Cycloheptapentylose

Endpoint summary

Description of key information

In a 52 week chronic feeding study with rats minimal effects on liver and minimal pigment deposition in the epthelium of cortical tubules were obseerved at converted doses of 1313 mg/kg b.w./day and 1743 mg/kg b.w. /day for male and females, respectiveley. At higher concetration these effects increased in incidence but not in severity.

In a thirteen week subchronic feeding study no such effects were observed at an equivalent dose of about 9000 mg/kg b.w.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD comparable guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Deviations:

yes

Remarks:

see principles

Principles of method if other than guideline:

Test performed according to Toxicological Principles for the Safety Assessment of Direct Food Additives and Colour Additives used in Foods, published by the US Food and Drug Administration Bureau of Foods, 1982.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

A total of 116 male and 116 female Cri: CD (SD) BR rats approximately 28 days old and within a weight range of 10 g for males and 13 g for females was received from Charles River Breeding Laboratories, Portage, Michigan, USA. For those animals selected for the study, their estimated age at the start of treatment was 6 weeks and their body-weights were in the range 170 to 198 g for malesand 123 to 160 g for females.
On arrival five males and five females selected at random were used for health check purposes.These animals were killed within 24 hours after arrival at testing laboratory and subjected to routine macroscopic examination. No abnormalities were seen. Lungs, liver, kidneys, spleen and heart were preserved in fixative, but not processed further.
The remaining rats were placed at random in suspended cages with wire mesh floors, according to sex, so that each cage contained 5 rats of the same sex. Each cage measured 35.8 cm wide, 53.0 cm deep and 25.7 cm high.
Animal room temperature and relative humidity controls were set at 21 ± 2°C and 55 ± 10 % respectively. Permanent weekly recordings of these parameters were made by a Foster CIearspan M206 recorder and these are archived with all other raw data for this study. Artificial lighting was controlled to give 12 hours continuous light and 12 hours continuous dark per 24 hours.
All rats had free access to tap water and powdered SDS Rat and Mouse No. 1 modified maintenance diet.
After an acclimatisation period of 7 days, each animal was weighed and the required number of animals was selected by discarding those animals furthest from the mean bodyweight. The remaining animals were then randomly assigned to cages, stratified by bodyweight, in such a way that the initial cage means were approximately equal. The appropriate numbers of cages were then allocated to each treatment group.
A further period of acclimatisation of 7 days was allowed between allocation of animals to groups and commencement of treatment. During this period a review of animal health was undertaken by a veterinary officer. The spare animals were retained during this acclimatisation period to replace any rat showing signs of ill health. On the day after commencement of treatment these spare rat discarded with no further investigations performed. Ten spare male and ten spare female rat were used to collect pre-treatment blood samples for vitamin assay. These rat were killed and discharged with no further investigations, immediately after the blood samples were withdrawn.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

A pre-mix was prepared each week by grinding the test substance directly into SDS Rat. and Mouse No. 1 modified maintenance diet and mixing in a Turbula mixer for a minimum period of 2 minutes. The required concentrations were then prepared by direct dilution of the pre-mix with further quantities of untreated diet, homogeneity being achieved by mixing in a double cone blender for a minimum period of 7 minutes

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations were tested to be both stable and homogenously mixed. No further details mentioned

Duration of treatment / exposure:

52 weeks

Frequency of treatment:

daily (feed)

Remarks:

Doses / Concentrations:
0, 12500, 25000 and 50000 ppm (equivalent to 650/860 , 1313/1743 and 2655/3614 mg/kg bw /day [male/female])
Basis:
nominal in diet

No. of animals per sex per dose:

20

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dosages used in this study (0, 12500, 25000, 50000 ppm) were selected by the Sponsor with reference to a preliminary study performed by the Sponsor (Report no. 89071).
- Rationale for animal assignment (if not random): random

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily.


BODY WEIGHT: Yes
- Time schedule for examinations: allocation to groups, commencement of treatment and one week thereafter including day of sacrifice.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption cal-culated as g food/kg body weight/day: Yes
The quantity of food left by individual animals was recorded on a daily basis throughout the experimental period. From these data the quantities consumed were calculated. From the mean weekly food consumption and bodyweight, the mean weekly intake of each group, in terms of mg/kg/day, was calculated.


