Cyclooctapentylose

Endpoint summary

Description of key information

The no-observed-adverse-effect level (NOAEL) of .gamma.-cyclodextrin in a chronic (52 weeks) study is placed at the highest dose level of 20% in the diet. This dietary level was equal to an overall intake of 8.7 and 10.8 g/kg body weight/day for males and females, respectively. 
Two chronic (52 weeks) feeding studies with dogs using the read-across substance .beta.-cyclodextrin have been performed; both result in a NOAEL of 50.000 ppm (highest dietary level tested).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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: Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Characterization:
The study was conducted with albino rats. The rat was used because this species is considered the most suitable for this type of study, and is usually required by regulatory agencies. Young, male and female Wistar rats (Crl:(WI)WU BR) were obtained from a colony maintained under SPF-conditions at Charles River Deutschland, Sulzfeld, Germany. The animals arrived on 5 February 1997 when they were about 3-4 weeks old. Upon arrival, the rats were taken in their unopened shipping boxes, checked for overt signs of ill health and anomalies, and kept in quarantine. During the quarantine period, the microbiological status of the rats was checked by serological investigation in random samples. On 11 February 1997, after the results of serology turned out to be satisfactory, the quarantine room was cleared for use as experimental room and the rats were further acclimatized to the conditions in this room until initiation of treatment on 20 February 1997. The body weights on the experimental start date were within ± 20% of the mean weight and ranged from 133 to 185 g (mean 157 g) for males and from 103 to 146 g (mean 126 g) for females.

Allocation:
On the day of arrival (5 February 1991) the rats were allocated to the various groups by computer randomization and identified with a temporary mark on their tail. Nine days (on 11 February 1997) and seven days (on 13 February 1997) before the start of the treatment the rats were weighed. In the pretreatment period (on day -7), a few rats were replaced by reserve animals because of clinical signs
or incorrect weight variation (exceeding ± 20% of the mean weight). On day -6, one rat died after blood sampling and was replaced by a reserve rat. On the starting day of the treatment (nominal day 0; 20 February 1997), the rats were weighed again. One rat was replaced because of inadequate growth, and subsequently the treatment was started. No rats were replaced by reserve animals after the start of the treatment. The remaining rats were kept as sentinel animals but not used in the study.

Identification
The study was identified as computer study no. 1882 and the different groups were coded by a letter and a colour. In the pretreatment period, the rats were identified with a temporary mark on their tail. From the day prior to the start of the treatment each rat was uniquely identified by an animal identification number (even for males and odd for females) that was tattooed in the ears. Each cage was provided with a card showing the study number, colour code, group letter, cage number and animal identification numbers.

Animal maintenance:
From their arrival in the animal room until the end of the study, the rats were group-housed (three rats per cage, separated by sex) in macrolon cages with sterilized wood shavings (Woody Clean; Type 3/4) as bedding material. The cages and bedding were changed weekly. The cages were divided over the cage racks stratified randomly. The control group and the test groups were housed in the same room. Housing conditions were conventional. The temperature in the animal room was targeted at 22 ± 3°C, and the relative humidity at 50 ± 20%. These conditions were monitored continuously2 by means of a thermohygrograph. The actual temperature was generally between 20-23°C and did not exceed the above target range, except on two occasions lasting 2-3 days, when the temperature reached a minimum of 18.5°C and maxima up to 28°C, respectively. The actual relative humidity was generally between 40-70%. Due to wet cleaning of the room, the relative humidity sometimes exceeded the upper value during periods of one or a few hours, reaching maxima up to c. 95%. On several other occasions, the relative humidity exceeded the upper value of 70% during periods lasting a few hours to a few days and, in one case eight days. During these periods, the relative humidity was generally between 70-80%, but occasionally reached maxima up to c. 90%. The number of air changes was about 10 per hour. Lighting was artificial by fluorescent tubes, time switch controlled at a sequence of 12 hours light, 12 hours dark.

