Cycloheptapentylose

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guiodeline study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

One hundred and forty-five male and one hundred and forty-three female Specific Pathogen Free rats, four weeks old (± 1 day) (CrJ: CR(SD) BR VAF/Plus strain) were received from Charles River Portage, Michigan, USA. Not more than four males or four females were used on the study from the same litter. An additional five males and five females were received for health check purposes. On arrival all animals were examined for abnormalities and for signs of overt ill health. Those designated as health check animals were killed within 24 hours after arri-val at HRC and subjected to routine macroscopic examination. Any abnormalities seen were processed immediately and examined microscopically. Lungs, liver, kidneys, spleen and heart were preserved in fixative, but not processed further. The remaining animals were weighed on arrival (males 65 - 98 g. females 54 - 95 g). After an acclimatisation period of one week they were weighed again and using these weights males only were assigned to four groups by com-puterised stratified randomisation to give approximately equal initial group mean bodyweights and to ensure that each litter sibling was allocated to different groups. A further acclimatisation period of 7 days was allowed before commencement of treatment. At the end of the 7th week of treatment for males, females were weighed and assigned to four groups as described for males, with treatment commencing after a further 7 days. Following allocation to groups, the animals were ear marked to give individual identification: for females prior to allocation, identification was made using an ink tail mark which was checked at least weekly and renewed as necessary.
Animal room controls for temperature and relative humidity were set at 21°C and 55% respec-tively; slight daily fluctuations in temperature and relative humidity were considered acceptable and continuous chart recordings of these data are retained with the other raw data. Lighting was controlled to give 12 hours light (8 am to 8 pm) and 12 hours dark per 24 hours. During the pre-mating period, males and females were housed separately, four to a cage. Cages of males were interspersed amongst those holding females to promote development of regular oestrous cycles (for F0 generation animals this only applied for the 3 weeks prior to the 1st mating). In addition, the cages constituting each treatment group were dispersed within the batteries so that possible environmental influences arising from their spatial distribution were equilibrated as far as possible for all treatments. Suspended galvanised metal cages (Bowman®) equipped with solid sides and back and wire mesh front, floor and top were used during the pre-mating. period except during the first week of the FIAgeneration when animals were housed in plastic cages (North Kent Plastics, RM-2 type). During the mating period, animals were housed on the basis of one male to one female in plastic breeding cages (North Kent Plastics, RM-2 type). At the end of the mating period the males where, possible were rehoused with their former cage mates in clean metal cages (Bowman®) and the females were housed in individual breeding cages (North Kent Plastics, RM-2 type) for the birth and rearing of young or for the sacrifice of animals on Day 20 of pregnancy. Suitable nesting material was provided. Throughout the study each cage was identified by a label coloured according to group and recording study schedule num-ber, animal number details of treatment and the name of the Study Supervisor and Study Direc-tor. All animals were given free access to diet and tap water.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

The test material. Beta-cyclodextrine was administered in the diet. Control animals received the basal diet throughout. The test material as supplied was weighed out and added to a weighed amount of sieved Biosure Laboratory Diet No. 2 and stirred with a mixer to give a pre-mix of suitable strength. The dietary concentrations required were obtained from this pre-mix by direct dilution with further quantities of diet. homogeneity being achieved by mixing for 7 minutes in a rotary double cone blender.
Fresh diets were prepared weekly and fed for no more than 14 days from the date of prepara-tion and stored at ambient temperature in labelled opaque bags until used. The dietary inclusion levels remained constant throughout the study. It was not the intention of this study to determine evidence of absorption of the test material.

Details on mating procedure:

The first generation animals (F0). 32 males/32 females per group were mated for the first time after 10 weeks of treatment for males, two weeks for females (both sexes same age). Pregnant females were allowed to give birth and the second generation (F1A). 28 males/28 females per group were selected from litters of this mating. Shortly after all females had weaned their litters, all first generation animals (F0) were remated using alternative pairings, pregnant females being sacrificed at Day 20 of pregnancy.
The second generation (F1A) were mated after 12 weeks of treatment and dams were allowed to give birth; direct treatment was considered to have commenced at nominal 4 weeks of age. although of course some exposure either directly or indirectly occurred prior to this in utero, through the milk during lactation and/or as weanlings made the transition to diet containing the test material. A limited number of second generation (F1A) animals were re-mated, including all those where proof of successful mating could not be established after the first mating. Dams of each generation which gave birth were allowed to rear their litters to weaning (Day 21 post partum) - culling was not performed. During the preweaning period a number of developmental landmark tests were performed on the F1A and F2A pups in excl. litter. All pups were subjected to macroscopic examination and additionally, for selected F1A pups selected organ weights were recorded.
For F0 generation dams mated for a second time and subsequently killed at Day 20 of pregnancy. their offspring (F1B foetuses) were examined for visceral and skeletal changes. Histopathological examinations were performed for the reproductive tract tissues of the F0 and F1A generations adults, and also the kidneys and liver of F1A generation adults treated at 0 (Control) and 50000 ppm. Additionally. tissues with macroscopic findings from adult animals of all groups including the controls, were also examined.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Satisfactory homogeneity and stability of the test compound in the diet for up to 18 days was determined by the Sponsor prior to the commencement of treatment. Analyses of the diet for achieved concentration were performed for each generation at the start of the pre-mating treatment period (including the start of treatment for males and females of the F0 generation), and a further three occasions during the study (approximately 90, 180 and 270 days).

Duration of treatment / exposure:

Treatment of F0 generation males and females by dietary inclusion began when the males were 6 weeks and the females 14 weeks of age, and continued up to and including the day of sacrifice.
Direct treatment of the F1A generation males and females was considered to have commenced when they were approximately 4 weeks of age and continued up to and including the day of sacrifice.

Frequency of treatment:

permanently: diet

Details on study schedule:

F0 generation
Rearing
Male animals of the F0 generation were approximately 6 weeks of age and females 14 weeks of age at the commencement of treatment. Males were maintained on their respective diets for at least 70 days and females for 14 days prior to mating, i.e. until they were approximately 16 weeks of age.
First mate
The animals were then mated on the basis of one male to one female for a period of 20 days. The dams were allowed to rear their young (F1A pups) to Day 21 post partum.
Selection
On Day 21 post partum 28 male and 28 female pups per group were selected to form the basis of the FIA generation. On Day 22 post partum, following selection of the F1A generation excess F1A pups were sacrificed and examined macroscopically where possible, for one male and one female pup per litter, specified organs were weighed and tissues preserved against contingency of histopathological examination.
Second mate
Shortly following (approximately 10 days) the weaning of F1A generation pups, F0 generation males and females were remated employing alternative pairings and mating previously non-pregnant females and males failing to induce pregnancy to animals which were successful at the first mating. They were mated for a period of 20 days. Females were then sacrificed on as-sumed Day 20 of pregnancy and foetuses (F1B) subjected to gross examination prior to de-tailed pathology.
Sacrifice
Shortly after the last Fa generation female had been sacrificed, F0 generation males were sacri-ficed and examined macroscopically. From all adults surviving to terminal sacrifice, specified organs were weighed and a full range of tissues preserved for histopathological examination.