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: Yes


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once before treatment started and during wk 13, 26, 39 and 52
- Dose groups that were examined: all, 10 animals per gruop


HAEMATOLOGY: Yes
- Time schedule for collection of blood: once before the start of treatment and again in wk 13, 26, 39 and 52 for analysis of haematological and biochemical parameters
- Anaesthetic used for blood collection: No data
- Animals fasted: yes
- How many animals: all
Packed cell volume (PCV), Haemoglobin (Hb), Red cell count (RBC), Total white cell count (WBC Total), Platelet count (pIts), Differential WBC count, Neutrophils (N) , Lym-phocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Cell Morphology, Prothrombin Time (PT), Activated Partial Thromboplastin Time (AP1T),

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once before the start of treatment and again in wk 13, 26, 39 and 52 for analysis of haematological and biochemical parameters
- Animals fasted: yes
- How many animals: all
Total Protein, Albumin (Alb), Globulin (Glob), ), Urea, Creatinine, Sodium (Na), Potas-sium (K), Calcium (Ca), Inorganic Phosphorus (P), Chloride (Cl), Total cholesterol (Chol) , Alkaline phosphatase (AP), Total bilirubin (Bilirubin), Glutamic-pyruvic transa-minase (GPT) - , Glutamic-oxaloacetic transaminase (GOT) , Gamma-glutamyl trans-ferase (GGT)., High density lipoprotein cholesterol (HDL), Triglycerides (Tri-glyc), Phos-pholipid (ph Lipid) , Ornithine carbamoyl transferase (OCT) , beta-Lipoprotein (ß-L) ,


URINALYSIS: Yes
- Time schedule for collection of urine: wk 13 and 52
- Metabolism cages used for collection of urine: No data
- Animals fasted: yes
Volume, pH: by pH meter, Specific Gravity (SG) , Protein (prot), Total reducing sub-stances (fRS) , Glucose, Ketones, Bile pigments, Urobilinogen, Haem pigments, Mi-croscopy
For microscopic examination, an aliquot of the urine sample was centrifuged at ap-proximately 1500 "g" for 10 minutes and the resulting deposit spread on a microscope slide. The deposit was examined for the presence of the following:
Epithelial cells (E)
Polymorphonuclear leucocytes (P)
Mononuclear leucocytes (M)
Erythrocytes (R)
Organisms (0)
Renal tubule casts (C)
Sperm (sp)
Other abnormal constituents (A)

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

TERMINAL STUDIES
Post mortem examination On completion of 52 weeks of treatment, all surviving rats were killed. Since the terminal procedures took 2 days to complete, the treated animals continued to receive the substance in their diet until the day on which they were killed. All rats were killed by carbon dioxide asphyxiation and subjected to the following de-tailed necropsy procedure:
All superficial tissues were examined visually and by palpation and the cranial roof re-moved to allow observation of the brain, pituitary gland and cranial nerves. After ventral mid-line incision and skin reflection all subcutaneous tissues were examined. The condi-tion of the thoracic viscera were noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastrointestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were re-moved and all pleural surfaces examined
suitable illumination. The liver was sectioned at intervals of a few millimetres; the kid-neys were incised and examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intraabdominal lymph nodes and accessory reproductive or-gans were recorded.
The following organs from all animals killed at the scheduled sacrifice were dissected free of fat and weighted: adrenals, brain, heart, kidneys, liver, lung, ovaries, pituitary
Prostate, salivary gland, seminal vesicles, spleen, testes (with epididymides), .thymus, thyroid, uterus.

Other examinations:

Vitamin assay from Liver samples

Statistics:

Data from male and female animals were analysed both together and separately.
Food consumption data were analysed using cumulative totals, expressed on a weekly basis. Bodyweight data were analysed using weight gains. The following sequence of statistical tests was used for food consumption, bodyweight, organ weight and clinical pathology data: If the data consisted predominantly of one particular value (relative frequency of the mode exceeded 75 %), the proportion of animals with values different from the mode was analysed. Otherwise: A test was applied to test for hetero-geneity of variance between treatments. Where significant (at the 1% level) heterogene-ity was found, a logarithmic transformation was tried to see if a more stable variance structure could be obtained. If no significant heterogeneity was detected (or if a satisfac-tory transformation was found), a one-way analysis of variance was carried out. If sig-nificant heterogeneity of variance was present, and could not be removed by a trans-formation, an analysis of ranks was used. Analyses of variance were followed by Stu-dent's't' test and WilIiams' test, although only the one thought most appropriate for the response pattern observed was reported. The Kruskal-Wallis analyses were followed by the non-parametric equivalents of these tests. For organ weight data, analysis of variance was performed using terminal bodyweight as covariate when the within-group relationship between organ weight and bodyweight was significant at the 10% level.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

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:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

MORTALITY
There were 4 deaths during the study, none of which were considered to be related to treatment with beta-cyclodextrin. One control group male was found dead in Week 41. Two control group males were sacrificed due to poor condition in Weeks 48 and 30 re-spectively. One female receiving 12500 ppm was sacrificed in Week 33 due to severely swollen limbs.