Feed and drinking water:
From the arrival of the rats until the end of the study, feed and drinking water were provided ad libitum, except during overnight fasting prior to certain laboratory examinations. The feed was provided as a powder in stainless steel cans, covered by a perforated stainless steel plate which prevented spillage. During the quarantine period, the rats were fed a closed formula diet (Rat & Mouse No. 3 Breeding Diet, RM3) obtained from SDS Special Diets Services, Witham, England. During the one-year treatment period, this diet was slightly modified. The modification consisted of the omission of 20% barley from the diet. The barley was replaced by the test substance and/or pregelatinized potato starch. Each batch of the modified diet was analyzed by the supplier for nutrients and contaminants. The drinking water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Tap water was suitable for human consumption (quality guidelines according to Dutch legislation based on the EEC Council Directive 80/778/EEC).

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

0, 5, 10, 20 %: 2.1, 4.3, 8.7 g/kg bw/day (males)
0, 5, 10, 20 %: 2.7, 5.4, 10.8 g/kg bw/day (females)

Duration of treatment / exposure:

12 months

Frequency of treatment:

7 days/week

Remarks:

Doses / Concentrations:
0, 5, 10, 20 %
Basis:
nominal in diet

No. of animals per sex per dose:

20 animals/sex/group

Control animals:

yes, plain diet

Details on study design:

Administration of the test substance:
The test substance was administered orally, because this is an anticipated route of human exposure. The test substance was administered in the feed for 12 months (7 days/week) at constant dietary levels of 0%, 5%, 10% or 20%. The test substance was incorporated in the feed at the expense of 20% barley. The control, low-dose and mid-dose diets were compensated by adding respectively 20%, 15% and 10%pregelatinized potato starch. Fresh batches of each test diet and the control diet were prepared 12 times in the study, viz on 19 February, 17 March, 28 April, 4 June, 1/2 July and 21 July, 10/11 September, 3 October and 23 October, 2 December and 16 December 1997 and 29 January 1998. Homogeneity was obtained by mixing in a mechanical blender (Lodige) for two minutes. After preparation the diets were stored in a refrigerator until use. The feed in the animal feeders was replaced by fresh portions once per week and topped up as needed.

Study design and dose levels:
The study comprised four groups of 20 rats/sex each, viz. one control group (A) and three treatment groups (B, C and D) receiving different levels of .gamma.-cyclodextrin. The feeding of the experimental diets was started on 20 February 1997 (nominal day 0). The study was terminated with the autopsy of the male rats on 2426 February 1998 (nominal days 369-371) and of the female rats on 26 and 27 February and 2 March 1998 (nominal days 371, 372 and 375).

Positive control:

not available

Observations and examinations performed and frequency:

Analyses of the test substance in the carrier:
Immediately after preparation of each batch of diets, samples were taken of each diet (0, 5, 10 and 20% .gamma.-cyclodextrin) and stored in a freezer.
The homogeneity of the test substance in the diet was assessed in the first batch of diets prepared (on 19 February 1997). For this purpose five samples per test diet (5, 10 and 20% .gamma.-cyclodextrin), taken at different places in the feed container, were analyzed.
For each dietary level of .gamma.-cyclodextrin, the content of the test substance was determined approximately once every three months (viz. in the diets prepared on 19 February, 4 June, 10/11 September and 2 December 1997, and on 29 January 1998. The stability of .gamma.-cyclodextrin in the diet after storage for one week in the animal room or for more than one month in a freezer was examined by analysing diets of the present toxicity study (study 1882; 50/0 and 20% level, prepared on 19 February 1997) or the concurrent carcinogenicity study (study 1883; 10% level, prepared on 3 February 1997).

Clinical signs:
Each animal was observed daily in the morning hours by careful observations. On working days, all cages were checked again in the afternoon. On Saturdays, Sundays and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.

Ophthalmoscopic examination:
Eye examination was conducted at the start of the study (day 0), in week i3 (day 90), in week 26 (day 181), in week 39 (day 272), and at the end of the study in week 52 (day 363). The examination was conducted in all rats of the control group (A) and the high-dose group (D). Since no treatment-related changes in the eyes were detected, examinations were not conducted in the intermediate-dose groups.
Eye examinations were carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulphate.