F1A generation
Rearing
The selected F1A generation animals were reared on their respective diets from nominal Week 4 (age at which direct treatment was considered to commence) for at least 84 days prior to mat-ing. i.e. until they were approximately 16 weeks of age.
First mate
Due to the death of male number 358, it was necessary to mate male number 357 with two fe-males; nos. 469 and 470. All other animals were mated on the basis of one male to one female for a period of 20 days (brother and sister pairings and other closely related pairings were avoided). The dams were allowed to rear their young (F2A pups) to Day 21 post partum. On Day 22 post partum. F2A pups were sacrificed and examined macroscopically:
Second mate
Following a review of mating performance, it was apparent that 1, 1, 5 and 6 females at 0 (Con-trol), 10000. 25000 and 50000 ppm respectively did not apparently produce a litter. A further female at 10000 ppm also showed total litter loss. To investigate mating performance and fertil-ity further these animals and their male partners were re-mated employing alternative pairings. In addition a further 10 males and 10 females selected from the control group were also re-mated to aid interpretation of findings.
Shortly following the weaning of F2A generation pups the unsuccessful F1A generation males and females described above were mated for a period of 14 days with vaginal smears being taken daily throughout. Females were allowed to rear their young (F2B pups) to Day 4 post par-tum. On Day 4 post partum, F2B pups were sacrificed and examined macroscopically.
Sacrifice
Those F1A generation males and females which were not re-mated were sacrificed shortly after the start of the re-mating period. For F1A generation re-mated animals. males were sacrificed shortly after the end of the mating period. females which gave birth were sacrificed with their litter on Day 4 post partum and apparently non-pregnant females sacrificed after the latest date on which they could have given birth.

Remarks:

Doses / Concentrations:
10000. 25000 and 50000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

32 (F0 generation), 28 (F1A generation)

Control animals:

yes, plain diet

Details on study design:

The test compound was administered in the diet at fixed concentrations of 0 (Control), 10000, 25000 and 50000 ppm through two generations; achieved intake values of test compound (mg/kg bodyweight/day) for the first week of treatment were:
within the F0 generation for males (6 weeks old): 1108, 2713 or 5444 and for females (14 weeks old): 655, 1584 or 3164.
within the F1A generation for males (4 weeks old): 1531, 3882 or 7996 and for females (4 weeks old): 1525, 3815 or 7819 respectively.

The dietary concentrations were chosen on the basis of a preliminary reproductive toxicity study in the rat in which a maximum inclusion level of 5% (50000 ppm) in the diet was well tolerated.

Positive control:

not applicable

Parental animals: Observations and examinations:

1. Adult animals
The following procedures were performed on F0 generation and F1A generation adults.

(a) Signs
All animals were regularly handled and examined for obvious changes or signs of reaction to treatment.

(b) Mortalities
Any rat which showed marked signs of ill health or reaction to treatment was isolated and/or killed to prevent cannibalism or autolytic degeneration. All animals that died or were killed for reasons of animal welfare were weighed and subjected to post mortem examination to estab-lish. where possible. the cause of death.

(c) Food consumption
Food intake of rats was recorded weekly on a cage basis. For F0 generation males and females measurement commenced in the week prior to the start of treatment and continued through the whole of the first pre-mating period. FO generation females received only two weeks of treat-ment prior to the first mate. F1A generation food consumption was measured during the first pre- mating period.
Food conversion ratios and achieved intake (mg/kg/day) of the test material were calculated for each sex for the periods of measurement of food consumption.

(d) Water consumption
Water consumption was measured on a daily basis during the initial two and final two weeks of the first pre- mating treatment periods for each generation.

(e) Bodyweight change
All animals were weighed at the start of each generation (i.e. 6 weeks of age for F0 generation, nominal 4 weeks of age for the F1A generation) and, subsequently at weekly intervals; weekly weights of FD generation females were only reported from age 13 weeks (allocation to groups). During the mating periods all females were weighed daily throughout and daily weighing contin-ued until parturition. The occurrence of a positive indication of mating (i.e. sperm or plug) was generally considered as Day 0 of pregnancy. Weights were reported for Days 0, 7, 14. 17 and 20 of gestation., Weights of pregnant animals without a positive indication of mating were re-ported for appropriate days taken retrospectively from birth. Dams that littered were weighed on Days 0, 7, 14 and 21 post partum: Days 0 and 4 for F1A generation re-mated females.

(f) Pregnancy rate
Pregnancy rate was determined as the percentage of surviving paired females that became pregnant.

(g) Mating performance
Vaginal smears were taken daily during the mating period to enable the number of animals that mated on specific days to be determined, this information was used:
(i) to detect whether or not pregnancy was interrupted after mating
(ii) to detect marked anomalies of the oestrous cycle
(iii) to determine the median pre-coital time for the group.
(iv) to determine, where applicable, the appropriate day for sacrifice (Day 20 of pregnancy).

(h) Gestation period
The gestation period (duration of pregnancy) for females which littered was taken as the time between the day of successful mating and the day on which pups were first seen.

(i) Terminal sacrifice
From all adults. specified organs were weighed and a full range of tissues preserved for histo-pathological examination. In addition, any tissue showing macroscopic abnormality was similarly preserved.
For F1A generation females which were not re-mated the uteri were examined and the number of implantation sites recorded. The uteri of apparently non-pregnant/no young born females (F0 or F1A generation) were examined by the Salewski techniquea prior to storage in fixative. The reproductive tract of all apparently infertile males and females was examined histologically. In addition the following procedures were carried out on the F1A generation.

(j) Post weaning development
The onset of vaginal opening was monitored in all females from 28 days post partum. until oc-currence in all animals. The onset of cleavage of the balanopreputial skinfold was monitored in all males from 35 days post partum,until occurrence in all animals.

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily during the mating period to enable the number of animals that mated on specific days to be determined, this information was used:
(i) to detect whether or not pregnancy was interrupted after mating
(ii) to detect marked anomalies of the oestrous cycle
(iii) to determine the median pre-coital time for the group.
(iv) to determine, where applicable, the appropriate day for sacrifice (Day 20 of pregnancy).

Sperm parameters (parental animals):

Parameters examined in [all/P/F1/F2] male parental generations: testis weight, epididymis weight

Litter observations:

2. Litter data - dams allowed to rear young to weaning (F0 and F1A generation first mate litters)
(a) Birth (Day 0) to Day 21 post partum
As soon as possible after parturition, the young were counted, individually identified within the litter by toe amputation, sexed. weighed and examined for external abnormalities. Keeping nest disturbance to a minimum all litters were examined daily for dead and/or abnormal young. The pups were also weighed on Days 4, 8, 12, 16 and 21 post partum.,Dead young were subjected to an autopsy. Pups with suspected,abnormalities were preserved either in Bouin's solution if it was considered that free-hand sectioning or histopathological examination would be of value or in alcohol for subsequent staining with alizarin if skeletal defects were suspected.