CLINICAL SIGNS
No clinical signs considered to have been attributable to treatment with beta-cyclodextrin were noted in any of the rats during the study.

BODYWEIGIITS
During the 52 weeks of the study, there were no effects on bodyweight gain attributable to treatment with beta-cyclodextrin.
Slightly reduced bodyweight gain was noted for males and females receiving 50000 ppm and females only, receiving 12500 ppm in comparison with controls. These reduc-tions in bodyweight gain are, however, not considered to be of any toxicological impor-tance due to the lack of dosage-relationship in the females, the marginal level of reduc-tion in the high dose group males and the lack of statistical significance throughout the study. Males receiving 25000 or 12500 ppm and females receiving 25000 ppm showed similar bodyweight gains to those of controls throughout the study.

FOOD CONSUMPTION
Cumulative food intakes for all treated and control• groups were similar throughout the 52 weeks of treatment.

EFFICIENCY OF FOOD UTILISATION
The efficiency of food utilisation was estimated by the calculation of food conversion ratios during the first 26 weeks of treatment. Overall, during the first 26 weeks of treat-ment, the efficiency of food utilisation by rats receiving beta-cyclodextrin was similar to that by controls.

ACHIEVED INTAKES
As was to be expected in a study with fixed dietary inclusion levels, the achieved in-takes in teerms of mg/kg/day of beta-cyclodextrin generally decreased as the body-weight of the animals increased. The interval between the achieved intakes for each group generally reflected the interval between the dietary inclusion levels.

WATER CONSUMPTION
Daily monitoring of the water bottles by visual appraisal throughout the study revealed no obvious differences in water intake between treated and control group rats.

OPIITHALMOSCOPY
Ophthalmoscopic examination of all allocated rats prior to the commencement of treat-ment revealed no findings of any significance. Ophtbalmoscopic examination of rats in the control and high dosage groups during Weeks 13, 26, 39 and 52 revealed no ab-normalities attributable to treatment with beta-cyclodextrin. Consequently ophthalmo-scopic examination was not extended to include animals from the low and untermediate dosage groups.

PHARMACOKINETIC SAMPLING
Urine
In Weeks 13 and 52, increasing amounts of beta-cyclodextrin were excreted into the urine with increasing dosage. It was noted that more beta-cyclodextrin was excreted in Week 13 for males than in Week 52, whilst for females a greater amount of beta-cyclodextrin was excreted in Week 52 than in Week 13.
Faeces
In Week 13, beta-cyclodextrin was detected, at below the limit of accurate detection (250 mg/kg), in the faeces of 5/10 males and 5/10 females receiving 50000 ppm. In 2/10 males receiving this dosage beta-cyclodextrin was detected at 330 or 560 mg/kg. No beta-eyclodextrin was detected in the faeces of control rats or the remaining rats receiv-ing 50000 ppm. In Week 52, no beta-cyclodextrin was detected in the faeces of control rats or rats receiving 50000 ppm.

HAEMATOLOGY
Investigation of haematological parameters during Weeks 13, 26, 39 and 52 of treat-ment did not reveal any differences between treated and control groups that were con-sidered to be related to treatment.
Although there were some differences from controls which attained statistical signifi-cance, they were minor, showed no clear dosage-relationship or consistency between the weeks and were within the historical control range. Therefore, they were considered to be of no toxicological importance.

BIOCHEMISTRY
Investigation of biochemical parameters during Weeks 13, 26, 39 and 52 of treatment revealed the following differences considered to be related to treatment.
In Weeks 13 and 26, a dosage-related increase in GPT and GOT (generally attaining a level of statistical significance) was noted for males and females receiving 50000 or 25000 ppm in comparisonwith controls. In these weeks, two animals in particular were affected, receiving 50000 ppm. In Week 39, only females receiving 50000 ppm demon-strated a statistically significant increase in GPT and GOT in comparison with controls. In Week 52, a statistically significant and dosage-related increase in GPT was noted for males receiving 50000 or 25000 ppm and for females receiving 50000 ppm only; also noted in this week was a statistically significant increase in GOT for males and females receiving 50000 ppm and an increase, though not statistically significant, in GOT for males receiving 25000 ppm. However, a high degree of individual variation was noted for these enzymes in each week investigated. Although a slight increase in GPT and GOT was noted for males receiving 12500 ppm in Week 52, a level of statistical signifi-cance was attained for GPT only and they were not associated with histopathological change, thus, the findings at this dosage are considered not to be of toxicological impor-tance. In Week 13, OCT was also increased for females receiving 50000 ppm and for males receiving 50000 or 25000 ppm, though not with a dosage-related trend. In Weeks 26 and 52, significantly increased OCR in comparison with control was noted only for males receiving 50000 or 25000 ppm. In Week 39, OCR activities were similar between treated groups and controls. In Weeks 26 and 52, a reduction in triglycerides was noted for males receiving 50000 ppm. A marginal reduction in triglycerides was also noted for males at this dosage in Week 39. In Weeks 39 and 52, a reduction in triglycerides was also noted for females receiving 50000 ppm in comparison with controls. However, the toxicological importance of this finding is not clear as during these weeks some control females showed abnormally high triglyceride concentrations.
Other differences from control, including those which attained statistical significance, were minor or showed no clear dosage relationship. Therefore they were considered to be of no toxicological importance.

