Cyclooctapentylose

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

three-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

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

Species:

rat

Strain:

other: Ico: OFA. SD (lOPS Caw)

Sex:

male/female

Details on test animals or test system 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.

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.

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

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);

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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.

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)

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)

Dose descriptor:

NOEL

Effect level:

1.25 other: % (874/1610 mg/kg bw/day [males/females])

Sex:

male/female

Basis for effect level:

other: reduction in perinatal pup growth and weight gain in males

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

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.

Reproductive effects observed:

not specified

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:

For this endpoint the read-across approach based on grouping of substances (category approach) was used.

The read-across substance .beta.-cyclodextrin 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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

874 mg/kg bw/day

Species:

rat

Quality of whole database:

Klimisch 1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

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.

Short description of key information:
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.

Justification for selection of Effect on fertility via oral route:
OECD Guideline study

Effects on developmental toxicity

Description of key information

On the basis of the results obtained it can be concluded that no effects on prenatal development were found in fetuses from rabbits fed .alpha.-cyclodextrin at levels up to 20 % (4900 - 7000 mg/kg bw) in the diet. 

Link to relevant study records

Reference

Endpoint:

developmental toxicity

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 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Virgin female New Zealand White rabbits obtained from ENKI Konijnenfarm, Someren, the Netherlands arrived on February 12, 1991 when they were about 6 months old.
Additional male New Zealand White rabbits, also obtained from ENKI Konijnenfarm, arrived May 29, 1990 and 15 males arrived October 23, 1990.
The last batch of males was used to replace some of the males of the first batch that appeared insufficient as supplier of motile sperm cells. The age of the males at arrival was about 26 weeks.

Upon arrival, all animals were housed and checked for overt signs of ill health and anomalies. An acclimatization period of 21 days in the study room was observed for the female rabbits. The body weights of the females at the start of the study (day 0 of gestation) ranged from 3248 to 4445 g.
Before the start of the study the females were weighed three times, on day 3, 10 and 17 of the acclimatization period. On basis of weight gain and mean body weight, the required number of female rabbits was selected for the study and assigned at random to the different groups. These data are available as raw data.

The remaining rabbits were kept in reserve, but were not used in the study. The female rabbits were housed individually in suspended galvanized cages (55 x 45 x 35 cm) fitted with wire mesh floors and fronts. The males were housed individually in PVC cages. Each cage was provided with a coloured card showing the animals identification number, the cage number, the group letter and the study number. Each rabbit was identified by a unique identification number. The number of the females was tattooed in the ears, the males were marked with felt pen in the ears. Housing conditions were conventional. The temperature in the animal room was 17.5 to 25 °C. The relative humidity generally fluctuated between 30 and 80 %. 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 (on: 07.30 h, off: 19.30 h), 12 hours dark. Before arrival of the animals the study room and cages were cleaned and disinfected. Room and excrement-collecting trays were cleaned weekly. No other test system was housed in the same room during the study.

Diets and drinking-water
From the arrival of the rabbits till the end of the study, feed and water were provided ad libitum. Tap-water was supplied in glass bottles which were cleaned and filled weekly. The bottles were checked daily, and replenished when necessary. All animals were maintained on pelleted basal diet during the acclimatization period. During the treatment period, control rabbits were given basal diet containing 20 % wheat starch, and rabbits of the test groups were fed the same diets containing .gamma.-CD at several levels, or lactose, incorporated at the expense of wheat starch. Homogeneity was obtained by mixing in a mechanical blender for about 2 minutes. All diets were pelletized after adding 5 % molasses and water 1:1. Three batches of the diets were prepared, which were stored-at 4 °c until use. The pelleted diets were provided in food hoppers. Food intake was measured by weighing the food hoppers.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

5, 10, 20 % .gamma.-cyclodextrin; 20% lactose

Details on mating procedure:

The females were artificially inseminated with about 40 million motile sperm cells, freshly obtained by "dummy" copulations of male breeders of proven fertility. Subsequently ovulation was induced by intravenous injection of a luteinizig hormone: 1 ml/kg body weight of a solution in saline, containing 50 IU/ml. The semen of one male was used for one or two females per day. If necessary, semen samples were pooled.