During the pre-weaning period all offspring in all litters were examined to determine the age at which the following developmental stages were attained:
(1) Surface righting reflex from Day 1 post partum to 100% success.
(2) Startle reflex from Day 11 to 100% success.
(3) Air righting reflex from Day 14 to 100% success.
(4) Pupil reflex once on Day 20 post partum.
Surface righting and air righting tests involve sensory and motor co-ordination systems. Auditory startle and pupil reflex were included as very simple indicators of auditory and visual function-ing. All these tests to some extent reflect physical development in the sense that physical de-velopment is an index of maturation. and the appearance of any of the above reflexes depends upon the maturation of underlying systems.
(b) Selection of pups for organ weight analysis and tissue .
preservation – F0 generation first mate offspring (F1A pups) only one male and one female weanling pup from each litter in the group was selected for organ weight analysis and preserva-tion of tissues .
(c) Terminal sacrifice of excess (F1A/F2A) pups
On Day 22 post partum all excess pups were sacrificed and examined externally and internally for abnormalities. Sex of the pups was confirmed by gonadal inspection. Any tissue showing macroscopic abnormality were preserved in neutral buffered 10% formalin to permit histological examination if required.

(d) Selection of pups for next generation
The following procedure applies to F0 generation first mate offspring (FIA litters).
On Day 21 post partum one male and one female pup in each of up to 28 litters per group were retained for further study. These litters were those weaned as close to the median weaning date as possible for the study or group but paying attention to retaining as wide a genetic pool as possible. Selection of pups was made on the basis of bodyweight at Day 21 post partum. Within each litter. the male pup chosen was as close as possible to the median weight of the males in the litter and the female pup chosen was as close as possible to the median weight of the females. Where less than 28 litters in a group were weaned or were within a reasonable range of weaning dates. or where all pups in a litter were of one sex. the complement was restored by taking a second pup from an appropriate litter. Records of lineage were maintained.

3. Litter data - dams allowed to rear young to Day 4 post parturn (FIA generation re-mate)
Birth (Day 0) to Day 4 post partum (F2B pups)
Pups from the partial remate of the F1A generation were sacrificed on Day 4 post partum and were examined macroscopically for external and internal abnormalities. Sex of pups was confirmed by gonadal inspection. Any tissues showing macroscopic abnormalities were preserved but not examined further.

4. Litter data – F0 generation second mate dams killed at Day 20 pregnancy
On Day 20 of pregnancy the females were killed by CO2 asphyxiation. dissected and examined for congenital abnormalities and macroscopic changes in maternal organs. The organs were weighed. The ovaries and uteri were examinedimmediately to determine:
(a) number of corpora lutea
(b) number and distribution of live young (FIB foetuses)
(c) number and distribution of embryofoetal deaths
(d) individual foetal weight from which the litter weight iscalculated
(e) foetal abnormalities
Embryofoetal deaths were classified as: Early: only placenta visible at termination; Late: both placental and embryonic remnants visible at termination

Live young were examined externally and weighed. Half the foetuses in each litter were pre-served in Bouin's solution for subsequent free-hand sectioning to discover visceral abnormali-ties (Wilson techniqueb)j the remainder were fixed in 74 OP industrial methylated spirit for sub-sequent macroscopic examination, evisceration, clearing and alizarin staining (modified Dawson technique C) for skeletal examination. Young showing suspected abnormalities were processed by the more appropriate technique for clarification of initial observations. All foetuses were sexed by gonadal inspection following preservation. Foetuses were uniquely identified to allow correlation of initial with subsequent findings. Structural changes are presented as: Malforma-tions: rare and/or probably lethal, e.g. exencephaly, anury. Anomalies: minor differences from 'normal' that are detected relatively frequently either by free-hand sectioning, e.g. increased renal pelvic dilatation, or at skeletal examination, e.g. bipartite centrum. Variants: alternative structures occurring regularly in the control population are classified as variants e.g. unossified sternebra(e).

Postmortem examinations (parental animals):

All animals were examined macroscopically.
The following organs were weighed in all F0, F1A generation adults and in selected FIA weanlings:
Adrenals; brain; heart; kidneys; liver; lungs; ovaries; prostate (with seminal vesicles and coagu-lating gland); - adults only; testes (with epididymides); thymus (if present). Where appropriate, organs were weighed as a pair. When one of a pair of organs was abnormal, both organs were weighed individually.
Histology was restricted to the following tissues of adult animals and was performed as follows:
1. Reproductive tract associated tissues all F0 and F1A generation adults of the control group and at the highest dietary concentrations; any apparently infertile male or female from the low-est
or intermediate dietary concentration.
2. Other identified tissues
F1A generation adults only of the control group and at the highest dietary concentration.
3. Macroscopically abnormal tissues
all F0 and F1A generation adults. The routine stain used was haematoxylin and eosin.
Histopathological examination of weanling tissues was not considered necessary.

Postmortem examinations (offspring):

All animals were examined macroscopically.
The following organs were weighed in all F0, F1A generation adults and in selected FIA weanlings:
Adrenals; brain; heart; kidneys; liver; lungs; ovaries; prostate (with seminal vesicles and coagu-lating gland); - adults only; testes (with epididymides); thymus (if present). Where appropriate, organs were weighed as a pair. When one of a pair of organs was abnormal, both organs were weighed individually.
Histology was restricted to the following tissues of adult animals and was performed as follows:
1. Reproductive tract associated tissues all F0 and F1A generation adults of the control group and at the highest dietary concentrations; any apparently infertile male or female from the low-est
or intermediate dietary concentration.
2. Other identified tissues
F1A generation adults only of the control group and at the highest dietary concentration.
3. Macroscopically abnormal tissues
all F0 and F1A generation adults. The routine stain used was haematoxylin and eosin.
Histopathological examination of weanling tissues was not considered necessary.

Statistics:

Statistical analyses were performed on litter data and other parameters as below. Significance tests were normally two-tailed.
Litter data
The basic sample unit was the litter. and due to the preponderance of non-normal distributions. non-parametric analyses were used. Mean values of litter size, pre- and post implantation loss, litter weight, mean foetal weight, mean pup weight, developmental landmarks and the incidence of anomalous offspring were analysed. Intergroup comparisons were made by the non-parametric equivalent of Williams' test. Only the testes considered to be appropriate are re-ported. Where 75% of the values for a given variable consist of one value, a Fisher's exact test was used.
Other parameters
Bodyweight, food, water and organ weight data were generally analysed using analysis of vari-ance (sometimes log transformed) followed by Williams' test. If significant heterogeneity of variance was present, and could not be removed by transformation, the Kruskal-Wallis analysis of ranks was used.

Reproductive indices:

Pregnancy rate
Pregnancy rate was determined as the percentage of surviving paired females that became pregnant.

Mating performance
Vaginal smears were taken daily during the mating period to enable the number of animals that mated on specific days to be determined, this information was used:
(i) to detect whether or not pregnancy was interrupted after mating
(ii) to detect marked anomalies of the oestrous cycle
(iii) to determine the median pre-coital time for the group.
(iv) to determine, where applicable, the appropriate day for sacrifice (Day 20 of pregnancy).

Gestation period
The gestation period (duration of pregnancy) for females which littered was taken as the time between the day of successful mating and the day on which pups were first seen.

