Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
three-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study report

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994
Reference Type:
publication
Title:
THE REPRODUCTIVE AND DEVELOPMENTAL TOXICITY PROFILE OF BETA·CYCLODEXTRIN IN RODENTS
Author:
PAUL C. BARROW, PHILlPPE OUVIER and DANIEL MARZlN
Year:
1995
Bibliographic source:
Reproductiuve Toxicology, 9 (4), 389- 398, 1995

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Similar to OECD 416 but three generations observed

US FDA : toxicological principles for the Safety Assessment of direct food additives and calor additives used in food, Bureau of foods, 1982.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
· Identification: beta-cyclodextrin.
· Supplier: ROQUETTE FRERES.
· Appearance : white crystalline powder.
· Storage conditions : at ambient temperature, away from light.
· Quantity received : 140 kg.
· Date of receipt : 15 January 1991.
· Expiry date: not applicable, stable.

Test animals

Species:
rat
Strain:
other: Ico: OFA. SD (lOPS Caw)
Sex:
male/female
Details on test animals and environmental conditions:
- Supplier: IFFA CREDO, Les Oncins - BP 0109/69592 L' ARBRESLE CEDEX, FRANCE
· Number of animals allocated to the study (P generation) : 240 (30 males and 30 females per group).
· Age at initiation of treatment : males : approximately 6 weeks, females : approximately 9 weeks.
· Body weight range of the animals at initiation of treatment : males: 158 to 197 g, females : 213 to 260 g.
The animals were housed in a single room per phase in an air-conditioned building (building K1/K2, barrier protected unit).
· temperature: 19 to 25°C (target range),
· relative humidity: 35 to 75 % (target range),
· air changes: minimum 8 air changes/hour,
· lighting : 12 hours light (artificial)/12 hours dark.
During the course of the study the environmental temperature occasionally increased or decreased by up to 2°C outside of the target range for short periods of time (up to 4 hours). Likewise, the relative humidity occasionally increased or decreased by up to 7 %: These minor deviations were not considered to have influenced the outcome of the study.

· Caging:
premating periods: 5 animals per cage - grid-floor metal cages (555 x 350 x 200 mm), mating period : one male and one female per cage - polycarbonate cages (cages MI : 365 x 225 x 180 mm) ; gestation and lactation one female (and its litter) per cage - polycarbonate cages (MI) with sterilized sawdust os bedding (analysed quarterly for chemical contaminants) ; after weaning from 3 to 6 weeks of age: male or female litter-mates in the same cage - grid-floor metal cages (260 x 350 x 200 mm).

Diet : commercial powdered complete rodent diet ref. A04C10 (Usine d' Alimentation Ration-nelle, 7 rue Gallienì Villemoisson, 91360 Epinay sur Orge, France) analysed for the absence of chemical and bacteriological contaminations. Distributed od libitum. Batch numbers 01017 and 01130 were used before the start of treatment.
Water : filtered (0.2 µm) mains water available from an automatic watering system or water bottles ad libitum. The water is analysed twice yearly for its chemical and bacteriological quali-ties by the "Service d'Hygiene et de Sante de 10 ville ae Lyon"

Animal health procedures : clinical examination for ill health on arrival.
Acclimatisation period : 7 days -for males and 8 days for females.
Allocation to treatment groups : performed during acclimatisation period using stratified randomisation accord-ing to body weight.
The F1 and F2 generation rats were randomly selected within groups at or shortly after
weaning from the Flb and F2b litters. Where possible, one pup of each sex was selected per litter.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
· Reason for the route of administration : route of human exposure.
· Method of administration : admixture in the powdered diet.