Duration of treatment / exposure:

30 days

Frequency of treatment:

daily

Duration of test:

50 days

Remarks:

Doses / Concentrations:
0, 5, 10, 20 % .gamma.-cyclodextrin; 20% lactose
Basis:
nominal in diet

No. of animals per sex per dose:

16 females per dose

Control animals:

yes, plain diet

other: 20% lactose

Details on study design:

The experiment was started on March 5 and lasted until April 23, 1991.

Maternal examinations:

Analyses of the test substance in the carrier:
In short, in batches prepared on March 4 and 5, 1991 the distribution of the test substances in the diets were checked, and the stability of the test substance was determined after a storage period of 7 and 28 days at 20°C and of 28 days at 4°C. Content of the test substances in the diets was determined in the batches prepared on March 4, 5, 21, and also in the batch prepared on April 5, 1991.

Clinical signs:
The general condition and behaviour of all animals were checked daily. Any sign of ill health or reaction to the treatment was recorded. During the treatment period the females were checked twice a day, viz. once in the morning and once in the afternoon, except in the weekends, when they were only checked in the morning.

Insemination of the females:
The females were artificially inseminated with about 40 million motile sperm cells, freshly obtained by "dummy" copulations of male breeders of proven fertility. Subsequently ovulation was induced by intravenous injection of a luteinizig hormone: 1 ml/kg body weight of a solution in saline, containing 50 IU/ml. The semen of one male was used for one or two females per day. If necessary, semen samples were pooled.
The day of insemination was considered to be day 0 of pregnancy.

Body weight:
During the study, females body weights were recorded on day 0, 6, 12, 19 and 29 of gestation.

Food consumption:
The quantity of food consumed by each female of each group was determined over the periods from day 0 - 6, 6 - 19 and 19 - 29 of gestation.

Water consumption:
The consumption of water was not recorded.

Intake of test substance:
The intake of the test substance per kg body weight is calculated on the basis of the nominal dietary level of the test substance, the food intake
and the mean body weight in the corresponding week.

Ovaries and uterine content:

The uterus and ovaries were removed for further examination.

Fetal examinations:

The fetuses were removed from the uterus, dried of amniotic fluids and examined grossly, upon which they were further processed.

Statistics:

Data handling and statistics:
All pairwise comparisons were two-tailed. Group mean differences with an associated probability of less than 0.05 were considered to be statistically significant.
Mean group values and SEM (standard error of the mean) were calculated from the individual means.

Adult data
Body weights and food consumption of the female rabbits were subjected to one-way analysis of (co-)variance (ANOVA), with the initial weights as co-variables, followed by Dunnett's Multiple Comparison test.

Mating data:
The mating data were calculated for the females from each group and compared by Fisher's exact probability test. The fertility index was calculated as follows:
fertility index = [(no. of pregnant rabbits)/ (no. of rabbits inseminated)] x 100.

Day 29 sacrifice data:
Numbers of corpora lutea, implantation sites, resorptions, and live and dead fetuses were subjected to one-way analysis of variance (Kruskal-Wallis), where necessary followed by the Mann-Whitney U-test. Resorption was classified "early" when only placental tissue was visible, and "late" when placental as well as embryonic tissue were visible at caesarian section.
The following indices were calculated for each litter and the results were analysed for each group by Kruskal-Wallis test followed by the Mann Whitney U-test:
pre-implantation loss (%) = (a-b)/a x 100
post-implantation loss (%) = (b-c)/b x 100
where a = number of corpora lutea
b = total number of implantation sites
c = number of live fetuses

Mean fetal body weights and lengths, and placenta weights were calculated for each litter and subjected to ANOVA + Dunnett-test. Visceral and skeletal anomalies were analysed by the Fisher's exact probability test.

Indices:

no data

Historical control data:

no data

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Maternal body weight:
Mean maternal body weight during gestation did not differ between the test groups and the control group.
Mean maternal body weight change of the 20 % .gamma.-CD group was statistically significantly different in the first week of gestation, but comparable in the other weeks of gestation. This observation was considered to be treatment-related.