URINALYSIS
Urine analyses performed during Weeks 13, 26, 39 and 52 of treatment did not reveal any differences between treated and control groups that were considered to be related to treatment. Although there were some differences from controls which attained statis-tical significance, they were either minor or showed no clear dosage-relationship and were within the historical control range. Therefore, they were considered to be of no toxicological importance.

ORGAN WEIGIITS
Analysis of organ weights following 52 weeks of treatment revealed no statistically sig-nificant differences between treated groups and controls.

MACROSCOPIC PATHOLOGY
The macroscopic examination performed following 52 weeks of treatment revealed no changes attributable to beta-eyclodextrin.

MICROSCOPIC PATHOLOGY
Liver
Male rats
Statistically significant increased incidences of single cell. necrosis, prominent sinusoi-dal lining cells/leucocytosis, centrilobular hepatocyte enlargement and portal inflamma-tory cell infiltration were seen in male rats receiving 50000 ppm when compared to con-trols. There was also a statistically significant increase in area/foci of hepatocytes with rarefied cytoplasm in male rats receiving 50000 ppm when compared to controls. How-ever, when rats with a single focus are included the increase is no longer statistically significant. This, therefore, probably represents an exacerbation of a background change by treatment with beta-cyclodextrin. Portal inflammatory cell infiltration was also statistically significantly increased in incidence in male rats receiving 25000 ppm.
Female rats
Statistically significant increased incidences of a single cell necrosis, centrilobular hepa-tocyte enlargement, portal inflammatory cell infiltration and focal basophilic hepatoeytes were seen in female rats receiving 50000 ppm. Single cell necrosis and focal basophilic hepatocytes were also statistically significantly increased in female rats receiving 25000 ppm. No treatment-related changes were seen in the livers of male or female rats re-ceiving 12500 ppm.
Kidneys
A statistically significant increased incidence of minimal/trace amounts of pigment in the epithelium of the cortical tubules was seen in female rats receiving 50000 or 25000 ppm. No treatment-related changes were seen in the kidneys of male rats nor in female rats receiving 12500 ppm.

Key result

Dose descriptor:

LOAEL

Effect level:

25 000 ppm

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

Remarks on result:

other: equivalent to 1313 [males] and 1743 mg/kg bw /day [females]

Dose descriptor:

NOAEL

Effect level:

12 500 ppm

Sex:

male/female

Remarks on result:

other: equivalent to 650 [males] and 860 mg/kg bw /day [females]

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 313 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Minimal effects on liver and minimal pigment deposition in the epthelium of cortical tubules were obseerved at converted doses of 1313 mg/kg b.w./day and 1743 mg/kg b.w. /day for male and females, respectiveley.

Executive summary:

Three groups of 20 male and 20 female Crl:CD (SD) BR rats received beta-cyclodextrin (>= 99 % pure), by dietary administration at dosages of 12500, 25000 or 50000 ppm for 52 weeks. A similarly sized group received untreated diet and acted as controls. The objective of the study was to assess the chronic toxicity of beta-cyclodextrin. Clinical signs, bodyweights and food consumption were monitored throughout the treatment period. Ophthalmoscopic, haematological, biochemical and urinalysis investigations were performed pre-treatment and in Weeks 13, 26, 39 and 52. Organ weights were obtained; macroscopic and microscopic pathological examinations were also performed. Urine and faeces samples were collected in Weeks 13 and 52 for pharmacokinetic analyses; serum samples were obtained pre-treatment and in Weeks 13, 26 and 52 for vitamins A, D and E analysis,. and liver samples were obtained at termination for analysis of vitamins A and E.

Dietary administration of beta-cyclodextrin to rats at dosages of 12500, 25000 or 50000 ppm for 52 weeks revealed no effects on mortality, clinical signs, bodyweight gains, efficiency of food utilisation, ophthalmoscopic findings, haematological and urine analysis investigations, organ weights or or macroscopic pathology.