DIET PREPARATION
Method : the test article was prepared in a form suitable for admixture with the basic powdered diet at the appropriate concentrations (assuming 100 % purity). . The test article was formulated every four weeks or more frequently as necessary, depending on the quantity required. It was stored at temperate room (<=. 15°C) before use. Stability of the test article in the vehicle at least 3 months
Details on mating procedure:
Each male was housed with one female, avoiding sibling matings, for a maximum cohabitation period of two weeks. Mating was detected by daily examination of the females for the presence of sperm in the vaginal smear. The day on which sperm were observed was designated day 0 of gestation.
Smearing of individual females ceased when sperm were found or at the end of the 14 day mating period.
Two weeks after weaning and termination of the first litter, the animals of each generation were repaired, where possible in the original mating combinations, to produce a second litter. The P and F2 generations were then repaired again in a similar manner tor a third mating.
The selected pups which formed the F1 and F2 generations were mated for the first time at least 10 weeks after selection.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The following samples were taken from the first diet formulation for the study :
1 sample (125 ml) of control diet;
3 samples (3 x 125 ml) of each dose concentration : one each from the top, middle and bottom of the mixer.
In addition, samples of each formulation of diet prepared for each group were taken and sent to the Sponsor. Chemical analysis for test article content was the responsibility of the Sponsor.
Duration of treatment / exposure:
The P males and the P females were dosed continuously for 10 and 2 weeks respectively before mating, throughout the mating period and up to necropsy. The F1 and F2 males and females were treated continuously until necropsy.
Frequency of treatment:
daily (diet)
Details on study schedule:
- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
Doses / concentrations
Remarks:
Doses / Concentrations:
P: 5 %, 2.5 %, 1.25 % and 0% in feed (3599/5697, 1758/3217, 874/1610, 0/0 mg/kg bw/day [males/females]), F1/F2: 1.25 %, 0.62 % and 0.31 %
Basis:
nominal conc.
No. of animals per sex per dose:
30 P generation, 25 in F1 and F2 generation
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: in previous toxicology studies in the rat no toxic effects were evident at a maximum dietary administration level of 10 %. The high dose level, of 5 %, for this study was considered to be the maximum administrable level for reproduction studies without incurring possible nutritional deficiency. The dose levels were reduced after compilation of the results for the first generation in order to ensure the identification of a no effect level.
- Rationale for animal assignment (if not random): random
Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
All adults were examined t\Nice daify at the beginning and at the end of the working day (including weekends and public holidays) to detect any which were dead or moribund.
All animals were examined at least once daily to detect any abnormalities in appearance, behaviour or signs of reaction to treatment. Towards the end of the gestation, pregnant females were examined twice daily for signs of parturition.
Individual body weights of the females were recorded :
· weekly during premating periods,
· on days 0, 7, 14 and 20 of gestation,
· on days 1, 4, 7, 14 and 21 of lactation.
. Individual body weights of the males were recorded weekly.

Food consumption was calculated weekly for males and for females during premating periods.
Five females with viable foetuses from each sub -group were used for the following
investigations :
· The pups were sacrificed after weighing on day 14 post-partum.
· The following day, maximal milk samples were collected from the lactating females. The samples were immediately deep frozen prior to dispatch in dry ice to the Sponsor for possible analysis.
· Blood samples were collected in dry tubes from the (non-fasted) adult females before
sacrifice on day 15 of lactation.
· One sample of serum per female was immediately deep frozen prior to dispatch in dry ice to the Sponsor for possible analysis.
Another blood sample was analysed at the testing facility for the following clinical
chemistry parameters : Sodium, Potassium, Chloride, Calcium, Inorganic phosphorus, Glucose, Blood urea nitrogen, Total cholesterol, Triglycerides, Total bilirubin, Total protein

Oestrous cyclicity (parental animals):
The following data were recorded for each female following each mating, as
appropriate :
· any abnormal mating behaviour
· date of mating
· date of parturition
· duration of gestation
· abnormalities of nesting or nursing behaviour
· number of implantation sites (at necropsy).
Sperm parameters (parental animals):
not examined
Litter observations:
For each female allowed to litter the following data were recorded
· number of pups born (live and dead)
· external abnormalities of the pups
· number, weight and sex of pups on days 1, 4, 7, 14 and 21 post-partum
· physical development of the offspring, as assessed by the intra -litter onset and duration of pinna unfolding, incisor eruption and eye opening.
· functional tests in two male and two female pups per litter as follows :
. pupillary reflex and auditory response on day 21 post-patium.
· external and necropsy findings of dead pups.
On day 4 post-partum the size of each litter was adjusted to 10 pups by eliminating extra pups by random selection to yield where possible 5 males and 5 females per litter. Following the third mating period, the F3 females were submitted to a caesarean examination.

P - F1c - ADDITIONAL LITTER EXAMINATIONS
This phase was performed to further investigate the observed effects on pup growth during the previous lactation phases. New data available to the Sponsor indicated that the test article mixed with the diet could prevent· absorption of lipid -soluble vitamins from the gastrointestinal tract. Vitamin D deficiency was suspected to be responsible for the observed effects on pup growth.
The mated females from each treatment group were divided into tvvo sub-groups. The P females of one sub-group from each treatment group were given 500 IU of vitamin D3 (B.O.N. -. Laboratoire DOMS-ADRIAN solute alcoolique) diluted 1/80 in 90 % ethanol administered by the intraperitoneal route on day 2 of lactation. The P males were sacrificed after completion of the mating period.
Food consumption of females was calculated for the following periods :
· gestation : days 0 to 7, 7 to 14, 14 to 20,
· lactation: days 1 to 4, 4 to 7, 7 to 14 and 14 to 21.
Food consumption was not· recorded :
· for parental males and females during mating periods,
· for parental males, during gestation and lactation of females.
Postmortem examinations (parental animals):
Surviving animals were killed and necropsied according to the following schedule :
Necropsy of parental animals
The parental animals were subjected to gross necropsy including an examination of all external surfaces. The males were sacrificed after completion of the last mating period and the females were killed after completion of the P - F1c, F1 - F2b or F2 - F3c examinations. The F2 females were sacrificed, where possible on day 20 post-coitum. Females that failed to mate during their final mating period were killed approximately 3 weeks after the last day of pairing and necropsied. P and F1 females that failed to produce a viable litter by day 26 +/- 1 post-coitum following the second mating, were killed and necropsied. The adult animals were killed by carbon dioxide inhalation followed by exsanguination.
ORGAN WEIGHTS OF PARENTAL ANIMALS
The epididymides and testes were weighed for all males. The paired organs were weighed together after dissection of fat and other contiguous tissues
.
ORGAN/TISSUE PRESERVATION AND HISTOPATHOLOGY
The following organs/tissues were sampled for all parental animals :
Coagulating gland; Epididymides; Ovaries; Pituitary; Prostate; Seminal vesicles; Testes; Uterus + cervix; Vagina; All macroscopic lesions.
Histopathology examinations were performed for all organs and tissues sampled in the control and high dose groups except for gross lesions for which diagnosis was not judged necessary by the pathologist.
Postmortem examinations (offspring):
Offspring
Dead and culled pups and all offspring sacrificed at weaning were subjected to a
macroscopic examination.
Organs showing macroscopic alterations were preserved but no histological examination
of the preserved tissues was performed in the first instance
NECROPSY
After the third mating oeriod. the F2 females were killed on day 20 post-coitum, in random group order, by carbon dioxide anaesthesia followed by exsanguination. They were submitted to a full macroscopic examination. Any abnormalities observed were recorded.
The ovaries and uteri were removed and examined and the following data were recorded: · pregnancy status, · number of corpora lutea, ··individual foetal weigths, · foetal sex.
number and distribution of intrauterine implantations classified as .
- implantation scar from previous pregnancy,
- live foetus,
- dead foetus,
- early intrauterine deaths,
- late intrauterine deaths,