Offspring viability indices:

Birth (Day 0) to Day 21 post partum:sexed. weighed and examined for external abnormalities; dead young were subjected to an autopsy. During the pre-weaning period all offspring in all lit-ters were examined to determine the age at which the following developmental stages were attained:
(1) Surface righting reflex from Day 1 post partum to 100% success; (2) Startle reflex from Day 11 to 100% success; (3) Air righting reflex from Day 14 to 100% success; (4) Pupil reflex once on Day 20 post partum.
Surface righting and air righting tests involve sensory and motor co-ordination systems. Auditory startle and pupil reflex were included as very simple indicators of auditory and visual function-ing. On Day 22 post partum all excess pups were sacrificed and examined externally and internally for abnormalities. Sex of the pups was confirmed by gonadal inspection.

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

At 50000 ppm:
- two deaths; one F0 female during parturition (first mate), one F1A male prior to mating. These deaths were not considered to be treatment-related
- marginally lower bodyweight gain during gestation for F0 (second mate) and F1A females (P>0.05)
- slightly longer median pre-coital time, F1A first mate only
- two total litter losses, F0 first mate (one of which was associated with the decedent female) and evidence which may suggest three total resorptions among F1A dams

At 25000 ppm:
- two mortalities; one F0 female killed during the first mating period, one FIA male died (both these animals had shown evidence of successful mating performance). Neither of these deaths were considered to be treatment-related
- marginally lower bodyweight gain during gestation for F0 (second mating) and F1A females (P>0.05)
- slightly longer median pre-coital time, F1A first mate only
- two total litter losses, one in the F0 and the other in the F1A generations and evidence which may suggest one total resorption among F1A dams

At 10000 ppm, all parameters inclusive of fertility and general reproductive performance were essentially similar to the controls throughout.

The pregnancy rate (% of pregnant females) at both 25000 and 50000 ppm was slightly lower than controls in the F1A generation. However, the differences were considered to be co-incidental since the numbers of animals of either sex which were of unproven fertility in two mat-ings of either generation was essentially similar compared with the controls.

For adults of the F0 and F1A generation, there were no macroscopic/microscopic findings at any treatment level which were considered indicative of an adverse effect of treatment with beta-cyclodextrin.

Dose descriptor:

NOEL

Effect level:

10 000 ppm

Sex:

male/female

Basis for effect level:

other: overall effects

Dose descriptor:

LOEL

Effect level:

25 000 ppm

Sex:

male/female

Basis for effect level:

other: body weight; litter losses; pup weight; survival index; sligfhtly longer precoital time

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings:

no effects observed

At 50000 ppm:
- slightly increased embryonic death, mainly early (P<0.05) F0 second mate
- slightly lower foetal bodyweight F0 second mate (P>0.05) and slightly lower pup bodyweight gain FO first mate (P<0.05)
- higher incidence of F0 second mate foetuses (F1B) with variant sternebrae (P<0.05), particularly unossified and/or reduced.

At 25000 ppm:
- slightly increased embryonic deaths, mainly early (P<0.05), F0 second mate
- slightly lower pup bodyweight gain, F0 first mate (P>0.05)

At 10000 ppm, all parameters inclusive of fertility and general reproductive performance were essentially similar to the controls throughout.

There was no effect on pup development tests and no macroscopic/ microscopic findings at any treatment level which were considered indicative of an adverse effect of treatment.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

10 000 ppm

Sex:

male/female

Basis for effect level:

other: overall effects

Dose descriptor:

LOEL

Generation:

F1

Effect level:

25 000 ppm

Sex:

male/female

Basis for effect level:

other: litter losses;

Reproductive effects observed:

not specified

Conclusions:

A dietary concentration of 10000 ppm beta-Cyclodextrin was considered to show no adverse effect upon either the treated parent animals or their offspring, in terms of growth, development, fertility and general reproductive performance

Executive summary:

The effect of Beta-cyclodextrine (99.3% pure) upon the growth and reproductive performance of therat was assessed in a two generations test according to OECD Guideline 416.The test compound was administered in the diet at fixed concentrations of 0 (Control), 10000, 25000 and 50000 ppm through two generations; achieved intake valuesof test compound (mg/kg bodyweight/day) for the first week of treatment were within the F0 generation for males (6 weeksold) was 1108, 2713 or 5444 and for females (14 weeksold) it was 655, 1584 or 3164 whereas within the F1A generation for males (4 weeks old) it was 1531, 3882 or 7996 and for females (4 weeks old) it was 1525, 3815 or 7819 respectively.

The first generation animals (F0)32 males/32 femalesper group were mated for the first time after 10 weeks of treatment for males, two weeks for females (both sexes same age). Pregnant females were allowed to give birth and the second generation(F1A).28 males/28 femalespergroupwere selected from litters of this mating. Shortly after all females had weaned their litters, all first generation animals (F0) were re-mated using alternative pairings, pregnant females being sacrificed at Day 20 of pregnancy.

The second generation (F1A) were mated after 12 weeks of treatment and dams were allowed to give birth; direct treatment was considered to have commenced at nominal 4 weeks ofage.although of course some exposure either directly or indirectly occurred prior to this inutero,through the milk during lactation and/or as weanlings made the transition to diet containing the test material. A limited number of second generation (F1A) animals were re-mated, including all those where proof of successful mating could not be established after the first mating. Dams of each generation which gave birth were allowed to rear their litters to weaning (Day 21 post partum) - culling was not performed. During the pre-weaning period a number of developmental landmarktestswere performed on the F1A and F2A pups in excl. litter. All pups were subjected tomacroscopic examination and additionally, for selected F1A pups selected organ weights were recorded.

For F0 generation dams mated for a second time and subsequently killed at Day 20 of pregnancy, their offspring (F1B foetuses) were examined for visceral and skeletal changes. Histopathological examinations were performed for the reproductive tract tissues of the F0 and F1A generations adults, and also the kidneys and liver of FIA generation adults treated at 0 (Control) and 50000 ppm. Additionally. tissues with macroscopic findings from adult animals of all groups including the controls, were also examined.

At 50000 ppm two deaths occurred. These deaths were not considered to be treatment-related. At this dose level marginally lower bodyweight gain during gestation (for F0 second mate and F1A females) as well as slightly longer median pre-coital time (F1A first mate only) and two total litter losses with evidence which may suggest three total resorptions among F1A dams were observed. Slightly increased embryonic death, mainly early at F0 second mate and slightly lower foetal bodyweight for F0 second mate as well as slightly lower pup bodyweight gain F0 first mate were seen in this group. Also a higher incidence of F0 second mate foetuses (F1B) with variant sternebrae was documented.

At 25000 ppm also two mortalities occurred, neither of these deaths were considered to be treatment-related. A marginally lower bodyweight gain during gestation (for F0 second mating and F1A females) was noted as well as slightly longer median pre-coital time (F1A first mate only). Further findings were two total litter losses(one in the F0 and the other in the F1A generations) and evidence which may suggest one total resorption among F1A dams, slightly increased embryonic deaths (mainly early, F0 second mate) and slightly lower pup bodyweight gain (F0 first mate).At 10000 ppm, all parameters inclusive of fertility and general reproductive performance were essentially similar to the controls throughout.

