/- 2°C, a relative humidity of at least 40% and a ventilation rate of c. 10/hour. Artificial light was provided for 12 hours/day continuously, from 7.30 a.m. till 7.30 p.m. Following the arrival of the rats till the initiation of the experimental period, the Institute's basal diet for rats and mice, and tap water were provided ad libitum.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The diets were prepared by mixing for 2 minutes in a mechanical blender. Batches of 4.5 kg of each of the diets were prepared twice during the study, viz. on 06-11-'86 and on 26-11-'86. The diets were stored in a refrigerator at c. 4°C until use. Immediately after. preparation of the diets, samples were taken and subsequently stored at -20°C

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 5% (4200 mg/kg bw males, 4162 mg/kg bw for females)
Basis:
nominal in diet

No. of animals per sex per dose:

5

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: acute tests
- Rationale for animal assignment (if not random): random

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

The general condi tion and behaviour of all rats were checked daily. All signs of ill health or reaction to treatment were recorded. The individual body weights of all rats were recorded initially, and on day 7, 14 ,21 and 28 of the study. Food intake was measured per cage (5 animals) weekly and the efficiency of food utilization was calculated and expressed as gram weight gain per gram food consumed. Water intake was measured daily, on a cage basis (5 animals) throughout the study. Samples of blood were collected from the tip of the tail of all rats on day 24, and examined for the following parameters: Haemoglobin, packed cell volume, red blood cells, white blood cells, differential white blood cell count, thrombocytes; mean corpuscular volume (MCV9; mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC),
on day 26, blood was collected from the tip of the tail of all animals after deprivation of water for 24 hours and of food during the last 16 hours of this period. In the blood samples, glucose was determined: At autopsy (on day 28), blood samples were collected from the aorta of all rats whilst under ether anaesthesia. Blood was sampled in heparinized plastic tubes. The blood samples were centrifuged at 2000 rpm for about 15minutes, using dispensers from General Di-agnostics for good
separation of the plasma. The following measurements were made in the plasma obtained from heparinized blood: alkaline phosphatase activity (ALP); glutamic-oxalacetictransaminase (GOT)/ aspartate amino transferase (ASAT) activity; glutamic-pyruvic transaminase (GPT)/ alanine amino transferase (ALAT) activity;: gamma glutamyl transpeptidase activity; total protein; albu-min, urea; creatinine, bilirubin total, sodium (Na), potassium (K), calcium (Ca), chloride (Cl), inorganic phosphate;
On day 25, freshly voided, early morning 3-hour urine samples were collected from unfasted rats. PH was determined in individual samples of all rats, with a pH meter. Subsequently, all rats were deprived of water for 24 hours and of food for 16 hours. Urine was collected in calibrated tubes from individual animals during the last 16 hours of the deprivation period. On day 26 the volume was measured, and the density of the urine was determined using a refractometer. The determinations were carried out in individual samples.

Sacrifice and pathology:

On day 28, the animals were killed after ether anaesthesia and bleeding by cannulating the aorta, and then examined macroscopically for pathological changes. Samples of the following tissues and organs of all animals were preserved in a neutral, aqueous, phosphate-buffered 4% solution of formaldehyde: adrenals, heart, kidneys, liver, mesenteric lymph nodes, ovaries, spleen, testes, thymus, thyroid (with parathyroids), caecum full, caecum empty, gross lesions

Other examinations:

none reported

Statistics:

Data on body weights were evaluated by one-way analysis of co-variance followed by Dunnett's multiple comparison test. Data on red blood cell
variables, total white blood cell counts, clinical chemistry values, volume and density of the urine and organ weights were evaluated by one-way
analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. Differential white blood cell counts and urinary pH values were analyzed by the Mann-Whitney u-test. The histopathological changes were examined by Fisher's exact probability test.

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:

effects observed, treatment-related

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

effects observed, treatment-related

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

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:

no effects observed

Details on results:

In rats fed 5% beta-cyclodextrin, soft faeces and slight to severe diarrhoea were observed from the start of the study up to day 7. Thereafter, only soft faeces were observed in these rats, or the pellets became fully normal. A swollen belly was observed in one female fed 5% beta-Cyclodextrin (wk 1). None of the rats died in the course of the study.
In males fed 5% beta_cyclodextrin, body weights were significantly lower than in controls in the first week only. Food intake of rats fed 5% test substance was also decreased at some stages. Water intake of males fed 5% test substance was generally slightly higher than in controls.
In males fed 5% cyclodextrin, red blood cell count was slightly though statistically significantly decreased, while mean corpuscular volume and mean corpuscular haemglobin were slightly increased. The percentage of monocytes was increased in females fed 5% test substance.
Garmma-glutamyl-transferase(GGT) activity was decreased in males fed 5% test substance. Urea concentration was relatively low in males and females fed 5% beta-Cyclodextrin. Glu-tamic-oxalacetic-transaminase (GOT) and glutamic-pyruvic-transaminase (GPT) activities were increased in females fed 5% beta-Cyclodextrin. The absolute and relative weight of the full and empty caecum were slightly increased in both sexes fed 5% beta-Cyclodextrin.