Body weights:
The body weight of each animal was recorded when starting the administration of the test substance (day 0), then weekly for the first 13 weeks and subsequently once every month. In addition, the animals were weighed on the scheduled sacrifice date in order to calculate their organ to body weight ratios.
Body weights were also recorded twice in the pre-treatment period to monitor adequate growth during the quarantine/acclimatization period. The latter data are not presented, but kept as raw data.

Food consumption and food conversion efficiency:
The quantity of food consumed by the animals of each cage was assessed on a cage basis, by weighing the feeders, over each 1-week period during the first 13 weeks of the study and subsequently over 1-week periods once every month. The results were expressed in gram per rat per day.
The efficiency of food utilization over periods of 7 days was calculated during the period of rapid growth (the first 13 weeks of the study) and expressed in g weight gain per g food consumed.

Intake of the test substance:
The intake of .gamma.-cyclodextrin per kg body weight per day was calculated from the nominal dietary concentration of the test substance, the mean food consumption and the mean body weight at the end of the pertaining week.
The overall test substance intake was calculated as a time-weighted average. To calculate this time-weighted average, a value was assigned to each week. If there was no calculated value on a given week, the subsequent value was used. The total of all values was divided by 52.

Water consumption:
The water consumption of the animals was measured per cage on seven consecutive days in or about week 1, week 12, week 24, week 36 and week 50 of the study.

Haematology, clinical chemistry and urinalysis:
Haematology, clinical chemistry and urinalysis were conducted on 10 rats/sex/group at the stages indicated in the paragraphs below. The rats were selected from as many different cages as possible (in each treatment group, the first one or two surviving rats were selected from each cage). The same rats were used at each stage for all determinations, with two exceptions, viz:
• one rat died after blood collection in the pre treatment period and was replaced by a reserve animal.
• one rat died as a result of the narcosis during blood collection on day 95 and was replaced by another rat of the same cage at subsequent collection stages.

Haematology:
Prior to the start of dosing (on day 6,), in week 14 (day 95) and in week 26 (day 179) blood samples were taken by orbita puncture.
At autopsy (day 369 and 370 for the selected males and day 371 and 372 for the selected females), blood samples were taken from the abdominal aorta. Blood was collected from the rats whilst under ether anaesthesia. In the pre-treatment period, samples were taken after deprivation of food (during approximately 16 hours); at all other stages, haematology was conducted in blood obtained from non-fasted rats. K2-EDTA was used as anticoagulant. The following measurements were made according to special methods:
Haemoglobin; prothrombin time; packed cell volume; thrombocyte count; red blood cell count; mean corpuscular volume (MCV); reticulocytes; mean corpuscular haemoglobin (MCH); total white blood cell count; mean corpuscular haemoglobin concentration; differential white blood cell count (MCHC).

Clinical Chemistry:
Fasting glucose:
Prior to initiation of treatment (on day 6), blood samples were taken by orbita puncture after deprivation of food during approximately 16 hours.
In week 13 (day 85), week 26 (day 176) and week 52 (day 358), blood samples were taken from the tip of the tail after deprivation of food during approximately 16 hours.
Other clinical chemistry determinations:
Prior to the start of dosing (on day 6,), in week 14 (day 95) and in week 26 (day 179) blood samples were taken by orbita puncture.
At autopsy (day 369 and 370 for the selected males and day 371 and 372 for the selected females) blood samples were taken from the abdominal aorta.
Blood was collected from the rats whilst under ether anaesthesia. In the pre-treatment period, samples were taken after deprivation of food (during approximately 16 hours); at all other stages, clinical chemistry was conducted in blood obtained from non-fasted rats.
The blood samples were collected in heparinized plastic tubes and plasma was obtained after centrifugation. The following analyses were conducted:
alkaline phosphatise; aspartate aminotransferase; alanine aminotransferase; gamma glutamyl transferase; ornithine carbamoyl transferase; total protein; albumin; ratio albumin to globulin; protein electrophoresis; urea; creatinine; bilirubin (total); cholesterol (total); triglycerides; phospholipids; calcium (Ca); sodium (Na); potassium (K); chloride (Cl); inorganic phosphate.