Each foetus was examIned for external defects and all live foetuses were killed by an intraperitoneal injection of sodium pentobarbitone. Dead foetuses were examined externally and preserved but not examined further. Approximately one half of each litter was examined for visceral abnormalitIes according to the standard operating procedures of the testing facility then eviscerated. The eviscerated foetal carcasses were fixed In
ethanol and processeti for skeletal examination.

The skeletal examinations were performed followiving digestion of the soft tissues with aqueous potassium hydroxide, staining of the skeleton with Alizarin red then passage into glycerol. The remaining foetuses were preserved in Bouin' s fluid for fixed visceral examination (modified Wilson-Barrow technique). Fixed-foetal examinations were performed "blind" (ie with the operator unaware of the identity of the litter) under low power magnification.
Statistics:
ANOVA and Dunnett's, ANOVA and t-test; ANOVA regression and Dunnett's, Kruskal- Wallis Terpstra - Jonckheere and Wilcoxon, Cochran - Armitage
and Fisher lrwin
Reproductive indices:
Pre-coital interval (in days); Insemination index; Fecundity index; Fertility index; Pre- birth loss; Postimplantation loss; Preimplantation loss
Offspring viability indices:
Gestation index, Live birth index, Viability indices; Weaning index; Sex ratio (proportion of male pups);

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

P-Generation:
One male from the group given 2.5 % beta -cyclodextrin was found dead during the 17th week of treatment : necropsy examination revealed 0 mass on the spleen and small nodules on the liver. All other P generation males in all groups survived to termi-nal sacrifice. A control female was sacrificed on day 2 of lactation during the third (P - F1c) mating phase following a traumatic injury. One female from the group treated with 1.25 % beta-cyclodextrin died giving birth during the third mating phase. Necropsy examination revealed dystocia. One female given the high dose level of 5 % died on day 18 of lactation during the first (P - F1a) mating phase: the cause of death could not be determined. Another high dose female died one week after weaning of the F1a litter, necropsy and histopathological examinations revealed raised areas on the lungs with moderate congestion and pale areas on the heart with slight infiltration of mononuclear cells. None of these deaths were considered likely to be treatment-related. The isolated nature of these findings did not suggest an effect of treatment. Clinical condition of the P generation mares and females was not adversely affected by treatment with beta-cyclodextrin. The three groups of males treated with beta-cyclodextrin showed a reduced rate of body weight gain during the first mating phase ; this difference showed a dose relationship during the first 8 weeks of treatment. The resulting differences in mean body weight of the P males with respect to the control group were still evident at terminal sacrifice after 33 weeks of treatment. Body weight profiles of the treated P generation females before mating and during all three gestation phases were superior or comparable with the control values in all groups. During the first (P - F1 a) mating phase the group of females given the high dose level of 5 % showed a slight, but statistically significant, reduction in mean body weight during lactation by comparison with the control group : the groups given the lower dose levels of 1.25 and 2.5 % were not affected. This difference was not observed during the two subsequent (P - F1 band P - F1 c) mating phases. Only equivocal inter-group differences in food consumption were observed for males and for females during each of the pre-mating and gestation periods.
The groups of P females given 5 % beta-cyclodextrin consumed slightly less food than the other groups during all three lactation periods, particularly during the last week. Mat-ing performance and fertility were not adversely affected by treatment with beta-cyclodextrin at any of the dose levels employed for the three mating phases of this generation. All treated males and females mated and produced viable pups during at least two out of three mating phases. Pregnancy rate, and gestation length, were comparable in all groups for all phases. Overall, when the three mating phases were considered, the incidence of stillbom pups was slightly higher in the three groups treated with beta-cyclodextrin than in the control group. These data did not show a dose-relationship, however, and the observed differences were generally caused by one or two -isolated females in the treated groups or by unusually low control incidences. Live litter sizes in the treated groups were comparable with the concurrent and historical control values for all three phases. Pup viability and clinical condition from birth to weaning and pup sex ratio were not adversely influenced by treatment. The rate of pup weight gain from birth until weaning showed a dose-related decrease during the first two mating phases with the resulting differences generally attaining statistical significance by comparison with the control group from days 7 or 14 post-partum. Only a marginal reduction was ob-served in the low dose group. During the final mating phase pup growth was reduced in the high dose group but was not significantly affected in the intermediate and low dose groups. The effect was not reversed by the administration of vitamin D. Pup develop-ment, as assessed by the day of occurrence of pinna unfolding incisor eruption and eye opening, and also by testing for auditory and pupil reflexes at weaning, was not adversely influenced by treatment during any of the three mating phases. All abnormalities found at necropsy examination of the F1a, F1b and F1c pups were incidental or minor in all groups. Necropsy and histopathological examinations and organ weights of the P generation adults did not reveal any indications of treatment-related toxicity. Cumulative pre-birth loss, determined by comparison of the total number of pups born for each phase of this generation with the number of uterine implantation sites found at necropsy of the females, did not show any treatment-related trends. The highest test article in-takes relative to body weight for females occurred during the last week of the first lacta-tion phase for groups 2 and 3 and during the same week of the second lactation phase for group 4; they were 2914, 5726 and 10123 mg/kg/day respectively. The values attained during gestation ranged between 735 and 1015 mg/kg/day in group 2, 1520 and 2052 mg/kg/day In group 3 and 3101 and 3968 mg/kg/day in group 4. The maximum intakes for males occurred during the first week of treatment and were 1614, 3160 and 6255 mg/kg/day in groups 2, 3 and 4 respectively.