Maternal food consumption and substance intake:
Maternal food consumption during gestation was similar in most groups, but it was statistically significantly reduced in the 20 % .gamma.-CD group during the first week of gestation. In the following weeks it remained slightly reduced, but the difference did not any more reach the level of statistical significance.
Substance intake expressed as gram per kg body weight per day ranged from 1.6-2.1 g/kg bw/d for the 5 % .gamma.-CD group, 2.9-4.2 g/kg bw/d for the 10 % .gamma.-CD group, 4.9 to 7.0 g/kg bw/d for the 20 % .gamma.-CD group and 5.4 to 8.6 g/kg bw/d for the lactose group.

Maternal necropsy findings:
All females were killed at the scheduled date. Upon necropsy no observations were made that could be related to the treatment.
Autopsy revealed a swollen, discoloured spleen and nephrotic kidneys in 1 animal of the control group and bilaterally ovary cysts in 1 animal of the 5 % .gamma.-CD group. In 2 animals, 1 of the 10 % .gamma.-CD and 1 of the 20 % .gamma.-CD group the uterus was found to be filled with haemorrhagic fluid. In 1 animal of the control group, the uterus was filled with pus. In 2 animals, 1 of the 20 % .gamma.-CO group and 1 of the 20 % lactose group, the uterus had a red appearance.

Maternal performance:
During gestation, 1 female of the control group and 1 of the 10 % .gamma.-CD group had an abortion.
At necropsy, a sufficient number of animals appeared to be pregnant, the fertility index ranging from 69 % to 94 %. All pregnant animals had live fetuses.

Dose descriptor:

NOAEL

Effect level:

10 other: % in the diet

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

> 20 other: % in the diet

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Abnormalities:

not specified

Developmental effects observed:

not specified

Reproduction findings, organ weights and litter data:

The mean number of corpora lutea was similar in all groups, as was the mean number of implantation sites and the mean number of life fetuses.

Pre- and postimplantation loss were variable, but the values did not reach the level of statistical significance in any of the groups. The number of dead fetuses and early and late resorptions were similar in all groups. The male/female ratio fluctuated around 50% for all groups. No statistically significant differences were observed in mean gravid and empty uterus weight and mean ovary weight.

 

Macroscopic examination of fetuses:

Placenta weights, fetal length and fetal body weights were similar in all groups, also when calculated per sex. Upon examination of the placentas of the control group and high dose groups, the incidence of placental cysts in the 20% lactose group was found to be higher than in the control group. This difference reached the level of statistical significance when the incidence was calculated per litter. The incidence of placental focal necrosis was distributed evenly amongst the groups. One fetus with a dysmature appearance was observed in a litter of the 20% lactose group.

 

Microscopic examination of fetuses: Visceral and skeletal examination

Visceral malformations:

Visceral malformations found at screening included the absence of the spleen, agenesis of testes and epididymides, absence of the subclavian artery, perforation of the dorsal aorta and a glassy spot in the common carotic artery. All this concerned single observations, occurring in a single group. None of the fetal visceral malformations was considered to be related to the treatment.

Visceral anomalies:

Visceral anomalies of the head, examined in all fetuses of the control, 20% .gamma.-CD group and the lactose group, included a folded retina or a soft lens, and in the brain dilatation of the lateral ventricles, haemorrhage between the lateral ventricles and meningial haemorrhage. All incidences were low. Visceral anomalies of the trunk, examined in all fetuses of all groups, included a pericard filled with haemorrhagic fluid, a small spleen, hyperfissured liver, round kidney, double gallbladder, haemorrhagic urinary bladder, small ovaries, cryptorchism and an enlarged testis. All incidences were very low.

The incidence of haemorrhagic fluid in the abdominal cavity reached the level of statistical significance in the 5% .gamma.-CD group, 20% .gamma.-CD group (also a statistically significantly high number of litters) and in the 20%lactose group. This increased incidence of haemorrhagic fluid in the abdominal cavity of fetuses might be due to the method of preservation of the fetuses after excision at -20°C. No definite opinion can be given on the relevance of this observation. However, since there were no other observations that could be considered as treatment-related and, moreover, it is very unlikely that a substance causes only one, very specific effect on foetal development, the toxicological relevance of the observed abdominal haemorrhages is at least questionable and probably of no significance.