The pregnancy rate (% of pregnant females) at both 25000 and 50000 ppm was slightly lower than controls in the F1A generation. However, the differences were considered to be coincidental since the numbers of animals of either sex which were of unproven fertility in two matings of either generation was essentially similar compared with the controls. There was no effect on pup development tests and no macroscopic/ microscopic findings at any treatment level which were considered indicative of an adverse effect of treatment. For adults of the F0 and F1A generation, there were no macroscopic/microscopic findings at any treatment level which were considered indicative of an adverse effect of treatment with beta-cyclodextrin.

At dietary concentrations of 50000 ppm beta-cyclodextrin and to a lesser extent at 25000 ppm, there was some evidence of adverse effects on the parent animals and offspring although in general the effects were slight and there were few findings consistent through both generations. Reproductive performance and fertility of adults treated at these dietary concentrations were not unduly compromised. A dietary concentration of 10000 ppm was considered to show no adverse effect upon either the treated parent animals or their offspring, in terms of growth, development, fertility and general reproductive performance.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

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 effect of Beta-cyclodextrin upon the growth and reproductive performance of the rat was assessed in a 2 generations test according to OECD Guideline 416.The test compound was administered in the diet at fixed concentrations of 0 (Control), 10000, 25000 and 50000 ppm through two generations; achieved intake values of test compound (mg/kg bodyweight/day) for the first week of treatment were within the F0 generation for males (6 weeks old) was 1108, 2713 or 5444 and for females (14 weeks old) it was 655,1584 or 3164 whereas within the F1A generation for males (4 weeks old) it was 1531, 3882 or 7996 and for females (4 weeks old) it was1525, 3815 or 7819 respectively.

The first generation animals (F0)32 males/32 females per group were mated for the first time after 10 weeks of treatment for males, two weeks for females (both sexes same age). Pregnant females were allowed to give birth and the second generation (F1A).28 males/28 females per group were selected from litters of this mating. Shortly after all females had weaned their litters, all first generation animals (F0) were re-mated using alternative pairings, pregnant females being sacrificed at Day 20 of pregnancy.

The second generation (F1A) were mated after 12 weeks of treatment and dams were allowed to give birth; direct treatment was considered to have commenced at nominal 4 weeks of age although of course some exposure either directly or indirectly occurred prior to this in utero, through the milk during lactation and/or as weanlings made the transition to diet containing the test material. A limited number of second generation (F1A) animals were re-mated, including all those where proof of successful mating could not be established after the first mating. Dams of each generation which gave birth were allowed to rear their litters to weaning (Day 21 post partum) - culling was not performed. During the pre-weaning period a number of developmental landmark tests were performed on the F1A and F2A pups in excl. litter. All pups were subjected to macroscopic examination and additionally, for selected F1A pups selected organ weights were recorded.

For F0 generation dams mated for a second time and subsequently killed at Day 20 of pregnancy, their offspring (F1B foetuses) were examined for visceral and skeletal changes. Histopathological examinations were performed for the reproductive tract tissues of the F0 and F1A generations adults, and also the kidneys and liver of FIA generation adults treated at 0 (Control) and 50000 ppm. Additionally. tissues with macroscopic findings from adult animals of all groups including the controls, were also examined.

At 50000 ppm two deaths occurred. These deaths were not considered to be treatment-related. At this dose level marginally lower bodyweight gain during gestation (for F0 second mate and F1A females) as well as slightly longer median pre-coital time (F1A first mate only) and two total litter losses with evidence which may suggest three total resorptions among F1A dams were observed. Slightly increased embryonic death, mainly early at F0 second mate and slightly lower foetal bodyweight for F0 second mate as well as slightly lower pup bodyweight gain F0 first mate were seen in this group. Also a higher incidence of F0 second mate foetuses (F1B) with variant sternebrae was documented.

At 25000 ppm also two mortalities occurred, neither of these deaths were considered to be treatment-related. A marginally lower bodyweight gain during gestation (for F0 second mating and F1A females) was noted as well as slightly longer median pre-coital time (F1A first mate only). Further findings were two total litter losses(one in the F0 and the other in the F1A generations) and evidence which may suggest one total resorption among F1A dams, slightly increased embryonic deaths (mainly early, F0 second mate) and slightly lower pup bodyweight gain (F0 first mate). At 10000 ppm, all parameters inclusive of fertility and general reproductive performance were essentially similar to the controls throughout.

The pregnancy rate (% of pregnant females) at both 25000 and 50000 ppm was slightly lower than controls in the F1A generation. However, the differences were considered to be coincidental since the numbers of animals of either sex which were of unproven fertility in two matings of either generation was essentially similar compared with the controls. There was no effect on pup development tests and no macroscopic/ microscopic findings at any treatment level which were considered indicative of an adverse effect of treatment. For adults of the F0 and F1A generation, there were no macroscopic/microscopic findings at any treatment level which were considered indicative of an adverse effect of treatment with beta-cyclodextrin.

At dietary concentrations of 50000 ppm beta-cyclodextrin and to a lesser extent at 25000 ppm, there was some evidence of adverse effects on the parent animals and offspring although in general the effects were slight and there were few findings consistent through both generations. Reproductive performance and fertility of adults treated at these dietary concentrations were not unduly compromised. A dietary concentration of 10000 ppm was considered to show no adverse effect upon either the treated parent animals or their offspring, in terms of growth, development, fertility and general reproductive performance.

 

Beta-cyclodextrin (> 99% pure) was administered by admixture in the diet at dose levels of 5 %, 2.5 % and 1.25 % to groups of 30 male and 30 female Ico: OFA. SD (lOPS Caw) rats in a three generation reproduction test. 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 21post 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.

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 %. 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:
In a three generation reproduction test in rats only slight effects on body weight gain of the offspring were observed in the medium (2.5% of feed) and high dose group (5%). No effect was observed in the 1.25% group (874/1610 mg/kg bw/day [P males/females]).

Effects on developmental toxicity

Description of key information

In a teratogenicity assay in rats no effect could be observed in the low and medium dose groups (1250 and 2500 mg/kg bw/day). Only in the high dose group of 5000 mg/kg bw/day the body weight gain of the pregnat rats was reduced. The NOEL is set to be 2500 mg/kg bw/day  for maternal toxicity and 5000 mg/kg bw for teratogenicity. 

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Animal species: rat
Strain: OFA originating from Sprague-Dawley
Source: IFFA CREDO (Les Oncin. France)
Sex: male and female
Age: 12 weeks
Weight: 257 - 288 grams
Number: sufficient number of virgin females in order to obtain 20 pregnant fe-males/group.
Rationale for species selection: Species usually used for this type of trial, and recom-mended by the OECD Guideline 414
Animal husbandry Housing: Makrolon cages, stainless-steel grid lid size 37.5 x 23.5 x 16 cm bedding of chopped maize cobs 5 animals/cage
Food: Pelleted diet UAR base 210 from Usine d'Alimentation Rafionnelle, Villemoisson-sur-Orge, France
Drinking water: Polypropylene water-bottles, Tap water, autoclaved for 10 minutes at 120 °C, changed 3 times a week.
Food and water were available at libitum. The quantities consumed were measured 3 times a week..
Environment: Temperature: 20 +/- 2 °C, humidity 50 +/- 10%, artificial lighting 12 hours/day, air changes 12 times/hour
The animals were housed under quarantine conditions for at least one week prior to the commencement of the study.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

The beta-Cyclodextrin suspensions were prepared daily at concentrations of 1.25, 2.5 and 5g/10ml of 1% CMC. They were stored at + 4°C after use and kept at the disposal of the laboratory for testing of the concentration. They were discarded after one week.

