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Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD comparable guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994
Reference Type:
publication
Title:
.beta.-Cyclodextrin: 52-Week Toxicity Studies in the Rat and Dog
Author:
M. E. Bellringer, T. G. Smith, R. Read, C. Gopinath and Ph. Olivier
Year:
1995
Bibliographic source:
Fd.Chem. Toxic. 33, 5, pp. 367-376. 1995

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identity: Beta-cyclodextrin
Chemical name: Cycloheptaamylose
Action: Molecular inclusion
Received from: Sponsor (Roquette Freres)
Date received: 15 January 1991
Lot/Batch number:E0165
Expiry date: January 1994.
Purity: >=99%
Appearance: White powder
Storage conditions: Room temperature protected from light
Stability of formulated diets: Up to 18 days at room temperature in the dark
A 1 g reserve sample of this batch of beta-cyclodextrin was retained in archive.

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals 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.

Administration / exposure

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)
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 12500, 25000 and 50000 ppm (equivalent to 650/860 , 2045/2187 and 3873/4249 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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
12 500 ppm
Sex:
male/female
Basis for effect level:
other: overall effects (equivalent to 650 [males] and 860 mg/kg bw /day [females])
Dose descriptor:
LOAEL
Effect level:
25 000 ppm
Sex:
male/female
Basis for effect level:
other: clinical chemistry; histopathology (equivalent to 2045 [males] and 2187 mg/kg bw /day [females]),

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Substance has liver as a target organ and a NOAEL of 12500 ppm is seen for this study.
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 (equivalent to 650/860 , 2045/2187 and 3873/4249 mg/kg bw /day [male/female]) 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.

In conclusion, the non-toxic dosage level based on the results in this study is considered to be 12500 ppm of beta-cyclodextrin (equivalent to 650 mg/kg bw /day [males] and 860 mg/kg bw/day [females]).