Effect levels (P0)

Key result
Dose descriptor:
NOEL
Effect level:
5 other: % (3599/5697 mg/kg bw/day [males/females])(1.25% for F1 and F2 after reduction of the dose)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: fertility, reproductive performance, in utero foetal development or physical pup development
Remarks on result:
other: Generation: all (migrated information)

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

F1 GENERATION
One male from the intermediate dose group was found dead 5 days after successfully mating (ie. 4 weeks after the dose level was reduced to 0.62 %). The cause of death could not be determined at necropsy. There were no other deaths of the F1 animals in any group. No treatment-related changes in clinical condition v/ere observed in the F1 males and females of any group. The F1 males and females in the groups given beta-cyclodextrin showed a reduced mean body weight by comparison with the control group after selection from the P - F1b litters; the difference, however was minor in the low and intermediate dose groups. Body weight gain during the next 7 weeks was reduced in the high dose group treated with 5 % beta-cyclodextrin but was comparable with the control group in the groups treated with 1.25 and 2.5 %. Following the reduction of the dose levels during week 8 the high dose group, now given 1.25 % beta -cyclodextrin, showed an accelerated rate of body weight gain such that the initial differences in mean body weight with respect to the control group were essentially recuperated. Thereafter, body weight profiles for the males and females during the gestation and lactation periods of both mating phases were comparable in all groups. The animals given the high dose level, of 5 % beta-cyclodextrin consumed less food than the control group during the first five weeks for males and the first seven weeks for females. A similar but much less marked reduction was also seen in the groups given the lower dose levels of 1.25 % and 2.5 % during the first two weeks. After reduction of the dose levels mean food con-sumption was comparable in all groups during the pre-mating, gestation and lactation periods of both mating phases. Two pairs of animals from each of the intermediate and high dose groups failed to mate during the first phase and three pairs from the low dose group and one pair from the high dose group failed to mate during the second phase. With the exception of one low dose female which did not give birth during either phase but had uterine implantation scars at necropsy, all pairs of animals produced viable off-spring during at least one of the two phases. Pre-coital interval was not adversely af-fected by treatment. One low dose female underwent total litter loss at birth during the second littering phase, otherwise, all females that gave birth raised viable pups to wean-ing. Gestation length was similar in all groups. The mean number of pups born was high (at least 14) in all groups following both matings. The incidence of stillborn pups was comparable in all groups. Pup viability from birth to weaning was not adversely influ-enced by treatment. The clinical condition of the litters was normal in all groups. Pup sex ratio did not show any treatment -related effects. Pup weight at birth before and af-ter covariance adjustment for litter size was marginally superior in the treated groups than in the control group for both littering phases. Thereafter, pup weight gain from birth to weaning tended to be marginally superior in the groups given beta-cyclodextrin but the resulting differences with respect to the control group were. minimal and did not at-tain statistical significance. Pup development, as assessed by the day of occurrence of pinna unfolding, incisor eruption and eye opening, and also by testing for auditory and pupil reflexes at weaning, was not adversely influenced by treatment.
Necropsy examination of the surviving pups after weaning revealed one intermediate dose pup from the second mating with agenesis of one kidney and another intermediate dose pup with occlusion of the colon. The isolated nature of these findings did not sug-gest an effect of treatment. All other abnormalities found at necropsy examination of the F2a and F2b pups were incidental or minor in nature in all groups. Necropsy and histo-pathological examinations and organ weights of the F1 generation adults did not reveal any indications of treatment-related toxicity. Cumulative pre-birth loss, determined by comparison of the total number of pups born for each phase of this generation with the number of uterine implantation sites found at necropsy of the females, did not show any treatment-related trends. The maximum weekly test article intake in males and females before mating occurred immediately after selection of the animals to form the F1 gen-eration and was approximately 2000, 4000 and 8000 mg/kg/day in groups 2, 3 and 4 respectively. The highest levels after reduction of the dose levels occurred for females during the first lactation period and were 714, 1498, and 2892 mg/kg/day in groups 2, 3 and 4 respectively. The values attained during gestation ranged between 195 and 246 mg/kg/day in group 2, 399 and 490 mg/kg/day in group 3 and 781 and 996 mg/kg/day in group 4.
