ß-CYCLODEXTRIN First draft prepared by Ms F D Pollitt Health Aspects of the Environment and Food (Medical) Division Department of Health London, England Explanation Biological data Toxicological studies Short-term toxicity studies Long-term toxicity/carcinogenicity studies Reproductive toxicity studies Special studies on interactions with bile acids and lipophilic nutrients Comments Evaluation References 1. EXPLANATION ß-Cyclodextrin was previously evaluated at the forty-first meeting of the Committee (Annex 1, reference 107), at which time a temporary ADI of 0-6 mg/kg bw was allocated, based on a NOEL of 2.5% in the diet (equal to 1230 mg/kg bw/day) in study in dogs and a safety factor of 200. The results of an ongoing 1-year oral toxicity study in dogs and information on the effects of ß-cyclodextrin on the bioavailability of lipophilic nutrients were required by 1995. These data, along with the results of a 1-year toxicity study in rats, a 3-generation reproductive toxicity study in rats and carcinogenicity studies in mice and rats were available for review at the present meeting, and are summarized in the following monograph addendum. 2. BIOLOGICAL DATA 2.1 Toxicological studies 2.1.1 Short-term toxicity studies 2.1.1.1 Rats In a 52-week study, groups of 20 male and 20 female CrI:CD(SD)BR rats were fed diets containing 0, 1.25, 2.5 or 5% ß-cyclodextrin (> 99% pure). Treatment commenced at 6 weeks of age. The rats were housed 5 to a cage and food and water were available ad libitum. Animals were observed daily for clinical signs. Body weight and food intake were recorded weekly. Haemato-logical (including RBC, WBC, haemoglobin and haematocrit), plasma biochemical (including protein, ASAT, ALAT, ALP, gamma-GT, sodium, potassium, calcium and triglycerides) and urine analyses (including pH and protein) were undertaken during weeks 13, 26, 39 and prior to termination at 52 weeks. Food was withdrawn overnight prior to sampling and water was also withdrawn overnight prior to urine sampling. Major organs were weighed. At termination, full macroscopic and microscopic examination of tissues and organs was undertaken. During the course of the study, 4 deaths were recorded, 3 males from the control group and 1 female which had received diet containing 1.25% ß-cyclodextrin. None of these deaths were attributed to treatment. No abnormal clinical signs were observed. Since the dietary concentration was fixed throughout the study period, the intakes of ß-cyclodextrin, expressed as mg/kg bw/day, decreased. The intakes of ß-cyclodextrin (mg/kg bw/day) at the beginning and end of the study are shown in Table 1. Table 1. Variation of intake in the 52-week study (HRC, 1994a) % ß-Cyclodextrin Intake, mg/kg bw/day in diet Male Female Week 1 Week 52 Week 1 Week 52 1.25 1506 395 1593 565 2.5 3292 797 3227 1146 5 6106 1639 6358 2391 In males, intake had decreased in week 52 to approximately 25% of that at week 1 and, in females, to approximately 33%. The mean intake in the 1.25% group over the course of the study was 650 mg/kg bw/day and 860 mg/kg bw/day in male and female rats, respectively. There was no statistically significant effect of treatment on body weight during the course of the study, although the body-weight gains in the high-dose males and females were slightly reduced in comparison to controls (4% and 7%, respectively). No effects due to ß-cyclodextrin were observed on haematological or urinalysis parameters. In the biochemical investigations, ALAT and ASAT were increased significantly compared to controls at week 13 in both males and females in the high- and mid-dose groups. The concentrations of these enzymes continued to be increased in both males and females in these groups throughout the study except at week 39, when the increase was only observed in the high-dose females. At week 13, significant increases in OCT levels were observed in mate and female animals receiving 5% ß-cyclodextrin and in males receiving 2.5% ß-cyclodextrin. Increased OCT concentrations were also observed at other times throughout the study, but these differences were not always statistically significant when compared with control values. At week 52, significant increases were observed in males receiving 5 and 2.5% ß-cyclodextrin. Triglyceride concentrations tended to be reduced compared to controls from week 26 onwards in the high-dose male rats and from week 39 in high-dose female rats, the difference occasionally reaching statistical significance. There was a tendency to reduced ß-lipoprotein concentrations at the high dose and, occasionally, the mid dose but the biological significance of this was unclear since statistically significantly