The liver was shown to be the target organ for toxicity with the following changes noted histopathologically: an increased incidence in single cell necrosis, portal inflammatory cell infiltration and focal basophilic hepatocytes in males and/or females receiving 50000 or 25000 ppm, and an increased incidence in centrilobular hepatocyte enlargement and prominent sinusoidal lining cells/leucocytosis in males and/or females receiving 5 סס oo ppm. These hepatic lesions were consistent with the increase in the liver enzymes GPT, GOT and OCT noted biochemically for males and/or females receiving 50000 or 25000 ppm, and the reduction in triglycerides noted for both sexes receiving 50000 ppm, with males appearing to be more affected. than females. Although a slight increase in GPT and GOT was noted for males receiving 12500 ppm in Week 52, no hepatic lesions were noted histopathologically, and as a level of statistical significance was attained for GPT only these findings at this dosage are considered not be of toxicological importance. Beta-cyclodextrin at a dosage of 50000 ppm was also noted to exacerbate the background incidence of area/foci of hepatocytes with rarefied cytoplasm. In addition in the kidney an increased incidence of minimal/trace amounts of pigment in the epithelium of the cortical tubules was noted histopathologically in females receiving 50000 or 25000 ppm.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1989-07-26 to 1990-06-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Gzuideline study report

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal species: rat
Strain: OFA originating from Sprague-Dawley
Source: IFFA CREDO (Les Oncin, France)
Sex: male and female
Weight: 150-250 grams
Number: sufficient number of virgin females in order to obtain 20 pregnant fe-males/group.
Rationale for species selection: Species usually used for this type of trial, and recom-mended by the OECD Guideline 408.
Animal husbandry Housing: Makrolon cages, stainless-steel grid lid size 37.5 x 23.5 x 16 cm bedding of chopped maize cobs 5 animals/cage
Food: Pelleted diet UAR base 210 from Usine d'Alimentation Rafionnelle, Villemoisson-sur-Orge, France
Drinking water: Polypropylene water-bottles, Tap water, autoclaved for 10 minutes at 120 °C, changed 3 times a week.
Food and water were available at libitum. The quantities consumed were measured 3 times a week.
Environment: Temperature: 20 +/- 2 °C, humidity 50 +/- 10%, artificial lighting 12 hours/day, air changes 12 times/hour
The animals were housed under quarantine conditions for at least one week prior to the commencement of the study.

Route of administration:

oral: gavage

Vehicle:

other: feed

Details on oral exposure:

The test compound is mixed with the semi-synthetic diet (base 210). Beta-cyclodextrin or lactose replacing a part (by weight) of the starch fraction in the diet.
At the beginning of lhe study, a sample of each batch of diet prepared is sent to the bio-chemistry and analytical laboratories. The diets must have the same composition, ex-cept for lactose or Beta-cyclodextrin contents, and the following parameters are ana-lysed: degree of dessication, protein, amino-acids, total lipids, total cholesterol, glucose,
Beta-cyclodextrin or lactose (as appropriate), starch, ash content (residue), total phos-phorus, organic phosphorus, chloride, calcium, manganese, potassium, sodium, magnesium, cobalt, iron, copper.
At the end of the study, Beta-cyclodxtrin or lactose content was re-determined, to test their stability in the feed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Beta-cyclodextrin levels in the feed, serum and urine were measured by High Performance Liquid Chromatography. The brand of the equipment used was WATERS (MILLIPORE-WATERS; MILFORO- Massachussets USA) and comprised: autosampler WISP, type 710; UV-visible dectector, type 481;
colulmn for the assay of :
- Beta-cyclodextrin in the feed : phenyl Microbondapak
- Beta-cyclodextrin (other samples): C18 Microbondapak
The mobile-phase was a mixture of methanol/water. The analysis was made according to the method of VICKMON (phenolphtatein). Results are expressed % beta-cyclodextrin in the food, and in g/l beta-cyclodextrin in serum and urine.

Duration of treatment / exposure:

daily for 13 weeks

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 1.25, 2.5, 5 and 10 %,
Basis:
nominal in diet

No. of animals per sex per dose:

20 animals per sex and dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: preliminary test
- Rationale for animal assignment (if not random): random
One additional group received 10 % lactose incorporated in the feed

Positive control:

not applicable

Observations and examinations performed and frequency:

All animals were investigated in the same way:

General state and behaviour daily
Weight growth weekly
Food intake weekly
Water intake weekly
Ophthalmological examinations 0 and 13 weeks


At Sacrifice::
Blood cell count, Differential leukocyte, Hematocrit, Hemoglobin, Platelet count, Prothombin time,

Blood biochemistry: Total cholesterol, Triglyceride, Glucose, Urea, Cretinine, Total bilirubin, Total protein, Albumin, GOT, GPT, Alkaline phosphatase, Sodium, Potassium, Chloride, Calcium