Visceral variations:

Visceral variation included haemorrhagic nasal conchae in 2 fetuses in the 20% .gamma.-CD group. The next variations were observed infrequently, and were distributed about equally over the groups: pale spleen, pale or spongy liver, enlarged, small or absent gallbladder, narrow urinary bladder, bent ureter, pale pancreas, haemorrhagic salivary glands and crooked fingers. The incidence of bent ureter was statistically significantly lower in the 20% .gamma.-CD group when compared to the control group; this was considered a fortuitous finding.

Minor variations in the aortic-arch area were observed in about half of the fetuses, but their occurrence was similar in all groups.

None of the visceral variation described is considered to be related to the treatment.

Skeletal malformations:

No skeletal malformations were found in the control group or any of the dose groups.

Skeletal anomalies:

The skeletal anomalies observed in the control group and high dose groups included reduced, dislocated, separated or fused sternebra and crooked hind phalanges. The incidence of all observations was about equal in the groups examined, apart from the observation concerning one dislocated sternebra that occurred statistically significantly less often in fetuses of the 20%lactose group. The total incidence of fetal skeletal anomalies was statistically significantly reduced in the 20% .gamma.-CD group and the 20%lactose group; this was considered not to be related to the treatment.

Skeletal variations:

The skeletal variations observed in the control group and high dose groups included the infrequently occurring supernumerary sternebrae and accessory cervical ribs. The incidence of accessory lumbar ribs was higher, occurring in about half of the fetuses observed. All observations were distributed about equally amongst the groups, and were considered not to be related to the treatment.

Variation in ossification of fetal skeletons:

Variations in ossification (incomplete or absent ossification) was found in the following bones: skull, sternebrae, ribs, metacarpals and phalanges. Most observations were present in equal quantities in both the control and high dose groups. In the 20% lactose group statistically significantly more front phalanges were ossified than in the control group, and the total incidence of variations in ossification of fetal skeletons also was statistically significantly reduced in the 20% lactose group. These observation are considered not to be related to the

treatment.

Conclusions:

Dietary administration of .gamma.-CD or lactose at levels up to 20 % of the diet from day 0 to 29 of gestation caused few adverse effects on pregnant rabbits and their fetuses.
2 animals of the 10 % .gamma.-CD group and 3 animals of the 20 % .gamma.-CD group had diarrhoea, during a few days of the study only.
During the first days of the treatment period (day 0-6 of pregnancy) body weight gain was statistically significantly decreased in the 20 % .gamma.-cyclodextrin group. No other differences in body weight gain and mean body weight were observed.
Food intake was not affected in most groups, apart from the .gamma.-cyclodextrin groups from day 0-6 of pregnancy. This effect was probably due to the palatibity of the high dose diets. None of the maternal necropsy findings was considered to be related to the treatment. Fertility index and other parameters did not show any treatment-related effects. The reproduction findings and litter data did not reveal any treatmentrelated effects.
At microscopic examination of the fetuses no visceral or skeletal malformations were observed that could be related to the treatment. In fact, skeletal examination did not reveal any finding, either major or minor, that could be related to the treatment.

On the basis of the results obtained it can be concluded that:
- No effects on prenatal development were found in fetuses from rabbits fed .gamma.-cyclodextrin at levels up to 20 % in the diet.
- Based on the decreased food intake during the first 6 days of treatment the no-adverse effect level of .gamma.-cyclodextrin for maternal effects is 10 % in the diet.

Executive summary:

The purpose of this study was to establish the embryo/fetotoxicity and teratogenicity potential of .gamma.-cyclodextrin in rabbits. In this study .gamma.-cyclodextrin was administered to mated female rabbits (16 animals per dose group) incorporated in the diet at the expense of wheat starch at levels of 0, 5 %, 10 % and 20 % and 20 % lactose from day 0 up to and including day 29 of gestation.

The study was started on March 5, 1991 and terminated on April 23, 1991.

During the study no mortality occurred in any of the test groups or in the controls.