F2 GENERATlON
One F2 male from each of the intermediate and high dose groups died after completion of the second (F2 - F3b) mating phase. One female from each of the low and intermedi-ate dose groups died before the first mating period, one of these (intermediate dose) was found trapped under the food hopper. None of these animals had any significant pathological abnormalities. No treatment-related changes in clinical condition were ob-served in the F2 mates and females of any group. Body weight profiles of the F2 males and females during all pre-mating, gestation and lactation periods were essentially simi-lar in all groups. Food consumption of the F2 males and females during all pre-mating, gestation and lactation periods was comparable in aft groups.
With the exception of one intermediate dose female, which either failed to mate or failed to become pregnant during all three mating phases, all surviving F2 males and females were fertile. The incidences of animals failing to mate and of females that copulated but failed to become pregnant during each mating phase did not indicate any adverse ef-fects of treatment. Pre-coital interval was not influenced by treatment during any of the three mating phases. All pregnant females gave birth to live pups during both littering phases. Gestation lengths were comparable in all groups. The mean numbers of pups born were high (at least 14) in all groups and were not adversely influenced by treat-ment. The incidence of stillborn pups was slightly higher than the control values in the intermediate and high dose groups during the first littering phase and in the high dose group only for the second phase. The overall incidences, however, were low in all groups and the individual data did not suggest an effect of treatment. During the second littering phase one high dose female gave birth to 15 stillborn pups and one live pup that died the following day. In addition, one female from each of the intermediate and high dose groups underwent total litter loss post-partum during one of the two littering phases. Pup survival from birth to weaning was similar or superior to that of the control group in all beta –cyclodextrin treated groups for both littering phases. Pup weight at birth was marginally superior in the treated groups than in the control group for both lit-tering phases : statistical significance was not attained. Thereafter, pup weight gajn from birth to weaning by comparison with the control group was comparable or margin-ally superior in the groups given beta-cyclodextrin. During the second mating phase a. slight increase in mean pup weight in the low dose group attained statistical significance for female pups on day 14 post-partum and for male and female pups at weaning. Pup development, as assessed by the day of occurrence of pinna unfolding, incisor eruption and eye opening, and also by testing for auditory and pupil reflexes at weaning, was not adversely influenced by treatment. All abnormalities found at necropsy examination of the treated F3a and F3b pups were incidental or minor in nature in all groups. Necropsy and histopathological examinations and organ weights of the F2 generation adults did not reveal any indications of treatment-related toxicity. Cumulative pre-birth loss, deter-mined by comparison of the total number of pups born for each phase plus the number of live foetuses at caesarean with the number of uterine implantation sites found at necropsy of the females, did not show any treatment-related trends. Implantation data re-corded at terminal caesarean examination did not show any treatment-related trends. One female from each of the control and low dose groups contained one or more late resorptions with no live foetuses. The incidences of total, early and late resorptions and post-implantation loss were tower in all treated groups than in the control group. Live litter size, mean foetal weight and foetal sex ratio were not adversely influenced by treatment. Fresh, fixed soft tissue and skeletal examinations of the F3c foetuses did not reveal any evidence of teratogenicity. The control group contained two malformed foe-tuses compared with one in each of the treated groups. The affected control foetuses had gastroschisis and a cardiac defect with marked generalised oedema respectively ; the low and intermediate dose foetuses both had gastroschisis and the high dose foetus had marked generalised oedema. The types and incidences of foetuses with minor soft tissue or skeletal abnormalities did not indicate any adverse effects of treatment with beta-cyclodextrin. The maximum weekly test article intake in males and females before mating occurred immediately after selection of the animals to form the F2 generation and were 477, 949 and 1891 mg/kg/day in groups 2. 3 and 4 respectively. The values attained during the three gestation periods and both lactation periods were comparable with those recorded during the previous (Fl) generation treated with the some dose levels.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
1.25 other: %
Sex:
male/female
Basis for effect level:
other: physical pup development