Urinalysis:
Renal concentration test:
In week 13 (day 84-85), 26 (day 175-176) and 52 (day 357-358), the selected rats were deprived of water for 24 hours and of food during the last c. 16 hours of this period. During the last 16 hours of deprivation, the rats were kept in metabolism cages (one rat per cage) and urine was collected. The concentrating ability of the kidneys was investigated by measuring the urinary volume and density in individual samples.

Analyses in urine collected in the concentration test:
In the urine samples collected from the rats in the renal concentration test, the following determinations were carried out in individual samples:
Appearance; ketones; glucose; protein; pH; bilirubin; occult blood; urobilinogen.
Subsequently, the samples were centrifuged and the following determinations were conducted:
- microscopy of the sediment
- electrolytes in supernatant (calcium, chloride, potassium and sodium)
- creatinine in supernatant.

Sacrifice and pathology:

The study was terminated with the autopsy of the rats on a number of successive working days (males on 24-26 February 1998 (nominal days 369-371) and females on 26 and 27 February and 2 March 1998 (nominal days.371, 372 and 375).
The rats were killed in such a sequence that the average time of killing was approximately the same for each group. The animals were killed by exsanguination from the abdominal aorta under ether anaesthesia. Subsequently they were examined macroscopically for pathological changes. A thorough autopsy was also performed on animal D49 that died intercurrently (on day 95).
At scheduled autopsy, the following organs were weighed (paired organs together) as soon as possible after dissection to avoid drying, and the relative weights (organ to body weight ratio's) were calculated from the organ weights and the terminal body weights:
Adrenals; brain; caecum (filled and empty); heart; kidneys; liver; ovaries; spleen; testes; thyroid (with parathyroids); thymus.
Samples of the following tissues and organs of all animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde (10% solution of formalin):
Adrenals; aorta; axillary lymph nodes; brain (brain stem, cerebrum and cerebellum); caecum; colon; epididymides; exorbitallachrymal glands; eyes; femur with joint (knee); parathyroids; rectum; seminal vesicles and coagulating glands; skeletal muscle (thigh); skin/ subcutis; small intestines (duodenum, ileum and jejunum); spinal cord (at three levels); spleen; GALT (Peyer's patches); Harderian gland; heart; kidneys; liver; lungs; mammary gland (both sexes); mandibular (cervical) lymph nodes; mesenteric lymph nodes; nerve-peripheral (sciatic nerve); oesophagus; ovaries; oviduct (fallopian tubes); pancreas; parotid salivary glands; pituitary; prostate; sternum with bone marrow; stomach (glandular and nonglandular stomach); sublingual salivary glands; submaxillary salivary glands; testes; tongue; thymus (or thymic region); thyroid; tracheal bronchi; urinary bladder; uterus (with cervix); vagina; Zymbal's gland all gross lesions/ tissues; showing abnormality.

Histopathological examination:
The tissues for light microscopic examination were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin. Histopathological examination was performed on all tissues and organs listed above of all animals of the control group and the high-dose group, including the animal that died intercurrently (on day 95).
The kidneys, liver, lungs and gross lesions/ tissues showing abnormality were examined in all animals of all groups.

Other examinations:

Neurotoxicity screening:
A functional evaluation battery of observations and tests selected to assess sensory activity to stimuli of different types, grip strength and motor activity was conducted on 10 animals/sex/group in week 48 (day 333-335) of the study.
Neurobehavioural screening was conducted on the rats that had also been selected for the conduct of laboratory determinations. In addition, the neurotoxicity screen included histopathological examination of tissue samples representative of major areas of the brain, spinal cord and peripheral nervous system.