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Concentration in vehicle: The test substance was suspended in carboxymethyl cellulose (CMC). This slurry was prepared by mixing 10 g CMC in 1 1 of water for injection (1%). and administered by oral gavage.
The doses are 0 ; 1.25 ; 2. 5 and 5 g/kg/d by gastric intubation in a volume of 10 ml/kg. These doses correspond to those ingested during a subchronic toxicity study in the rat.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

B-cyclodextrin levels in the suspension were measured by High Performance Liquid Chromatography (HPLC).
The brand of the equipment used was WATERS (MILLIPORE - WATERS. USA) and comprised:an isocratic pump (type WATERS 510),a sampler (type WATERS 710B) or any injection system (type Rheodyne) with a loop of 10 microlitres, a differential refractometer (type WATERS R410), a recorder-integrator (type Shimadzu CRSA).The analytical column was SUPERCOSIL LC-18 5 microns (SUPELCO ref 5-B985).

Details on mating procedure:

Each female was cage with a male on day 0, from the end of the afternoon until the following morning. A cervical smear was performed the day after mating. The presence of spermatozoa) detected .by microscopic examination. reveals the coitus had occured. The female was thus considered mated and at day 1 of gestation. The gestation is confirmed on day 14 by abdominal palpation .

Duration of treatment / exposure:

from day 7 to day 16 the period of organogenesis

Frequency of treatment:

once daily

Duration of test:

On day 21 of gestation, the females were sacrificed (ether) and subjected to a post-mortem examination .

No. of animals per sex per dose:

20 pregnant females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: These doses correspond to those ingested during a subchronic toxicity study in the rat. Thus the highest dose was considered as close to the No Observable Effect Level .
- Rationale for animal assignment (if not random): random

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked in table were included.


DETAILED CLINICAL OBSERVATIONS: no


BODY WEIGHT: Yes
- Time schedule for examinations: days 1, 3, daily from 7 to 16, 21


FOOD CONSUMPTION : Yes

WATER CONSUMPTION: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [half per litter ]
- Skeletal examinations: Yes: [half per litter ]
- Head examinations: Yes: [ half per litter ]

Statistics:

The following statistical methods were performed by means of Statgraphics and SAS programs.
1. Preliminary
From the data collected, three other variables were calculated: By Litter
VAR 13 - Percentage of resorptions over the number of implants
VAR 14 - Percentage of males over the number of live foetuses
VAR 15 - Ratio number af females/number of males.
2. Descriptive statistics:
The same calculations were done for each of the four groups as follows
• For the variables concerning the dams or litters (var. 1 to 9, 13, 14, 15)
• Firstly, calculation of some caracteristics (mean, minimum, maximum, standard devia-tion. standard error) on all dams of the group.
• On the other hand, only for var. 4 to 9. determination of the distribution of all dams ac-cording to the various values of the variable studied.
• For the variables concerning the foetuses (var. 10, 11, 12) :
• First, for each litter, calculation of the mean (and standard deviation) body weight. size. and determination of the distribution of the sex on all foetuses of the litter.
• On the other hand. on all dams of the group. calculation of the caracteristics previously cited and determination of the distribution of sex .
3. Groups comparison :
A univariate comparison (one variable at a time) between the 4 groups was first done; then, if a significant difference was observed. a multivariate comparison was performed. The tests used depend upon the type of variable analysed. The different tests used are : • One-factor variance analysis on the raw data or after arc sin Vp transformation on the percentage.
• Nested two~factor analysis of variance and n repetitions on raw values.
• Nested three-factor analysis of variance and n repetitions on raw values. For each variance analysis. if a significant difference is observed. then group homogeneity is de-termined by the test of Ouncan, Scheffe and LSD.
• Non-parametric Kruskall and Wallis test
• Chi-square test comparing several distributions observed

Indices:

no data

Historical control data:

given in report

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
At the high dose level only, a statistically significant reduction of body weight gain in females, accompanied by a reduction of food consumption.

Dose descriptor:

NOAEL

Effect level:

2 500 mg/kg bw/day (actual dose received)

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEL

Effect level:

5 000 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

5 000 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
not applicable

Dose descriptor:

NOAEL

Effect level:

5 000 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Substance induced slight reduction of body weight gain in the high dose group but no embryotoxic or fetotoxic effects in any of the tested groups.

Executive summary:

A teratogenicity study of .beta.-cyclodextrin (92.5 % pure) according to OECD 414 was carried out in Sprague-Dawley rats. Four groups, each comprising 20 females, beta Cyclodextrin was administered by oral gavage at levels of 0; 1250; 2500 and 5000 mg/kg body weight/d respectively from day 7 to day 16. The day the presence of spermatozoa was observed on the vaginal smear was considered as day 1 of gestation and the females were sacrificed on day 21 of gestation.

In the high dose group five females died from gavage accidents between day 7 and day 17. Necropsy revealed oesophageal perforation for the 5 females. On day 1, there were no statistically significant differences between control and each treated group concerning the body weight variability or mean. The treated animals presented a reduced body weight gain from the beginning of the treatment period. The difference was statistically significant for the high dose group from day 8. At cessation of treatment, the body weight gain of the animals of this group was similar to that of the controls but their body weight at day 21 was still significantly decreased

During the first and third weeks of the study there were no significant . differences between control ·and treated groups concerning food consumption. During the second week which corresponds to the treatment period, a significant decrease in food consumption was· noted for mid and high dose groups and ( - 7.4 and - 19,2 %).

No statistical differences were noted on food conversion efficiency during the 3 weeks, between control and treated groups, despite an increase observed during week 2 for mid and high dose groups. 

Water consumption was similar in all groups during the first week.

The visceral examination by's technique and skeletal examination by alizarin technique of rat fetuses. from darns treated during the sensitive phase of embryogenesis with the test substance at the oral doses of 1250, 2500 and 5000 mg/kg/day did not reveal any detectable embryotoxic or teratogenic effect.

Following parameters were examined in litters: weight of full and empty uterus, weight of placenta, number of live and dead foetuses, number of implantation sites and resorptions, number of corpora lutea, ratio - resorption/implantation. No differences are noted between the groups.

The following parameters were examined in foetuses: weight and size of the foetuses, sex, ratio male/live foetuses, ratio female/male. The only significant difference in both sexes is the increased size of the foetuses in groups low and mid dose groups, compared with controls: +0 0.3 (0.84%) and + 0.4 mm (1.14%) respectively for males and females in low dose group ; + 0.6 (1.68%) and + 0.6 mrn (1.72%) respectively for males and females in the mid dose group. 