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The highest dietary concentration of 5 % beta-cyclodextrin tested in this study caused a slight reduction in perinatal pup growth. The highest no adverse effect level, identified under the defined experimental conditions of this study was 1.25 %. No adverse effects on fertility, reproductive performance, in utero foetal development or physical pup development were found with any of the dietary concentrations of beta-cyclodextrin tested in this study.
Executive summary:

Beta-cyclodextrin (> 99% pure) was administered by admixture in the diet at dose levels of 5 %, 2.5 % and 1.25 % P: 5 %, 2.5 %, 1.25 % and 0% in feed (3599/5697, 1758/3217, 874/1610, 0/0 mg/kg bw/day [males/females of P generation])to groups of 30 male and 30 female Ico: OFA. SD (IOPS Caw) rats (groups 4, 3 and 2 respectively). The parental (P) generation males and females were maintained on the treated diets for ten and two weeks respectively before pairing and during the gestation and lactation periods of three successive mating periods. A similar group of rats received the untreated basal diet over the same periods and served as a control group. Two subsequent generations, comprised of 25 males and 25 females, randomly selected from the F1b and F2b litters, were treated with dietary concentrations of 1.25 %, 0.62 % and 0.31 % of beta-cyclodextrin (groups 4, 3 and 2 respectively). The dose levels were reduced three weeks before mating of the F1 generation in order to confirm the definition of a no effect level.

The offspring from the third mating of the P generation (P - F1c) were used to investigate the effects of vitamin D supplementation on pup growth. 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 foetuses. 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 the pups. Non-selected pups were submitted to a necropsy examination on or soon after day 21 post partum.At terminal necropsy the adult males and females of each generation were given a detailed macroscopic examination ; selected organs were weighed and selected tissues were examined histopathologically.

P-Generation:

One male from the group given 2.5 % beta -cyclodextrin was found dead during the 17th week of treatment : necropsy examination revealed 0 mass on the spleen and small nodules on the liver. All other P generation males in all groups survived to terminal sacrifice. A control female was sacrificed on day 2 of lactation during the third (P - F1c) mating phase following a traumatic injury. One female from the group treated with 1.25 % beta-cyclodextrin died giving birth during the third mating phase. Necropsy examination revealed dystocia. One female given the high dose level of 5 % died on day 18 of lactation during the first (P - F1a) mating phase: the cause of death could not be determined. Another high dose female died one week after weaning of the F1a litter, necropsy and histopathological examinations revealed raised areas on the lungs with moderate congestion and pale areas on the heart with slight infiltration of mononuclear cells. None of these deaths were considered likely to be treatment-related. The isolated nature of these findings did not suggest an effect of treatment. Clinical condition of the P generation mares and females was not adverselyaffectedby treatment with beta-cyclodextrin. The three groups of males treated with beta-cyclodextrin showed a reduced rate of body weight gain during the first mating phase ; this difference showed a dose relationship during the first 8 weeks of treatment. The resulting differences in mean body weight of the P males with respect to the control group were still evident at terminal sacrifice after 33 weeks of treatment. Body weight profiles of the treated P generation females before mating and during all three gestation phases were superior or comparable with the control values in all groups. During the first (P - F1 a) mating phase the group of females given the high dose level of 5 % showed a slight, but statistically significant, reduction in mean body weight during lactation by comparison with the. control group : the groups given the lower dose levels of 1.25 and 2.5 % were not affected. This difference was not observed during the two subsequent (P - F1 band P - F1 c) mating phases. Only equivocal inter-group differences in food consumption were observed for males and for females during each of the pre-mating and gestation periods.

The groups of P females given 5%beta-cyclodextrin consumed slightly less food than the other groups during all three lactation periods, particularly during the last week. Mating performance and fertility were not adversely affected by treatment with beta-cyclodextrin at any of the dose levels employed for the three mating phases of this generation. All treated males and females mated and produced viable pups during at leasttwoout of three mating phases. Pregnancy rate, and gestation length,werecomparable in all groups for all phases. Overall, when the three mating phases were considered, the incidence of stillbom pups was slightly higher in the three groups treated with beta-cyclodextrin than in the control group. These data did not show a dose-relationship, however, and the observed differences were generally caused by one or two -isolated females in the treated groups or by unusually low control incidences. Live litter sizes in the treated groups were comparable with the concurrent and historical control values for all three phases. Pup viability and clinical condition from birth to weaning and pup sex ratio were not adversely influenced by treatment. The rate of pup weight gain from birth until weaning showed a dose-related decrease during the first two mating phases with the resulting differences generally attaining statistical significance by comparison with the control group from days 7 or 14 post partum.Only a marginal reduction was observed in the low dose group. During the final mating phase pup growth was reduced in the high dose group but was not significantly affected in the intermediate and low dose groups. The effect was not reversed by the administration of vitamin D. Pup development, as assessed by the day of occurrence of pinna unfolding incisor eruption and eye opening, and also by testing for auditory and pupil reflexes at weaning, was not adversely influenced by treatment during any of the three mating phases. All abnormalities found at necropsy examination of the F1a, F1b and F1c pups were incidental or minor in all groups. Necropsy and histopathological examinations and organ weights of the P generation adults did not reveal any indications of treatment-related toxicity. Cumulative pre-birth loss, determined by comparison of the total number of pups born for each phase of this generation with the number of uterine implantation sites found at necropsy of the females, did not show any treatment-related trends.