lower levels than in controls were also observed in predose high-dose animals and, in males, in mid-dose blood samples. Analysis of the urine for ß-cyclodextrin at weeks 13 and 52 demonstrated the presence of a low level of unchanged compound. Analysis of the faeces showed virtual absence of ß-cyclodextrin. Analysis of organ weights at 52 weeks revealed no statistically significant differences between treated groups and controls. No effects attributable to ß-cyclodextrin were observed at macroscopic examination of the tissues. However, histological examination revealed treatment-related changes in the liver and kidney. In the liver, significantly increased incidences of single cell necrosis, centrilobular hepatocyte enlargement and portal inflammatory cell infiltration were seen in both males and females fed 5% ß-cyclodextrin compared to controls. Females from this same dose group were also observed to have an increased incidence of focal basophilic hepatocytes. Males fed 2.5% ß-cyclodextrin had an increased incidence of portal inflammatory cell infiltration, while females of this group showed an increased incidence of single cell necrosis and focal basophilic hepatocytes. Examination of the kidneys revealed an increased incidence of pigment in the epithelium of the cortical tubules in females fed 2.5% or 5% ß-cyclodextrin, but no treatment- related changes were observed in males. No other treatment-related abnormalities were observed in any of the other tissues examined. The NOEL in this study was 1.25% in the diet, equal to 650 mg/kg bw/day (HRC, 1994a). 2.1.1.2 Dogs The oral toxicity of ß-cyclodextrin (purity >99%) was studied in beagle dogs for a period of 52 weeks. The animals were acclimatized for a minimum of 4 weeks during which time they were inoculated and received anthelmintic treatment. Following acclimatization, the dogs were divided into 4 groups each of 4 males and 4 females and were administered by inclusion in the diet, 0%, 0.62%, 1.25%, or 5.0% ß-cyclodextrin. The age of the animals at the start of treatment was 35-36 weeks and their body weights ranged from 8.4-11.8 kg. The dogs were fed once daily and had free access to water. They were housed in kennels capable of accommodating 2 animals of the same sex and dose group. However, dogs were segregated for the assessment of clinical signs and food consumption which were recorded daily. Body weights were recorded weekly. Ophthalmic, haematological (including haematocrit, haemoglobin and blood cell counts), plasma biochemical (including protein, urea, inorganic salts, ALAT, ASAT and gamma-GT) and urine (including volume, pH and protein) analyses were undertaken after 13, 26, 39 and 52 weeks of treatment. Urine and faecal samples were collected during weeks 13 and 52 for toxicokinetic purposes. Serum vitamin A, D and E were determined at 13, 26, 39 and 52 weeks while vitamins A and E were determined in aliquots of liver at termination of the study. Major organs were weighed but the caecum was not routinely weighed. A full microscopic examination of H and E stained sections was performed. Liver sections were also stained with Oil Red O (for fat) and PAS (for glycogen). There were no deaths during the course of this study. All animals receiving ß-cyclodextrin showed a slightly higher incidence of intermittent liquid faeces compared to controls. Body-weight gain in both sexes in the high-dose group was reduced in comparison with controls, although the difference was not statistically significant. One female from the high-dose group showed a weight loss of 0.9 kg from week 31, although food consumption was, for the most part, maximal. The average intakes of ß-cyclodextrin over the study period were equal to 230, 470 and 1900 mg/kg bw/day for the low-, intermediate- and high-dose groups, respectively. There was no effect of treatment on food intake, haematological measurements or the results of ophthalmic examination. There was a statistically significant increase in plasma potassium levels in females in the mid- and high-dose dogs at weeks 39 and 52 compared to controls. However, there were no differences in plasma biochemistry parameters between the control and treated groups which were considered to be conclusively related to treatment. Other differences which were statistically significant showed a lack of consistency and/or dose-relationship or could be attributed to abnormal group mean values for control dogs. Urinalysis revealed an increase in protein concentration in males fed 5.0% ß-cyclodextrin. Although at week 13 this was due to one high individual value, a trend developed over the remainder of the treatment period with 3/4 males showing