Immunotoxicity screening:
Primary indicators of possible immune toxicity derived from routine measurements and examinations in this study were used as an immunotoxicity screen.
Primary indicators of an effect on the immune system were:
- haematology (total and differential white blood cell counts)
- clinical chemistry (total protein, albumin, albumin/globulin ratio, protein electrophoresis)
- body weights and organs weights (thymus, spleen)
- gross and microscopic examination of lymphoid tissues (spleen, lymph nodes, thymus, GALT (Peyer's patches), bone marrow)
- microscopic examination of non-lymphoid organs.

Statistics:

The statistical procedures used to evaluate the results were as follows:
- Body weights: one-way analysis of covariance using pre-exposure (day 0) weights as the covariate. When group means were significantly different (p<0.05), individual pair wise comparisons were made using Dunnett's multiple comparison method;
• Food consumption, food conversion efficiency, water intake, red blood cell and coagulation variables, total- and absolute differential white blood cell counts, clinical chemistry, urinary volume, density and electrolyte concentration and excretion, and organ weights: analysis of variance (Anova) followed by
Dunnett's multiple comparison tests;
• Total- and relative differential (percentages) white blood cell counts, semi quantitative observations in urine and microscopy of the urinary sediment:
Kruskal-Wallis nonparametric one-way analysis of variance. When this analysis yielded a significant difference, pair wise comparisons between the control- and treatment groups were made by means of Mann-Whitney U-tests;
• Histopathological changes: Fisher's exact probability test. Incidental gross lesions, verified in the intermediate dose groups, were not subjected to statistical evaluation.
All analyses were two-sided. Group mean differences with an associated probability of less than 0.05 were considered to be statistically significant.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

one female rat died as a result of the narcosis during blood collection

Mortality:

mortality observed, treatment-related

Description (incidence):

one female rat died as a result of the narcosis during blood collection

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

statistically significantly decreased (males)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

intake of test substance gradually decreased (males)

Food efficiency:

no effects observed

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

effects observed, treatment-related

Description (incidence and severity):

decreased (males+females)

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

prothrombin time slightly increased (females)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

alkaline phosphatase activity increased (both sexes); alanine aminotransferase activity increased (males); ornithine carbamoyl transferase activity increased (both sexes); creatinine concentration increased (females); etc.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

urinary volume slightly increased (females)

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

absolute weight+the relative weight of the filled caecum were slightly/statistically significantly increased (males); relative weight of the testes was statistically significantly increased; absolute weight of the thymus was decreased (females)

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Details on results:

Analysis of the test substance in the carrier:
In summary, .gamma.-cyclodextrin was homogeneously distributed in the first batch of diets prepared in the study. Diets containing 5%, 10% or 20% .gamma.-cyclodextrin were stable after storage for one week in the animal room or for more than one month in a freezer. The content of .gamma.-cyclodextrin in all diets analyzed was close to intended.

Intake of the test substance:
Because of the lower food intake per kg body weight with increasing age of the rats, the intake of the test substance per kg body weight gradually decreased.
The overall intake (time-weighted average) was 2.1, 4.3 and 8.7 g/kg bw/day for males and 2.7, 5.4 and 10.8 g/kg bw/day for females of the low-, mid- and high dose group, respectively.

Symptomatology and survival:
There were no treatment-related differences in general condition or behaviour between the treated rats and the controls. The incidence of visible or palpable masses was low and did not suggest any treatment-related effect (a mass was detected in only two male rats of the low-dose group).
One female rat of the high-dose group died as a result of the narcosis during blood collection on day 95.

Ophthalmoscopic examination:
Ophthalmoscopic examination in week 13, week 26, week 39 and week 52 did not reveal any treatment-related changes.

Body weights:
Mean body weights were statistically significantly decreased in males of the mid- and high-dose group in the first week of the study only. Otherwise there were no statistically significant differences in mean body weights among the groups.

Food intake and food conversion efficiency:
Food intake was relatively low in males of the mid- and high-dose group in the first week of the study only. There were no noticeable differences in overall food intake or in food conversion efficiency among the groups.

Water intake:
Water consumption tended to be decreased in males of all treatment groups at all stages examined, and in females of all treatment groups in weeks 1, 12, 24 and 36. The differences with the controls reached the level of statistical significance in males of all dose groups and in females of the mid- and/or the high dose
group at several occasions.