During the first days of the treatment period (day 0-6 of pregnancy) body weight gain was statistically significantly decreased in the 20 % .gamma.-cyclodextrin group. No other differences in body weight gain and mean body weight were observed.

Food intake was not affected in most groups, apart from both the 20 % .gamma.-cyclodextrin groups from day 0-6 of pregnancy.

No statistically significant differences were observed in the maternal performance and reproduction parameters between any of the groups.

Upon macroscopic fetal examination no test substance-related defects were observed.

Upon microscopic fetal examination no test substance-related visceral and skeletal malformations or anomalies were observed that could be related to the treatment with .gamma.-cyclodextrin.

On the basis of the results obtained it can be concluded that:

- No effects on prenatal development were found in fetuses from rabbits fed .gamma.-cyclodextrin at levels up to 20 % in the diet.

- Based on the decreased food intake during the first 6 days of treatment the no-adverse effect level of .gamma.-cyclodextrin for maternal effects is 10 % in the diet.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

4 900 mg/kg bw/day

Species:

rabbit

Quality of whole database:

Klimisch 1

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The purpose of the study was to establish the embryo/fetotoxicity and teratogenicity potential of .gamma.-cyclodextrin in rabbits. In this study .gamma.-cyclodextrin was administered to mated female rabbits (16 animals per dose group) incorporated in the diet at the expense of wheat starch at levels of 0, 5 %, 10 % and 20 % and 20 % lactose from day 0 up to and including day 29 of gestation. During the study no mortality occurred in any of the test groups or in the controls. During thefirst days of the treatment period (day 0-6 of pregnancy) body weight gain was statistically significantly decreased in the 20 % .gamma.-cyclodextrin group. No other differences in body weight gain and mean body weight were observed.Food intake was not affected in most groups, apart from both the 20 % .gamma.-cyclodextrin groups from day 0-6 of pregnancy. No statistically significant differences were observed in the maternal performance and reproduction parameters between any of the groups. Upon macroscopic fetal examination no test substance-related defects were observed. Upon microscopic fetal examination no test substance-related visceral and skeletal malformations or anomalies were observed that could be related to the treatment with .gamma.-cyclodextrin. On the basis of the results obtained it can be concluded that:

- No effects on prenatal development were found in fetuses from rabbits fed .gamma.-cyclodextrin at levels up to 20 % in the diet.

- Based on the decreased food intake during the first 6 days of treatment the no-adverse effect level of .gamma.-cyclodextrin for maternal effects is 10 % in the diet.

 

An oral embryotoxicity/ teratogenicity study with .gamma.-cyclodextrin (.gamma.-CD) was carried out in pregnant Wistar rats. These test substances were administered to mated female rats (25 animals per dose group) by incorporation into a powdered diet at dose levels of 1.5%, 5%, 10% and 20% (w/w) from day 0 of gestation up to and including day 21 of gestation. Data obtained for the groups of rats fed .gamma.-CD were compared with a control group (fed powdered diet only) and with another control group fed 20% lactose in the diet. On day 21 of gestation the female rats were killed. In the present study administration of .gamma.-CD at dose levels up to 20% (w/w) in the diet did induce toxicologically adverse effects in the dams. No effects were observed on reproductive performance in any of the treatment groups. Upon macroscopical examination of the foetuses no effects were found that could be attributed to the test substances. Upon visceral examination of the foetuses only one treatment-related effect could be detected that was attributed to one of the test substances: a significantly increased incidence of renal pelvic cavitation was found in foetuses from rats fed 20% lactose. No adverse effects with respect to skeletal malformations, anomalies, variations or retardations could be found in any of the treated groups that could be attributed to the test substances. On basis of the results obtained in the present study it can be concluded that for rats, treated under the conditions described in this report:

- No effects with respect to maternal toxicity were found in pregnant rats fed .gamma.-CD at levels up to 20% in the diet.

- No effects on prenatal development were found in foetuses from rats fed .gamma.-CD at levels up to 20% in the diet.

Justification for selection of Effect on developmental toxicity: via oral route:
OECD Guideline study

Justification for classification or non-classification

Conclusive but not sufficient for classification.