During the study, it was observed that the administration of beta-cyclodextrin at doses of 0, 1250, 2500 or 5000 mg/kg induced at the high dose level only, a statistically significant reduction of body .weight gain in females, accompanied by a reduction of food consumption. On the other hand, these females showed difficulty in swallowing the gavage suspension, and five of them died following oesophageal perforation. The dose level of 5000 mg/kg in a volume of 10 ml/kg is the limit of ingestion in this type of study . The lower dose of 2500 mg/kg per day had no toxic effect on the females. However. this produced no observable differences in the weight of the uteri and placentae, the number of live (100%) or dead (0%) foetuses, the number of implantation sites, resorptions and corpora lutea The same applies to the following foetal parameters: size, body weight, sex ratio. visceral and skeletal examinations. which indicate no embryotoxic or teratogenic effects ascribable to the treatment.

No embryotoxic or teratogenic effects were observed. It is concluded that beta-cyclodextrin administration at levels up to 2500 mg/kg bw/d had no adverse effect observable on pregnant females and at levels up to 5000 mg/kg bw/d had no adverse effect observable in that kind of study on litters and foetuses.

 

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with limited documentation

Qualifier:

according to guideline

Guideline:

other: 1. Cook, M.J., Fairweather, F.A. and Hardwick, M. (1969): Further thoughts of teratogenic testing. In: Bertelli, A. and Donati, L., eds. Teratology. Excerpta Medica Found., Amsterdam, pp.34-42.

Qualifier:

according to guideline

Guideline:

other: 2. Registration of pharmaceutical specialties (1974): Instructions for the application, etc. Pharmacological and toxicological data pp.7-10, ed. by Socialstyrelsen, National Board of Health and Welfare of Sweden.

Principles of method if other than guideline:

Doses: Pilot study (non-pregnant females) : 250, 500, and 1000 mg/kg/day
Main study (pregnants): 150, 300, and 600 mg/kg per day
Solvent: 1 ml/kg of 1.25 % aqueous methylcellulose
Route of administration: oral, by gavage
Treatment period: Pilot study daily, for 13 days
Main study daily, from the 7th to the 19th gestational day
Control: untreated
Group size: Pilot study : 3 females/group
Main study 12-14 dams/group

GLP compliance:

no

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Thalidomide-sensitive New Zealand white rabbits were used in the experiments (breeder: Agricultural Combinate, Kornye, Tornyópuszta, Hungary). Females, weighing 3- 4 kg, were artificially inseminated by the breeder.

Route of administration:

oral: gavage

Vehicle:

other: methylcellulose

Details on exposure:

Pregnant animals were transported to the Institute for Drug Research (Dunakeszi Facility) in the early forenoon hours on the same day, on their 4th, 5th, 6th and 7th gestational days. The day of insemination was considered as the first day of pregnancy. After transportation the dams were placed into individual cages according to the insemination days in separate rooms of the building. Pelleted rabbit food (55-3/2 rabbit food, Környe) and tap water were provided ad libitum. Dams were weighed on the 7th gastational day, thereafter twice weekly and on the day of autopsy (28th day of pregnancy).

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: artificial insemination

Duration of treatment / exposure:

Treatment period: Pilot study daily, for 13 days
Main study daily, from the 7th to the 19th gestational day

Frequency of treatment:

daily

Duration of test:

11 days

No. of animals per sex per dose:

12-14 (main study)

Control animals:

no

Details on study design:

After transportation the dams were placed into individual cages according to the insemination days in separate rooms of the building. Pelleted rabbit food (55-3/2 rabbit food, Környe) and tap water were provided ad libitum. Dams were weighed on the 7th gastational day, thereafter twice weekly and on the day of autopsy (28th day of pregnancy).

Maternal examinations:

During the experiment the dams were observed daily and the symptoms were recorded individually. The pregnants were exsanguinated to death and dissected on the 28th day of pregnancy. Viscera were examined and the observed pathological alterations were recorded.

Ovaries and uterine content:

Uterine horns and ovaries were removed together with the fetuses. The following parameters were determined per uterine horn: preimplantation loss, implantation sites, implants died during the intrauterine development (early and late embryonic, as well as fetal mortality) and viable fetuses. Uteri were then opened to release the viable fetuses and placentae, and the symptoms of life (movement, respiratory movement, umbilical cord bleeding) were examined. The number of corpora lutea per ovaries were counted. The fetuses and placentae were weighed individually, the length of fetuses was measured (crown-rump length).

Fetal examinations:

all viable and dead fetuses, if maceration was not advanced, were examined for external, visceral and skeletal abnormalities. External and visceral examinations were performed on the day of autopsy, the found alterations were recorded and the fetuses were fixed in 96% ethanol for further skeletal examination. About half of the heads from each litter were fixed in Bouin's solution for sensory organ and brain examination. According to Wilson's technique ca. 1 mm thick frontal sections were prepared by means of a razor blade from the heads. Skeletons were examined after KOH alizarin red S staining. Both the viscera and skeletons were studied by dissecting microscopy. All pathological alterations observed during external, visceral and skeletal examinations were recorded.

Statistics:

Mean, standard error, Student's t test
Body weight and body weight gain of dams to the 28th gestational day; Body weight and length of fetuses, placental weight, No. of corpora lutes, implants and viable fetuses per litter.

Percentual mean, standard error, Student's t test
Intrauterine mortality (preimplantation loss, early and late embryonic, as well as fetal mortality; total mortality) per litter, Fetal abnormalities per litter (minor anomalies and/or retardations, major malformations) according to the type of examination (external, vis-ceral, skeletal), Sex distribution per litter .

Chi-square test
Intrauterine mortality (preimplantation loss, early and late embryonic, as well as fetal mortality; total mortality) per litter, Fetal abnormalities per litter (minor anomalies and/or retardations, major malformations) according to the type of examination (external, vis-ceral, skeletal), Sex .distribution per litter.

Hypergeometric test
Mortality of dams, Dams with complete intrauterine death and/or with spontaneous abortion.

Indices:

no data

Historical control data:

no data

Details on maternal toxic effects:

Maternal toxic effects:no effects

Dose descriptor:

NOEL

Effect level:

>= 600 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOEL

Effect level:

>= 600 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

The applied doses induced no toxic symptoms. Two days after the last treatment transitory diarrhoea occurred in one animal (250 mg/kg). Pathological alterations were not found at autopsy apart from kidney changes induced by Nosema infection and the foci in the liver due to coccidiosis. The body weight gain of females in each treatment group corresponded to the species and strain applied. 1000 mg/kg Induced no toxic symptoms or pathological changes in the pilot study still, because of the high density of the suspension of the 1000 mg/kg dose, for the main study the highest dose was reduced: to 600 mg/kg/day.

Clinical symptoms due to the treatment were not observed in the treated groups. Transitory, mild diarhhoea was sporadically observed in all dose groups. During the study two dams were lost, one of them from the 150 mg/kg group due to purulent metritis, the other one from the control group following abortion .

Spontaneous abortion occurred sporadically. One animal of the 150 mg/kg aborted her fetuses on the 27th gestational day, an other one of the 600 mg/kg on the 23rd day of pregnancy and two further animals of the control group ended their pregnancy with abortion on the 25th and 22nd days (the last animal died two days later).