elevated urinary protein concentrations at week 52. Fractionation of the proteins revealed most of the protein to be globular fractions with statistically significantly increased globulin levels usually mirroring those of increased total protein. This was considered to be an effect of treatment. There was also an increase in urinary protein concentrations in high-dose females compared to controls, achieving statistical significance at week 26, but this effect was not observed at termination of the study. Urinary calcium concentration was also increased in the high-dose male group from week 13 onwards. The same trend was also observed in females from week 13 onwards, but again was not observed at week 52. Urinary gamma-GT and N-acetyl-ß-D-glucos- aminidase (NAG) levels were measured at week 52 only. The gamma-GT level was higher than for the control group in mid- and high-dose males, but the difference was only statistically significant for the high-dose group. NAG levels were also raised in these groups compared to controls but the differences were not statistically significant nor was there a dose-related effect. No similar effect was seen in females. Toxicokinetic analysis demonstrated the presence of unchanged ß-cyclodextrin in urine and, to a lesser extent, in faeces. During the 24 h that the dogs were in the metabolism cages for collection of urine, the amount of ß-cyclodextrin excreted varied from 1.3 to 6.2% of the daily dose on week 13, and from 1.4 to 3.3% of the daily dose on week 52 (group mean data). There was no effect of treatment on serum vitamin A, D or E levels nor on vitamin A and E levels in the liver. At necropsy, group mean thymus weights in all treated male groups were statistically significantly higher than in the control group. No such effect was seen in females and no histological changes were observed in the thymus. This effect was therefore not considered to be related to treatment. In high-dose males, group mean prostate weight was statistically significantly reduced compared to the control group. No treatment-related abnormalities were recorded at histopathological examination in this or any other tissue examined at termination of this study. The Committee concluded that the NOEL in this study was 1.25%, equal to 470 mg/kg bw/day, based on urinary effects in males and the slightly reduced body-weight gain seen at the high-dose level (HRC, 1994b). 2.1.2 Long-term toxicity/carcinogenicity studies 2.1.2.1 Mice ß-cyclodextrin (purity > 86.6%) was administered in the diet to groups of Crl-CD-I(ICR)BR mice (52/sex/dose) at dose levels of 0, 25, 75, 225 or 675 mg/kg bw/day. Treatment commenced at about 5 weeks of age and continued for 93 weeks (males) or 105 weeks (females). The mice were housed 4 to a cage and food and water were available ad libitum. All animals were observed daily for clinical signs. There were no treatment-related effects on survival nor on the incidence or type of clinical signs. Body weights were recorded weekly for the first 13 weeks of treatment, biweekly until week 73 and weekly thereafter. Food consumption was measured weekly for the first 13 weeks and biweekly thereafter, and water intake on week 1 and monthly thereafter for the control and high-dose groups only. There was no effect of treatment on body-weight gain, food consumption or food conversion ratio. There was a general trend to lower water consumption by the high-dose group than by the controls, but the difference was not statistically significant. At 12 and 18 months and prior to terminal sacrifice, blood was taken from the tail of all animals and smears for differential white blood cell counts were prepared. Data were presented for the control and high-dose groups only. A number of sporadic, statistically significant differences were seen at 12 and 18 months between the control and high-dose groups, but no such differences were seen in the blood samples taken prior to terminal kill. These sporadic cases of statistically significant findings were not considered to be treatment-related. All mice dying during the course of the study, killed in moribund condition, or surviving to terminal sacrifice were subjected to a gross necropsy. In the control and high-dose groups, histopathological examination was conducted on all organs. At 25, 75 and 225 mg/kg bw/day, only abnormalities and major organs were examined histopathologically. With the exception of one high-dose male, treatment-related lesions were seen only in decedent animals. Macroscopically, these lesions comprised: fluid, soft, mucoid or foamy and yellowish contents in caecum, colon and rectum; lumen of colon distended and mucosa