Effect levels

Dose descriptor:

NOAEL

Effect level:

>= 4 160 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: only reversible effects

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

 The ingestion of beta-cyclodextrin by rats for a period of 4 weeks in the present study was accompanied by a number of changes. Slight caecal enlargement occurred in males and females fed 5% beta-Cyclodextrin. Many other poorly digestible carbohydrates (e.g. chemically modified starches, lactose) tend to induce caecal enlargement . The phenomenon is ascribed to an increased load of osmotically active substances (such as volatile fatty acids or lactic acid) in the large intestine, arising from microbial fermentation of carbohydrates that were incompletely digested or absorbed in the small intestine. It is known from the literature that cyclodextrins are not absorbed from the stomach and the small intestine, while the microflora of the large intestine has an important part in the metabolism of cyclodextrins. In the present study, microbiological degradation of unabsorbed beta-Cyclodextrin may explain the observed distention of the caecum, the swollen bellies, the increased water intake and the diarrhoea observed at levels of 5% beta-cyclodextrin.

The above intestinal changes are not deemed to be of toxicological significance but are rather adaptation phenomena.

The relatively low plasma urea concentrations in rats fed 5% beta-cyclodextrin, are ascribed to increased microbial activity as well. In the rat, blood urea is related to the ammonia levels in the caecum. Bacteria may utilise ammonia nitrogen (derived from the hydrolysis of urea) for protein synthesis. As a result of increased bacterial population, less ammonia is reabsorbed to be reconverted to urea in the liver. This may result in lower blood urea levels. Therefore no toxicological significance is attached to this finding.

It is not clear from the present study whether the other changes in clinical chemistry variables in rats fed 5% beta-cyclodextrin are of toxicological significance, or whether they represent reversible adaptive responses associated with the ingestion of large quantities of a poorly digestible carbohydrate.

Diarrhoea was observed already on the first day of the study. From day 7 the rats apparently adapted to the feeding of beta-cyclodextrin since the signs of diarrhoea disappeared.

In rats fed 5% beta-cyclodextrin a few changes in haematology or clinical chemistry variables were observed which were generally slight and possibly of doubtful toxicological significance.

 

Applicant's summary and conclusion

Conclusions:

Test substance cause only slight and reversible effects not relevant for classification.

Executive summary:

In a sub-acute (4-wk) feeding study performed according to OECD Guideline 407, the toxicity of beta-cyclodextrin (> 98 % pure) was examined by administering the test substance at a five percent level in the diet to Wistar rats (4200 mg/kg bw males; 4160 mg/kg bw for females). The substance was mixed with the diet without an analytical verification. Clinical observations, growth, food and water intake, haematology, clinical chemistry, urinalysis, organ weights , macroscopic examination, and microscopic examination of the liver, kidneys, heart, adrenals, spleen, caecum and mesenteric lymph nodes were used as criteria for disclosing possible harmful effects.

 The ingestion of beta-cyclodextrin by rats for a period of 4 weeks in the present study was accompanied by a number of changes. Slight caecal enlargement occurred in males and females fed 5% beta-Cyclodextrin. Many other poorly digestible carbohydrates (e.g. chemically modified starches, lactose) tend to induce caecal enlargement . The phenomenon is ascribed to an increased load of osmotically active substances (such as volatile fatty acids or lactic acid) in the large intestine, arising from microbial fermentation of carbohydrates that were incompletely digested or absorbed in the small intestine. It is known from the literature that cyclodextrins are not absorbed from the stomach and the small intestine, while the microflora of the large intestine has an important part in the metabolism of cyclodextrins. In the present study, microbiological degradation of unabsorbed beta-Cyclodextrin may explain the observed distention of the caecum, the swollen bellies, the increased water intake and the diarrhoea observed at levels of 5% beta-cyclodextrin.

The above intestinal changes are not deemed to be of toxicological significance but are rather adaptation phenomena. The relatively low plasma urea concentrations in rats fed 5% beta-cyclodextrin, are ascribed to increased microbial activity as well. In the rat, blood urea is related to the ammonia levels in the caecum. Bacteria may utilise ammonia nitrogen (derived from the hydrolysis of urea) for protein synthesis. As a result of increased bacterial population, less ammonia is reabsorbed to be reconverted to urea in the liver. This may result in lower blood urea levels. Therefore no toxicological significance is attached to this finding.

It is not clear from the present study whether the other changes in clinical chemistry variables in rats fed 5% beta-cyclodextrin are of toxicological significance, or whether they represent reversible adaptive responses associated with the ingestion of large quantities of a poorly digestible carbohydrate. Diarrhoea was observed already on the first day of the study. From day 7 the rats apparently adapted to the feeding of beta-cyclodextrin since the signs of diarrhoea disappeared. In rats fed 5% beta-cyclodextrin a few changes in haematology or clinical chemistry variables were observed which were generally slight and possibly of doubtful toxicological significance. As a result and since all other changes were only slight and/or reversible within the study period, the NOAEL for both sexes is set to be > 4160 mg/kg bw.