F1 GENERATION

One male from the intermediate dose group was found dead 5 days after successfully mating (ie. 4 weeks after the dose level was reduced to 0.62 %). The cause of death could not be determined at necropsy. There were no other deaths of the F1 animals in any group. No treatment-related changes in clinical condition v/ere observed in the F1 males and females of any group. The F1 males and females in the groups given beta-cyclodextrin showed a reduced mean body weight by comparison with the control group after selection from the P - F1b litters; the difference, however was minor in the low and intermediate dose groups. Body weight gain during the next 7 weeks was reduced in the high dose group treated with 5%beta-cyclodextrin but was comparable with the control group in the groups treated with 1.25 and 2.5%.Following the reduction of the dose levels during week 8 the high dose group, now given 1.25 % beta -cyclodextrin, showed an accelerated rate of body weight gain such that the initial differences in mean body weight with respect to the control group were essentially recuperated. Thereafter, body weight profiles for the males and females during the gestation and lactation periods of both mating phases were comparable in all groups. The animals given the high dose level, of 5 % beta-cyclodextrin consumed less food than the control group during the first five weeks for males and the first seven weeks for females. A similar but much less marked reduction was also seen in the groups given the lower dose levels of 1.25%and 2.5 % during the first two weeks. After reduction of the dose levels mean food consumption was comparable in all groups during the pre-mating, gestation and lactation periods of both mating phases. Two pairs of animals from each of the intermediate and high dose groups failed to mate during the first phase and three pairs from the low dose group and one pair from the high dose group failed to mate during the second phase. With the exception of one low dose female which did not give birth during either phase but had uterine implantation scars at necropsy, all pairs of animals produced viable offspring during at least one of the two phases. Pre-coital interval was not adversely affected by treatment. One low dose female underwent total litter loss at birth during the second littering phase, otherwise, all females that gave birth raised viable pups to weaning. Gestation length was similar in all groups. The mean number of pups born was high (at least 14) in all groups following both matings. The incidence of stillborn pups was comparable in all groups. Pup viability from birth to weaning was not adversely influenced by treatment. The clinical condition of the litters was normal in all groups. Pup sex ratio did not show any treatment -related effects. Pup weight at birth before and after covariance adjustment for litter size was marginally superior in the treated groups than in the control group for both littering phases. Thereafter, pup weight gain from birth to weaning tended to be marginally superior in the groups given beta-cyclodextrin but the resulting differences with respect to the control group were. minimal and did not attain statistical significance. Pup development, as assessed by the day of occurrence of pinna unfolding, incisor eruption and eye opening, and also by testing for auditory and pupil reflexes at weaning, was not adversely influenced by treatment.

Necropsy examination of the surviving pups after weaning revealed one intermediate dose pup from the second mating with agenesis of one kidney and another intermediate dose pup with occlusion of the colon. The isolated nature of these findings did not suggest an effect of treatment. All other abnormalities found at necropsy examination of the F2a and F2b pups were incidental or minor in nature in all groups. Necropsy and histopathological examinations and organ weights of the F1 generation adults did not reveal any indications of treatment-related toxicity. Cumulative pre-birth loss, determined by comparison of the total number of pups born for each phase of this generation with the number of uterine implantation sites found at necropsy of the females, did not show any treatment-related trends. The maximum weekly test article intake in males and females before mating occurred immediately after selection of the animals to form the F1 generation and was approximately 2000, 4000 and 8000 mg/kg/day in groups 2, 3 and 4 respectively. The highest levels after reduction of the dose levels occurred for females during the first lactation period and were 714, 1498, and 2892 mg/kg/day in groups 2, 3 and 4 respectively. The values attained during gestation ranged between 195 and 246 mg/kg/day in group 2, 399 and 490 mg/kg/day in group 3 and 781 and 996 mg/kg/day in group 4.

F2 GENERATlON

One F2 male from each of the intermediate and high dose groups died after completion of the second (F2 - F3b) mating phase. One female from each of the low and intermediate dose groups died before the first mating period, one of these (intermediate dose) was found trapped under the food hopper. None of these animals had any significant pathological abnormalities. No treatment-related changes in clinical condition were observed in the F2 mates and females of any group. Body weight profiles of the F2 males and females during all pre-mating, gestation and lactation periods were essentially similar in all groups. Food consumption of the F2 males and females during all pre-mating, gestation and lactation periods was comparable in aft groups.