Haematology:
There were no statistically significant changes in red blood cell variables or thrombocyte counts.
In females, prothrombin time was slightly increased in the mid- and high-dose group in week 14 and in the high-dose group in week 53 of the study.
These changes were well within the range of historical mean control data for females (viz: range 33.0- 40.8, overall mean 36.5 sec for the 14-wk stage; and range 33.9-42.5, overall mean 38.3 sec for subsequent stages). In week 26, there were no significant changes in prothrombin time.
There were no statistically significant changes in total white blood cell counts. Differential white blood cell counts showed some incidental differences with the controls.
Since these changes were neither consistent nor dose-related they are not ascribed to the treatment with .gamma.-cyclodextrin.

Clinical chemistry:
A number of statistically significant changes in clinical chemistry variables was observed, viz:
- alkaline phosphatase activity was increased in the high-dose group in both sexes in week 14
- alanine aminotransferase activity was increased in males of the high-dose group in week 14 and 26
- ornithine carbamoyl transferase activity was increased in the high-dose group in males in week 14, and in females in week 14 and 26. Ornithine carbamoyl transferase activity was also increased in two females of the high-dose group in week 53, but this did not result in statistically significant differences with the controls
- creatinine concentration was increased in females of the high-dose group in week 14 and 26, and also in females of the mid-dose group in week 14
- triglycerides values were decreased in males of the high-dose group in week 14 and 53. Triglycerides were also relatively low in males of this group in week 26, but the difference with the controls was not statistically significant
- phospholipids were decreased in males and females of the high-dose group in week 14.
There were no other noticeable changes in clinical chemistry variables including the results of protein electrophoresis.

Urinalysis:
In the renal concentration test, urinary volume was slightly increased in females of all treatment groups in week 52, but similar findings were not obtained at earlier stages, there was no dose-response relationship and the urinary density was not affected.
There were no treatment-related changes in urinary electrolytes, semiquantitative observations in the urine or in the microscopy of the urinary sediment.

Organ weights:
The absolute weight and the relative weight of the filled caecum were slightly, though statistically significantly increased in males of the high-dose group.
The relative weight of the testes was statistically significantly increased in the high-dose group.
The absolute weight of the thymus was decreased in females of the high-dose group, but this change was not reflected in a significant change in the relative weight of this organ.
There were no other statistically significant changes in organ weights.

Pathology
Macroscopic examination:
Gross examination at autopsy did not reveal any treatment-related abnormalities. The macroscopic changes observed are common in rats of this strain and age, and occurred in one or a few animals only or at comparable incidences in test groups and controls.

Microscopic examination:
Microscopic examination did not reveal any treatment-related histopathological changes.
The changes observed are common findings in rats of this strain and age. The incidence of some of these changes showed a statistically significant difference between a treatment group and the controls, viz brown pigment accumulation and multifocal tubular dilatation in the kidneys occurred less frequently in males of the mid-dose group, and transitional cell hyperplasia in the kidneys and mononuclear cell infiltrate in the liver were seen more frequently in females of the mid-dose group. Since these findings were not confirmed at the high-dose level, they are considered to be fortuitous. All other histopathological changes were about equally distributed among the treatment groups or occurred in one or a few animals only.
Only a few neoplastic changes were found. They are common findings in rats of this age and their incidence in the various groups did not indicate a treatment-related effect.

Neurotoxicity screening:
The results of neurobehavioural observations did not indicate any neurotoxic potential of the test substance. Some statistically significant differences between test groups and controls were observed (e.g. impaired righting reflex and decreased total distance run during motor activity assessment in males of the highdose group), but they were not considered to be treatment related.
In addition, histopathological examination of tissue samples representative of major areas of the brain, spinal cord and peripheral nervous system did not reveal any changes that pointed to neurotoxicity.

Immunotoxicity screening:
The administration of the test substance did not produce immunotoxicity as evidenced by the absence of treatment-related changes in the relevant data from haematology, clinical chemistry, organ weights and pathology.