oedematous. Microscopically, these lesions comprised: serious atrophy of fat pads in the abdominal cavity; active desquamation of surface epithelium in the caecum, colon and/or rectum; flattening of the mucosa, atrophy of intestinal glands and/or acute catarrhal typhlitis in the caecum; and mucosa covered by mucous secretion containing exfoliated epithelial cells in the colon and/or rectum. One or more of these symptoms were seen in the following animals where death was considered to be caused by treatment: 1 male at 75 mg/kg bw/day, 3 males and 2 females at the high dose. In other animals exhibiting these symptoms (1 male at 75 mg/kg bw/day, 1 male at 225 mg/kg bw/day, 4 males and 4 females at the highest dose tested) the cause of death was considered to be an incidental spontaneous lesion. The high-dose male animal sacrificed at terminal kill and whose symptoms were considered to be treatment-related exhibited caecal mucosal flattening associated with atrophy of the intestinal glands. No other treatment-related non-neoplastic lesions were seen in this study. The NOEL based on the inflammatory changes seen in the lower gastrointestinal tract was 25 mg/kg bw/day. Among the neoplastic findings, uterine endometrial polyps occurred as follows in the female mice: 0/51 in controls, 3/48 in the low-dose group, 2/52 at 75 mg/kg bw/day, 3/52 at 225 mg/kg bw/day and 3/51 in the high-dose group. This increased incidence of uterine endometrial polyps showed no dose-related trend and it was concluded that this was not a treatment-related effect. Phaeochromocytoma of the adrenal gland was seen in 1/52 high-dose males and 1/51 high-dose females, and in 1/52 females at 75 mg/kg bw/day. No phaeochromocytomas were seen in the control animals, nor in 228 historical control animals from the same laboratory. The authors concluded that this was not a treatment-related effect. Although an increased incidence of phaeochromocytoma has been seen in rats fed polyol sweeteners which, like ß-cyclodextrin, are poorly digestible substances, they were not seen in mice fed similarly high levels of polyols. In view of the lack of association in mice and the lack of a dose-related trend in the incidence of this tumour in this study, the Committee agreed with the authors' conclusion. There were no other neoplastic findings of note (Gur et al., 1993a). 2.1.2.2 Rats ß-cyclodextrin (> 86.6% pure) was administered in the diet to groups of 50 male and female Fischer 344 rats at concentrations sufficient to provide nominal doses of 0, 25, 75,225 or 675 mg/kg bw/day. Treatment commenced at 4-5 weeks of age and continued for 122 weeks (males) or 130 weeks (females). The rats were housed 5 to a cage and food and water were available ad libitum. All animals were observed daily for clinical signs. Body weights were recorded weekly for the first 15 weeks of treatment and biweekly thereafter. Achieved dosages (mg/kg bw/day) in the first 2 weeks were only approximately half the nominal level but were close to the required level for the rest of the study. There were no treatment-related effects on survival nor on the incidence or type of clinical signs. There was no effect of treatment on body-weight gain, food consumption or food conversion ratio. Water consumption in the high-dose female rats was slightly lower than in controls throughout the study. The authors did not consider this to be a treatment-related effect. At 12, 18 and 24 months and prior to terminal sacrifice, blood was taken from the tail of all animals and smears for differential white blood cell counts were prepared. Data were presented for the control and high-dose groups only. There was no adverse effect of treatment. All rats dying during the course of the study, killed in moribund condition, or surviving to terminal sacrifice were subjected to gross necropsies. In the control and high-dose groups, histopathological examination was conducted on all organs. In the other groups, only abnormalities and major organs were examined histopathologically. There was no effect of treatment on organ weight nor on the incidence of macroscopic lesions or non-neoplastic microscopic lesions. Historical control data for the F344 rats was provided from the testing facility for both life-span studies (n = 60 of each sex) and 2-year carcinogenicity studies (n = 305 of each sex) and from the US National Toxicology Program for life-span studies (n = 529 of each sex) and 2-year studies. The incidence of a few types of neoplasm showed a trend of statistical significance with treatment but the findings were not considered to be related to treatment with ß-cyclodextrin because of the lack of a clear dose-relationship and/or because