With the exception of one intermediate dose female, which either failed to mate or failed to become pregnant during all three mating phases, all surviving F2 males and females were fertile. The incidences of animals failing to mate and of females that copulated but failed to become pregnant during each mating phase did not indicate any adverse effects of treatment. Pre-coital interval was not influenced by treatment during any of the three mating phases. All pregnant females gave birth to live pups during both littering phases. Gestation lengths were comparable in all groups. The mean numbers of pups born were high (at least 14) in all groups and were not adversely influenced by treatment. The incidence of stillborn pups was slightly higher than the control values in the intermediate and high dose groups during the first littering phase and in the high dose group only for the second phase. The overall incidences, however, were low in all groups and the individual data did not suggest an effect of treatment. During the second littering phase one high dose female gave birth to 15 stillborn pups and one live pup that died the following day. In addition, one female from each of the intermediate and high dose groups underwent total litter losspost-partumduring one of the two littering phases. Pup survival from birth to weaning was similar or superior to that of the control group in all beta –cyclodextrin treated groups for both littering phases. Pup weight at birth was marginally superior in the treated groups than in the control group for both littering phases : statistical significance was not attained. Thereafter, pup weight gajn from birth to weaning by comparison with the control group was comparable or marginally superior in the groups given beta-cyclodextrin. During the second mating phase a. slight increase in mean pup weight in the low dose group attained statistical significance for female pups on day 14post-partumand for male and female pups at weaning. Pup development, as assessed by the day of occurrence of pinna unfolding, incisor eruption and eye opening, and also by testing for auditory and pupil reflexes at weaning, was not adversely influenced by treatment. All abnormalities found at necropsy examination of the treated F3a and F3b pups were incidental or minor in nature in all groups. Necropsy and histopathological examinations and organ weights of the F2 generation adults did not reveal any indications of treatment-related toxicity. Cumulative pre-birth loss, determined by comparison of the total number of pups born for each phase plus the number of live foetuses at caesarean with the number of uterine implantation sites found at necropsy of the females, did not show any treatment-related trends. Implantation data recorded at terminal caesarean examination did not show any treatment-related trends. One female from each of the control and low dose groups contained one or more late resorptions with no live foetuses. The incidences of total, early and late resorptions and post-implantation loss were tower in all treated groups than in the control group. Live litter size, mean foetal weight and foetal sex ratio were not adversely influenced by treatment. Fresh, fixed soft tissue and skeletal examinations of the F3c foetuses did not reveal any evidence of teratogenicity. The control group contained two malformed foetuses compared with one in each of the treated groups. The affected control foetuses had gastroschisis and a cardiac defect with marked generalised oedema respectively ; the low and intermediate dose foetuses both had gastroschisis and the high dose foetus had marked generalised oedema. The types and incidences of foetuses with minor soft tissue or skeletal abnormalities did not indicate any adverse effects of treatment with beta-cyclodextrin. The maximum weekly test article intake in males and females before mating occurred immediately after selection of the animals to form the F2 generation and were 477, 949 and 1891 mg/kg/day in groups 2. 3 and 4 respectively. The values attained during the three gestation periods and both lactation periods were comparable with those recorded during the previous (Fl) generation treated with the some dose levels.

 

 

The initial high dose level of 5 % beta-cyclodextrin caused minimal toxicity in the parental males only, characterised by a minor reduction in body weight gain. This dose level also caused a slight reduction in the rate of growth of the pups of the treated dams. A corresponding reduction in maternal food consumption during lactation was observed, but the magnitude of this difference was considered insufficient to account for the effect on pup growth. The available evidence would suggest a maternally-mediated nutritional influence arising from the physical and chemical nature of the test article rather than a direct toxic effect on the litter. Beta-cyclodextrin is intended for use in food as a carrier of smaller lipid-soluble molecules. It is possible that the observed effects in this study were caused by the test article in the diet encapsulating or otherwise interfering with the absorption of lipid-soluble vitamins or nutrients from the gastrointestinal tract. In the present study, however, the effect was not prevented by vitamin D supplementation, suggesting the involvement of another phenomenon.

Administration of the lower initial dose levels, of 2.5 % and 1.25 %. resulted in only minimal differences in pup growth. No clear no effect level was determined with these doses, but all of the observed differences with respect to the control group could be regarded as both minimal and equivocal. Further investigations on the two subsequent generations (each composed of two or three mating phases) did not reveal any indications of treatment-related toxicity with dose levels of 1.25 % or lower. Therefore. after consideration of all of the data generated during this study, the dose level of 1.25 % was considered to represent a no adverse effect level.

The highest dietary concentration of 5 % beta-cyclodextrin tested in this study caused a slight reduction in perinatal pup growth. However, there were no subsequent adverse influences on the reproductive performance and development of the two subsequent generations derived from the affected litters, which were then continuously treated with 1.25 % beta-cyclodextrin. Moreover, the affected offspring of the first generation generally recovered the difference in body weight with respect to the control group after reduction of the dietary concentration to 1.25%.

The highest no adverse effect level, identified under the defined experimental conditions of this study was 1.25 % (874/1610 mg/kg bw/day for males/females of parenteral generation). No adverse effects on fertility, reproductive performance in utero foetal development or physical pup development were found with any of the dietary concentrations of beta-cyclodextrin tested in this study.