Dose descriptor:

NOAEL

Effect level:

20 other: % in the diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Substance intake for 20 % .gamma.-CD, male: 8700 mg/kg bw/d 20 % .gamma.-CD, female: 10800 mg/kg bw/d

Critical effects observed:

not specified

Conclusions:

In conclusion, .gamma.-cyclodextrin was tolerated without any specific adverse effect at all dose levels tested. Therefore, the no-observed-adverse-effect level (NOAEL) of .gamma.-cyclodextrin in the present study is placed at the highest dose level, viz. 20% in the diet. This dietary level was equal to an overall intake of 8.7 and 10.8 g/kg body weight/day for males and females, respectively.

Executive summary:

The oral toxicity of .gamma.-cyclodextrin was examined in a chronic (12 months) toxicity study with groups of 20 male and 20 female Wistar rats. The test substance was incorporated in the feed of the rats at constant levels of 0% (control), 5%, 10% and 20%. These dietary levels were equal to an overall intake (time-weighted average) of 2.1/2.7, 4.3/ 5.4 and 8.7/10.8 g/kg bw/day for males/ females of the low-, mid- and high-dose group, respectively.

General condition and behaviour were not adversely affected by the test substance. Results of neurobehavioural observations did not indicate any neurotoxic potential of the test substance. There were no test substance related deaths during the study. Ophthalmoscopic examination did not reveal any treatment-related changes.

There were no noticeable differences in mean body weights, food intake or food conversion efficiency among the groups. Water consumption tended to be decreased in all treatment groups throughout the study.

Haematology was conducted in 10 rats/sex/group prior to the start, at 3 months, at 6 months and at the end of the study. There were no treatment related changes in red blood cell variables, or in total- or differential white blood cell counts. Prothrombin time was slightly increased in mid- and high dose females after 3 months, and in high-dose females at the end of the study.

Clinical chemistry was conducted in 10 rats/sex/group prior to the start, at 3 months, at 6 months and at the end of the study. A number of changes in clinical chemistry variables was observed in the high-dose group, but they were inconsistent and were not considered to be a direct consequence of exposure to .gamma.-cyclodextrin.

Urinalysis was conducted in 10 rats/sex/group at 3 months, at 6 months and at the end of the study. No treatment-related changes in urinary volume and density were observed in a renal concentration test. There were no differences among the groups in urinary electrolytes, semi-quantitative observations in the urine or in the microscopy of the urinary sediment.

The absolute and relative weight of the filled caecum was slightly increased in males of the high-dose group. The relative weight of the testes was increased in males of the high-dose group.

Macroscopic- and microscopic examination did not reveal any treatment related abnormalities.

It was concluded that .gamma.-cyclodextrin was tolerated without any specific adverse effect at all dose levels tested. Therefore, the no-observed-adverse-effect level (NOAEL) of .gamma.-cyclodextrin in the present study is placed at the highest dose level, viz. 20% in the diet. This dietary level was equal to an overall intake of 8.7 and 10.8 g/kg body weight/day for males and females, respectively.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

8 700 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Klimisch 1

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

.GAMMA.-CYCLODEXTRIN

The oral toxicity of .gamma.-cyclodextrin was examined in a chronic (12 months) toxicity study with groups of 20 male and 20 female Wistar rats. The test substance was incorporated in the feed of the rats at constant levels of 0% (control), 5%, 10% and 20%. These dietary levels were equal to an overall intake (time-weighted average) of 2.1/2.7, 4.3/ 5.4 and 8.7/10.8 g/kg bw/day for males/ females of the low-, mid- and high-dose group, respectively. General condition and behaviour were not adversely affected by the test substance. Results of neurobehavioural observations did not indicate any neurotoxic potential of the test substance. There were no test substance related deaths during the study. Ophthalmoscopic examination did not reveal any treatment-related changes. There were no noticeable differences in mean body weights, food intake or food conversion efficiency among the groups. Water consumption tended to be decreased in all treatment groups throughout the study. Haematology was conducted in 10 rats/sex/group prior to the start, at 3 months, at 6 months and at the end of the study. There were no treatment related changes in red blood cell variables, or in total- or differential white blood cell counts. Prothrombin time was slightly increased in mid- and high dose females after 3 months, and in high-dose females at the end of the study.