the incidence in treated groups was well within the background rate for this strain of rat. These included: testicular interstitial cell tumours, subcutaneous fibroma, and endometrial stromal polyp and sarcoma combined by disease severity. The incidence of parathyroid adenoma also reached a low trend of significance in males and in the data combined by sex (males: 0, 0, 3.6, 6.9 and 7.5%; combined: 0, 2, 2, 6.5 and 5.6% for the control, 25, 75, 225 and 675 mg/kg bw/day groups, respectively). The incidences in the 225 and 675 mg/kg bw/day dose male groups exceeded those reported for all groups of historical controls but, given the lack of a clear dose-relationship in the data combined by sex, this finding is considered unlikely to be associated with treatment. The finding of a renal neoplasms (one adenoma and one carcinoma) in two rats from the high-dose male group and none in any other group was not statistically significant. The incidence was high compared with background control data from the testing facility but within the range seen in the NTP studies. There was no increase in renal hyperplasia in treated groups (Gur et al., 1993b). 2.1.3 Reproductive toxicity studies 2.1.3.1 Rats In a 3-generation reproductive toxicity study, ß-cyclodextrin (> 99% pure) was administered in the diet at dose levels of 0, 1.25, 2.5 or 5% to groups of 30 male and 30 female Ico:OFA.SD.(IOPS Caw) rats. The parental (P) generation males and females were maintained on these diets for 10 and 2 weeks respectively before pairing and during the gestation and lactation periods of three successive mating periods. Male rats were approximately 6-week old at the start of treatment and female rats were approximately 9-week old. Two subsequent generations, comprised of 25 males and 25 females, randomly selected from the F1b and F2b litters, were treated with dietary concentrations of 0, 0.31, 0.62 or 1.25% ß-cyclodextrin: the dose levels were reduced 3 weeks before mating of the F1 generation to confirm the definition of a NOEL. The F1 and F2 generations were each mated twice and allowed to raise their offspring to weaning. The study was terminated with a third mating phase of the F2 animals with caesarean examination of the pregnant females and soft tissue and skeletal examination of the fetuses. The offspring from the third mating of the P generation (F1c animals) were used to investigate the effects of vitamin D supplementation on pup growth, because vitamin D deficiency was thought to be responsible for effects on pup growth. The mated females were divided into 2 sub-groups and 1 sub-group was given 500 IU vitamin D3 diluted 1/80 in 90% ethanol i.p. on day 2 of lactation. Milk and blood samples were taken from 5 females with viable foetuses from each sub-group on day 15 post-partum for clinical chemistry analyses. Animals were housed 5 to a cage during the premating periods, 1 male and 1 female per cage during the mating period, 1 female (and its litter) to a cage during gestation and lactation, and with all male or female litter-mates in the same cage after weaning from 3 to 6 weeks of age. Litters were culled to 10 pups each on day 4 post-partum, to yield, where possible 5 males and 5 females. Food and water were available ad libitum throughout. All animals were observed daily for clinical signs of toxicity. Body weight and food consumption were monitored during the respective pre-mating, gestation and lactation periods. Fertility and reproductive performance of the P, F1 and F2 generations were assessed by evaluation of mating performance, duration of gestation, parturition and viability, growth and development of pups. Pups which had not been selected for mating were submitted to a gross necropsy examination on or soon after day 21 post-partum. At terminal necropsy, the adult males and females of each generation were given a macroscopic examination; selected organs were weighed and selected tissues were examined histopathologically. Because ß-cyclodextrin was fed at a fixed rate in the diet, intake on a mg/kg bw/day basis varied considerably during the study. This was particularly marked in the first pre-mating period and, in females, during lactation, when intakes varied 2-3-fold within each dose group. P Generation Body-weight gains in male groups receiving ß-cyclodextrin were slightly lower than in the control group, in a dose-related manner, at most times, but the differences were not statistically significant. Conversely, body-weight gain in the high-dose female group was statistically significantly higher than in controls during the premating period but statistically significantly less during the first lactation. During the first