Urine biochemistry: Total protein, Urea, Uric acid, Sodium, Potassium, pH, Ketone bod-ies, Bilirubin, Hemoglobin, Glucose,. Protein

Sacrifice and pathology:

Macroscopic observations
At Sacrifice:
Removal and fixation of organs: Eye, Hypophysis, Encephalon, Spinal cord, Thyroid, Thymus, Salivary glands, Parathyroide gland, 1 mesenteric ganglion, Liver, Adrenal glands, Kidneys, Spleen, Heart, Esophagus, Stomach, Duodenum, Jejunum, Ileum, Caecum, Colon, Epididymis, Urinary bladder, Prostate, Seminal vesicles, Uterus, Gonads

Other examinations:

In addition to the standard tests described the following were also performed:
serum Beta-cyclodextrin at sacrifice
urinary Beta-cyclodextrin during the 6th week and at sacrifice

Statistics:

* All parameters
Mean
Standard deviation
* Parameters measured on all animals
Student's t-test
Correlation coefficient in case of dose-related effect
* Parameters measured by cage: food and water consumption
non-parametric Mann-Whitney U-test.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

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:

no effects observed

Details on results:

The animal behaviour, body weight, food and water consumption were not modified by the treatment. Ophthalmological examinations disclosed no treatment-induced changes. Macroscopical examinations performed at necropsy, histopathological and histochemical examination, revealed no lesions or functional modification attributable to treatment. The only treatment-related modification was an increase In the caecal weight in both sexes, also observed in the group treated with lactose. This is an observation commonly described in rats receiving fermentable carbohydrates which are not completely di-gested in the smaII intestine. One biochemical parameter, the plasma triglyceride level was significantly decreased, in a dose-related manner, in all male animals receiving beta-cyclodextrin. No other modifications attributable to treatment were observed in plasma or urine parameter examined. The few variations were within normal biological limits for the species used. The same applies to the hematological parameters. In con-clusion, administration of beta-Cyclodextrin to Sprague-Dawlev rats for 13 weeks did not induce any modification or anomalv indicative of a toxic effect.

Key result

Dose descriptor:

NOAEL

Effect level:

10 other: % in diet

Sex:

male/female

Remarks on result:

other: equivalent to 9000mg/kg b.w./day

Key result

Critical effects observed:

no

Conclusions:

Substance did not show an adverse effect up to dose group receiving 10% in the diet.

Executive summary:

A subchronic (13 weeks) toxicity of beta-cyclodextrin (89.9% pure) was carried out in Sprague-Dawley rats according to OECD Guideline 408. Six groups of animals each comprising 20 males and 20 females were used. The test substance was incorporaled into the semi-synthetic diet in place of the equivalent fraction of starch (by weight). at doses of 0, 1.25, 2.5, 5 and 10 % respectively. The last group constituted received a diet containing 10 % lactose incorporated in the feed as for the other groups. The substance was stable during the course of this test as was shown by HPLC analysis at the beginning and the end of the study.

The animal behaviour, body weight, food and water consumption were not modified by the treatment. Ophthalmological examinations disclosed no treatment-induced changes. Macroscopical examinations performed at necropsy, histopathological and histochemical examination, revealed no lesions or functional modification attributable to treatment. The only treatment-related modification was an increase In the caecal weight in both sexes, also observed in the group treated with lactose. This is an observation commonly described in rats receiving fermentable carbohydrates which are not completely digested in the small intestine. One biochemical parameter, the plasma triglyceride level was significantly decreased, in a dose-related manner, in all male animals receiving beta-cyclodextrin. No other modifications attributable to treatment were observed in plasma or urine parameter examined. The few variations were within normal biological limits for the species used. The same applies to the hematological parameters. In conclusion, administration of beta-Cyclodextrin to Sprague-Dawlev rats for 13 weeks did not induce any modification or anomalv indicative of a toxic effect.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

1 313 mg/kg bw/day

Study duration:

chronic

Species:

rat

Organ:

kidney

liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a sub-acute (4-wk) feeding study performed according to OECD Guideline 407, the toxicity of beta-cyclodextrin 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.