Clinical chemistry was conducted in 10 rats/sex/group prior to the start, at 3 months, at 6 months and at the end of the study. A number of changes in clinical chemistry variables was observed in the high-dose group, but they were inconsistent and were not considered to be a direct consequence of exposure to .gamma.-cyclodextrin. Urinalysis was conducted in 10 rats/sex/group at 3 months, at 6 months and at the end of the study. No treatment-related changes in urinary volume and density were observed in a renal concentration test. There were no differences among the groups in urinary electrolytes, semi-quantitative observations in the urine or in the microscopy of the urinary sediment.

The absolute and relative weight of the filled caecum was slightly increased in males of the high-dose group. The relative weight of the testes was increased in males of the high-dose group. Macroscopic- and microscopic examination did not reveal any treatment related abnormalities.

It was concluded that .gamma.-cyclodextrin was tolerated without any specific adverse effect at all dose levels tested. Therefore, the no-observed-adverse-effect level (NOAEL) of .gamma.-cyclodextrin in the present study is placed at the highest dose level, viz. 20% in the diet. This dietary level was equal to an overall intake of 8.7 and 10.8 g/kg body weight/day for males and females, respectively.

 

The oral toxicity of .gamma.-cyclodextrin was examined in a sub-chronic (90day) feeding study with four groups of four male and four female beagle dogs, which received the test substance in the diet at dose levels of 0 (control), 5, 10, or 20% for a period of 3 months.

Body weights and food consumption were recorded throughout the study at weekly intervals. Ophthalmoscopic observations were made in all dogs in week 0 and 13. Blood was collected prior to the start of the study and in week 6 and 12 for routine haematological and clinical chemical investigations. In week 13, urinalyses were carried out. All animals were killed in week 14, and subjected to a thorough gross examination, and several organs were weighed. Detailed histopathological examination of a great number of organs and tissues was performed on all dogs. During the last phase of the administration period, the male dogs of the 20% group gained less weight than did the controls, which was accompanied by a somewhat lower food intake in this group. Diarrhoea, caecal enlargement and increased acidity of the urine, which occurred in dogs fed 20% .gamma.-cyclodextrin, are probably a result of intestinal fermentation of unabsorbed test substance. Diarrhoea and caecal enlargement were observed also in the 10% group. However, no treatment-related histopathological changes were detected in the caecum. No treatment-related changes were noted in behaviour and appearance of the dogs, survival or haematology. Some isolated changes observed in the clinical chemistry values were not considered to be of toxicological significance.

The relative weights of the liver in males and of the ovaries in females showed dose-related increases in the 10 and 20% diet groups. Since there wase no evidence of histopathological changes in these organs, the increased weights were not considered of toxicological importance.

Gross examination at autopsy did not reveal treatment-related changes. Microscopically, no changes were found that could be related to the administration of the test substance. The results of the present study failed to show obvious toxic effects of .gamma.-cyclodextrin at dietary levels up to 20%. The only changes considered to be treatment-related, were slight caecal enlargement associated with diarrhoea and increased acidity of the urine. These findings are considered to represent an adaptive response since they were not associated with any histopathological alteration. All other changes were considered of no toxicological significance.

 

Justification for classification or non-classification

The chronic oral treatment with .gamma.-cyclodextrin did not induce signs of adverse effects in rats. It can be concluded that the test substance .alpha.-cyclodextrin shows no signs of toxicity up to dietary levels of 20% .alpha.-cyclodextrin which is equal to an overall intake of 8.7 and 10.8 g/kg body weight/day for males and females, respectively. The read-across substance .beta.-cyclodextrin showed no adverse effects or signs of toxicity in a chronic study in dogs up to 50.000 ppm. The data are conclusive but not sufficient for classification.