and second lactations, females receiving 5% ß-cyclodextrin consumed statistically significantly less food than the other groups; a trend to lower food consumption was also seen at times in the 1.25 and 2.5% female groups. There was no effect of treatment on mating performance nor on gestation at any of the three matings. There were consistently more dams with stillborn young in the treated groups. However, although the proportion of pups which were stillborn was also consistently higher in the treated groups, there was no dose-relationship and this finding can be attributed to an abnormally low incidence of stillbirths in controls. Litter size overall was large, averaging 14-15 live young. Pup viability during lactation was not affected by treatment but mean pup weight was statistically significantly reduced in the high-dose group from about day 7 to day 14 post-partum until weaning at all three matings. Eye opening was slightly delayed in the high-dose group compared to controls at all three matings but this can be attributed to lower pup body weight in the second half of lactation. Other markers of pup development, i.e. the day of occurrence of pinna unfolding and incisor eruption, and testing for auditory and pupil reflexes at weaning, were not adversely affected by treatment. The administration of vitamin D on day 2 of lactation had no effect on any parameter. There were no clear differences in clinical chemistry parameters of the maternal blood and milk following treatment with ß-cyclodextrin. F1 Generation Body weights of intermediate- and high-dose animals were statistically significantly lower than controls at the start of this phase but began to recover when the doses of ß-cyclodextrin were reduced, although body weights in the high-dose group remained lower than other groups during both mating periods. Food consumption showed a similar trend although the differences from controls were only statistically significant in the high-dose group during gestation after the second mating. There was no effect of treatment on female body weight or food consumption during gestation or lactation after the first mating. After the second mating, high-dose female body weights were statistically significantly less than controls on days 1 and 14 of lactation only. There was no effect of treatment on reproductive performance or litter parameters, nor on pup viability, body-weight gain or development. F2 Generation There was no effect of treatment at any of the three matings on paternal or maternal body-weight gain or food consumption. No adverse effect was seen on reproductive performance, pup viability, body-weight gain or development. A slightly higher proportion of dams with stillbirths and of pups which were stillborn was seen in the intermediate- and high-dose groups at the first and second matings but there was no clear dose-response and this was not considered to be a treatment-related effect. The incidence of malformations and anomalies in fetuses delivered by Caesarian section after the third mating were similar in dosed and control groups. The NOEL in this study was 1.25% ß-cyclodextrin in the diet, equal to a mean achieved ß-cyclodextrin intake which ranged from 560 - 2900 mg/kg bw/day over the different phases of the study (Pharmakon Europe, 1994; and Personal communication from Dr Ph. Olivier, Roquette Frères, Lestrem, France to F. D. Pollitt, Department of Health, UK, submitted to WHO by F. D. Pollitt). 2.1.4 Special studies on interactions with bile acids and lipophilic nutrients An in vitro study was carried out, using NMR spectrometry, to evaluate interactions of ß-cyclodextrin with key bile acids, including sodium lithocholate, which are present in human bile and to study the competition between bile acids and the lipophilic vitamins A and D3 for ß-cyclodextrin inclusion. It was found that, in water solutions, sodium cholate, sodium taurocholate, sodium chenodeoxycholate and sodium lithocholate give 1:1 complexes with ß-cyclodextrin, the affinity of sodium cholate and sodium taurocholate for ß-cyclodextrin being weaker than that of sodium chenodeoxycholate or sodium lithocholate i.e. the affinity increased with an increase in the hydrophobicity of the bile acid. The complex of vitamin A with ß-cyclodextrin was shown to have a low solubility in water. When sodium cholate or sodium taurocholate entered into competition with vitamin A in the presence of a sub-stoichiometric quantity of ß-cyclodextrin, there was incomplete complexation of bile acids, confirming that competition occurs between these acids and vitamin A. However, when sodium lithocholate or sodium chenodeoxycholate were mixed with a vitamin A/ß-cyclodextrin complex in solution, the NMR spectrum of the mixture was identical to that of the lithocholate-ß-cyclodextrin complex, showing that vitamin A does not interact with ß-cyclodextrin in the presence of these bile acids. Similar results were seen with vitamin D3. It was concluded that these findings, together with the fact that the quantity of bile acids present during digestion is far higher than the ingested amounts of vitamins, indicated that depletion of lipophilic vitamins will not occur when ß-cyclodextrin is used as an ingredient in foods or orally administered drugs (Comini et al., 1994). 