 

A subchronic (13 weeks) toxicity of beta-cyclodextrin was carried out in Sprague-Dawley rats according to OECD Guideline 408. Six groups of animals each comprising 20 males and 20 females were used. The test substance was incorporaled into the semi-synthetic diet in place of the equivalent fraction of starch (by weight). at doses of 0, 1.25, 2.5, 5 and 10 % respectively. The last group constituted received a diet containing 10 % lactose incorporated in the feed as for the other groups. The substance was stable during the course of this test as was shown by HPLC analysis at the beginning and the end of the study. The animal behaviour, body weight, food and water consumption were not modified by the treatment. Ophthalmological examinations disclosed no treatment-induced changes. Macroscopical examinations performed at necropsy, histopathological and histochemical examination, revealed no lesions or functional modification attributable to treatment. The only treatment-related modification was an increase In the caecal weight in both sexes, also observed in the group treated with lactose. This is an observation commonly described in rats receiving fermentable carbohydrates which are not completely digested in the smaII intestine. One biochemical parameter, the plasma triglyceride level was significantly decreased, in a dose-related manner, in all male animals receiving beta-cyclodextrin. No other modifications attributable to treatment were observed in plasma or urine parameter examined. The few variations were within normal biological limits for the species used. The same applies to the hematological parameters. In conclusion, administration of beta-Cyclodextrin to Sprague-Dawlev rats for 13 weeks did not induce any modification or anomalv indicative of a toxic effect.

 

Groups of 17 (19 at the highest dose) male and female Sprague-Dawley rats, 4 weeks old at commencement of the study, were given beta-cyclodextrin by gavage in aqueous suspension at daily dose levels of 0, 100, 400 or 1 600 mg/kg bw After 3 months administration an interim sacrifice was made as follows: Control, 5 males & 4 females; 100 mg/kg bw/d group, 3 males & 5 females; 400 mg/kg bw/d group, 5 males & 4 females; 1600 mg/kg bw/d group, 2 males & 4 females. The remaining animals were maintained on the same dosing regime to 6 months. Food and water intake and body weights were determined weekly. At termination, urinalysis (pH, protein, glucose, ketone bodies, blood, bilirubin, urobilinogen) was carried out on 5 animals of each sex in each group; haematological (RBC, WBC, haemoglobin and haematocrit) and serum biochemical analyses (protein, albumin/globulin, GOT, GPT, Alk-P-ase, BUN, bilirubin, total cholesterol and glucose) were carried out on all survivors. At autopsy, the following organs were weighed: brain, pituitary, thyroid, thymus, heart, lung, liver, kidneys, adrenals, spleen, pancreas, testes or ovaries; these organs and stomach and intestinal tract were examined histologically (haematoxylin & eosin). A total of 18 animals died during the study as follows: 100 mg/kg bw/d, 3 males; 400 mg/kg bw/d, 2 females; and 1600 mg/kg bw/d, 6 males and 7 females. It was claimed that these deaths were due to misdosing, as no abnormalities were detected other than "pneumonia-like" lung pathology. There was a small decrement of weight gain of both sexes in the top-dose group only, otherwise weight gain, food and water intake were similar to controls and there were no treatment-related effects on organ weights, urinalysis or haematological parameters. Serum biochemical indices generally were within the normal range although some significant differences from controls were observed, notably a dose-related increase in alk-P-ase in males and a decrease in blood glucose in females of the top two dose groups. Gross and histopathological examination did not reveal any treatment-related abnormalities. If the deficit in weight gain at the top-dose level is considered an adverse effect in the absence of other, pathological, changes, the NOAEL for this study is 400 mg/kg bw/day by gavage.

The toxicity of Beta-cyclodextrine was assessed in the dog following its administration in the diet at levels of 0 (Control), 10000. 25000 and 50000 ppm for 52 weeks according to a guideline equivalent to OECD guideline 452. 4 dogs per sex and dose group were used for this study which were administered daily. There were no deaths and no clinical signs considered to be related to treatment. There was no effect of treatment on the bodyweight or bodyweight gain of the dogs. There was no effect of treatment on food consumption. There were no treatment-related changes on the eyes of the animals. There were no changes in hematology and biochemistry considered to be of toxicological importance. At dietary inclusion levels of 25000 and 50000 ppm there was a trend for increased urinary protein concentrations for males and females. There were no treatment-related changes seen in the bone marrow examination. Also no effect of treatment was seen on organ weights or at macroscopic post mortem evaluations. There were no treatment-related findings seen in histology. It is concluded that the level of 50000 ppm represents the no observed adverse effect level (NOAEL) in this study.