3. COMMENTS The Committee considered that the further toxicity studies confirmed the low systemic toxicity of ß-cyclodextrin. The NOEL in the 1-year studies in rats and dogs was 1.25% in the diet, equal to 650 and 470 mg/kg bw/day, respectively. At higher doses there were minor changes in blood biochemical and/or urine analytical parameters and, in the rat, minor necrotic and inflammatory changes in the liver and an increased incidence of pigment in conical tubular epithelium in the kidney. The NOEL was also 1.25% in the diet (equal to between 560 and 2900 mg/kg bw/day, depending on the stage of the study) in the 3-generation reproductive toxicity study in rats, in which the only adverse effect seen at higher doses was impaired pup growth during lactation, which was probably secondary to reduced food consumption and body-weight gain in the dams at this dose level. In the carcinogenicity study in mice, ß-cyclodextrin caused inflammatory changes in the lower gastrointestinal tract, which were considered to be the cause of death of some animals. The lowest dose level at which this occurred was 75 mg/kg bw/day (1/52 male affected) and the NOEL was 25 mg/kg bw/day. The Committee considered that these lesions probably represent a species-specific reaction to ß-cyclodextrin in some mice. No such effects were seen either in the carcinogenicity study in rats, in which the concentration of ß-cyclodextrin in the diet was about three times that in the diet of mice or in shorter-term studies in rats and dogs at far higher ß-cyclodextrin concentrations in the diet. No treatment-related neoplastic lesions were observed in the carcinogenicity studies. An in vitro study indicated that ß-cyclodextrin is unlikely to deplete lipophilic vitamins when used as an ingredient in food, since it has a higher affinity for bile acids than for vitamins A and D3. The fact that laboratory animals experienced no apparent nutritional deficiencies affecting clinical status or survival following lifetime exposure to high levels of ß-cyclodextrin in the diet also provides reassurance that the use of this additive will not adversely affect the nutritional status of humans. 4. EVALUATION The Committee allocated an ADI of 0-5 mg/kg bw for ß-cyclodextrin, based on the NOEL of 1.25% in the diet (equal to 470 mg/kg bw/day) in the 1-year study in dogs and a safety factor of 100. 5. REFERENCES COMINI, S., OLIVIER, P., RIOTTOT, M., & DUHAMEL, D. (1994). Interaction of ß-cyclodextrin with bile acids and their competition with vitamins A and D3 as determined by 1H-NMR spectrometry. Clinica Chimica Actam, 228: 181-194. GUR, E., NYSKA, A., & WANER, T. (1993a). ß-cyclodextrin: Oncogenicity study in the mouse by dietary administration. LSRI project no. CHS/066/BCD. Unpublished report from Life Science Research Israel Ltd, Ness Ziona 70 451, Israel. Submitted to WHO by Roquette Frères, Lestrem, France. GUR, E., NYSKA, A., & WANER, T. (1993b). ß-cyclodextrin: Oncogenicity study in the rat by dietary administration. LSRI project no. CHS/065/BCD. Unpublished report from Life Science Research Israel Ltd, Ness Ziona 70 451, Israel. Submitted to WHO by Roquette Frères, Lestrem, France. HRC (1994a). Beta-cyclodextrin: Toxicity to rats by dietary administration for 52 weeks. Unpublished report no. ROQ 4/931090 from Huntingdon Research Centre Ltd, Huntingdon, Cambridgeshire, UK. Submitted to WHO by Roquette Frères, Lestrem, France. HRC (1994b). Beta-cyclodextrin: Toxicity to dogs by repeated dietary administration for 52 weeks. Unpublished report no. ROQ 3/931848 from Huntingdon Research Centre Ltd, Huntingdon, Cambridgeshire, UK. Submitted to WHO by Roquette Frères, Lestrem, France. Pharmakon Europe (1994). Beta-cyclodextrin: Three generation oral (dietary administration) reproduction toxicity study in the rat. Study no. 430/006. Unpublished report from Pharmakon Europe, L'Arbresle, France. Submitted to WHO by Roquette Frères, Lestrem, France.
See Also: Toxicological Abbreviations Beta-Cyclodextrin (WHO Food Additives Series 32) beta-CYCLODEXTRIN (JECFA Evaluation)