Beta-cyclodextrin was assessed for its toxicity to dogs by repeated dietary administration over a period of 52 weeks. Thirty-two pure-bred beagle dogs were divided into 4 groups, each containing 4 males and 4 females. Three groups received the test substance, Beta-eyclodextrin, by inclusion in the daily diet at levels of 6200, 12500 or 50000 ppm for 52 weeks. The fourth group received untreated basal diet and acted as controls. Clinical signs, bodyweight and food consumption were monitored throughout the study. Ophthalmoscopy and laboratory investigations were performed at intervals. On completion of 52 weeks treatment, the animals were killed; a bone marrow smear was taken, macroscopic examination of all tissues was performed, organ weights were recorded and an extensive list of tissues was processed and examined microscopically. The following comments in relation to the principal findings during the study are made in summary. There were no unscheduled deaths. There was a slightly higher incidence of intermittent liquid faeces in animals receiving Beta-cyclodextrin compared to concurrent controls but no dosage relationship was apparent and overall incidences were low. There were no other signs considered to be related to treatment. Bodyweight and food consumption showed no conclusive effects of treatment. For ophthalmoscopy all changes were considered spontaneous in origin and unrelated to treatment. There was no conclusive effect of treatment for haematology and biochemistry. At Weeks 13, 26, 39 and 52, group mean urinary protein levels were increased for males and slightly increased for females receiving 50000 ppm. The majority of protein was found to be the globulin fractions. Group mean urinary calcium levels were increased at 50000 ppm in Weeks 13, 26 and 39 particularly for males. At Week 52, calcium values for males only were increased at 50000 ppm. In the additional analyses performed at Week 52 only, 1 male receiving 12500 ppm and the majority receiving 50000 ppm showed slightly increased GGT .levels. Group mean NAG levels were also considered to be slightly increased for males receiving 12500 or 50000 ppm, though not in a dosage related fashion. There were no other noteworthy findings. All marrow smears were considered normal. Minor intergroup differences in organ weights and pathological findings were considered ·to be spontaneous in origin and unrelated to treatment. A marginally higher incidence of liquid faeces was noted in treated groups, but overall incidences were so low as to be considered of no toxicological importance. Urinary protein (particularly globulin) and urinary calcium levels were increased at 50000 ppm, and slightly increased urinary GOT and NAG levels were noted at 12500 and 50000 ppm. In the absence of pathological changes considered to be related to treatment, it is concluded that administration of beta-cyclodextrin to dogs by dietary inclusion at levels of up to 50000 ppm for 1 year produced no clear signs of systemic toxicity.

In the key study, three groups of 20 male and 20 female Crl:CD (SD) BR rats received beta-cyclodextrin (>= 99 % pure), by dietary administration at dosages of 12500, 25000 or 50000 ppm for 52 weeks. A similarly sized group received untreated diet and acted as controls. The objective of the study was to assess the chronic toxicity of beta-cyclodextrin. Clinical signs, bodyweights and food consumption were monitored throughout the treatment period. Ophthalmoscopic, haematological, biochemical and urinalysis investigations were performed pre-treatment and in Weeks 13, 26, 39 and 52. Organ weights were obtained; macroscopic and microscopic pathological examinations were also performed. Urine and faeces samples were collected in Weeks 13 and 52 for pharmacokinetic analyses; serum samples were obtained pre-treatment and in Weeks 13, 26 and 52 for vitamins A, D and E analysis,. and liver samples were obtained at termination for analysis of vitamins A and E. Dietary administration of beta-cyclodextrin to rats at dosages of 12500, 25000 or 50000 ppm for 52 weeks revealed no effects on mortality, clinical signs, bodyweight gains, efficiency of food utilisation, ophthalmoscopic findings, haematological and urine analysis investigations, organ weights or or macroscopic pathology. The liver was shown to be the target organ for toxicity with the following changes noted histopathologically: an increased incidence in single cell necrosis, portal inflammatory cell infiltration and focal basophilic hepatocytes in males and/or females receiving 50000 or 25000 ppm, and an increased incidence in centrilobular hepatocyte enlargement and prominent sinusoidal lining cells/leucocytosis in males and/or females receiving 50000 ppm. These hepatic lesions were consistent with the increase in the liver enzymes GPT, GOT and OCT noted biochemically for males and/or females receiving 50000 or 25000 ppm, and the reduction in triglycerides noted for both sexes receiving 50000 ppm, with males appearing to be more affected. than females. Although a slight increase in GPT and GOT was noted for males receiving 12500 ppm in Week 52, no hepatic lesions were noted histopathologically, and as a level of statistical significance was attained for GPT only these findings at this dosage are considered not be of toxicological importance. Beta-cyclodextrin at a dosage of 50000 ppm was also noted to exacerbate the background incidence of area/foci of hepatocytes with rarefied cytoplasm. In addition in the kidney an increased incidence of minimal/trace amounts of pigment in the epithelium of the cortical tubules was noted histopathologically in females receiving 50000 or 25000 ppm.

Justification for classification or non-classification

From the key studies the 90 day NOEAL can be considered as greater than 1000 mg/kg b.w., the limit dose in repeated dose toxitiy studies. The substance therefore is non-hazardous for repeated dose toxicity according to CLP.