Autopsy revealed no alterations indicating any effect of the compound under test. Independently from the applied dose in the kidneys of treated and control animals signs of Nosema infection, in the livers symptoms of Coccidium infection and sporadically fatty infiltration were recorded. Five cases with purulent processes like peritonitis, metritis, abscess in the omentum, mesometrium of uterine cervix were detected independently from the dose with uniform occurrance among the treated and control groups. These animals and litters were excluded from the evaluation.

The body weight gain of dams corresponded in all experimental groups to that characteristic of the species and strain used in this study.

The average number of corpora lutea and implantations in dams was similar in all groups. The intrauterine mortality in the treated groups did not exceed the control level The body weight and length of fetuses, as well as the placental weight showed similar values in the treated and control groups. The number of viable fetuses per dam was similar in the treated and control groups. This was the case with the sex ratio of the viable fetuses, too. The number of minor anomalies (retardations in weight and length) found during the external examination was at the control level in all treated groups. Major malformations were recorded only sporadically and accidentally .

The incidence of minor anomalies observed in the course of visceral examination was insignificantly and dose-independently increased in the treated groups, which might be related to the higher occurrence of the supernumerary arteries originating from the aortic arch and carotid arteries. Skeletal examination revealed a slight, insignificant increase in the number of retardations in the 600 mg/kg group. The occurrence of malformations was sporadic and accidental. As shown by the cumulative evaluation of abnormalities the incidence of minor anomalies and malformations was rather similar in the treated groups and controls. The abnormalities were regarded as sporadic and accidental ones.

Conclusions:

Substance did not show teratogenic nor maternal toxicity under the conditions of the main study.

Executive summary:

The teratological study of beta-cyclodextrin was performed on white rabbits. The compound was given orally in doses of 150, 300 and 600 mg/kg/day, suspended in 1ml/kg of 1.25 % aqueous methylcellulose. Pregnants were treated daily from the 7th to the 19th gestational day (the day of arteficial insemination was the first day of pregnancy).Untreated dams served as controls.

For the 150 mg/kg treatment group no clinical symptoms were detected on the dams, their body weight gain was adequate.Fetal parameters, like mortality, body weight and length, placental weight, were not different from the control values. Out of the fetal abnormalities only the incidence of minor anomalies was insignificnatly higher than in controls. The 300 mg/kg group showed that the body weight gain of dams was similar to control. They were free of symptoms during the treatment period. The intrauterine mortality, the weight and length of fetuses and the placental weight corresponded to the control values, however, the incidence of minor Visceral anomalies was slightly but statistically insignificantly higher. For the high dose group (600 mg/kg) clinical symptoms were not observed throughout the study and the body weight gain of darns was satisfactory. Intrauterine mortality, body weight and length of fetuses, and the placental weight were at the control level. The frequency of visceral and skeletal anomalies was slightly and insignificantly higher. Beta-cyclodextrin did not influence the normal intrauterine development of white rabbits in the experimental conditions used. Neither an intrauterine mortality increasing effect, nor teratogenicity was recorded. Based on these results the developmental toxicity of beta-caclodextrine in the doses applied can be excluded in white rabbits.

 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

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

A teratogenicity study of .beta.-cyclodextrin (92.5 % pure) according to OECD 414 was carried out in Sprague-Dawley rats. Four groups, each comprising 20 females, beta Cyclodextrin was administered by oral gavage at levels of 0; 1250; 2500 and 5000 mg/kg body weight/d respectively from day 7 to day 16. The day the presence of spermatozoa was observed on the vaginal smear was considered as day 1 of gestation and the females were sacrificed on day 21 of gestation. In the high dose group five females died from gavage accidents between day 7 and day 17. Necropsy revealed oesophageal perforation for the 5 females. On day 1, there were no statistically significant differences between control and each treated group concerning the body weight variability or mean. The treated animals presented a reduced body weight gain from the beginning of the treatment period. The difference was statistically significant for the high dose group from day 8. At cessation of treatment, the body weight gain of the animals of this group was similar to that of the controls but their body weight at day 21 was still significantly decreased. During the first and third weeks of the study there were no significant differences between control and treated groups concerning food consumption. During the second week which corresponds to the treatment period, a significant decrease in food consumption was noted for mid and high dose groups and (- 7.4 and -19,2 %). No statistical differences were noted on food conversion efficiency during the 3 weeks, between control and treated groups, despite an increase observed during week 2 for mid and high dose groups. Water consumption was similar in all groups during the first week. The visceral examination by technique and skeletal examination by alizarin technique of rat fetuses. from darns treated during the sensitive phase of embryogenesis with the test substance at the oral doses of 1250, 2500 and 5000 mg/kg/day did not reveal any detectable embryotoxic or teratogenic effect. Following parameters were examined in litters: weight of full and empty uterus, weight of placenta, number of live and dead foetuses, number of implantation sites and resorptions, number of corpora lutea, ratio - resorption/implantation. No differences are noted between the groups. The following parameters were examined in foetuses: weight and size of the foetuses, sex, ratio male/live foetuses, ratio female/male. The only significant difference in both sexes is the increased size of the foetuses in groups low and mid dose groups, compared with controls: +0 0.3 (0.84%) and + 0.4 mm (1.14%) respectively for males and females in low dose group + 0.6 (1.68%) and + 0.6 mrn (1.72%) respectively for males and females in the mid dose group.  During the study, it was observed that the administration of beta-cyclodextrin at doses of 0, 1250, 2500 or 5000 mg/kg induced at the high dose level only, a statistically significant reduction of body weight gain in females, accompanied by a reduction of food consumption. On the other hand, these females showed difficulty in swallowing the gavage suspension, and five of them died following oesophageal perforation. The dose level of 5000 mg/kg in a volume of 10 ml/kg is the limit of ingestion in this type of study . The lower dose of 2500 mg/kg per day had no toxic effect on the females. However this produced no observable differences in the weight of the uteri and placentae, the number of live (100%) or dead (0%) foetuses, the number of implantation sites, resorptions and corpora lutea The same applies to the following foetal parameters: size, body weight, sex ratio, visceral and skeletal examinations. which indicate no embryotoxic or teratogenic effects ascribable to the treatment. No embryotoxic or teratogenic effects were observed. It is concluded that beta-cyclodextrin administration at levels up to 2500 mg/kg bw/d had no adverse effect observable on pregnant females and at levels up to 5000 mg/kg bw/d had no adverse effect observable in that kind of study on litters and foetuses.

 

Following a pilot investigation at dose levels of 500 and 2 500 mg/kg bw/day a teratology study was performed in Wistar rats given beta-cyclodextrin as a suspension in 1.25% aqueous methyl cellulose by gavage at doses of 0, 100, 500 and 2 500 mg/kg bw/day on days 7-16 of pregnancy. The group sizes were 26-28 sperm positive females, of which 22-25/group proved to be pregnant. The dams were sacrificed on day 21 and the following parameters monitored: number of corpora lutea and implantations, preimplantation loss, embryonic and late fetal mortality, and number of viable fetuses. About 50% of the fetuses from each litter were examined for soft tissue defects ('s technique) and the remainder were cleared, stained (Alizarin Red or Alcian Blue/Alizarin red) and examined for skeletal anomalies. The doses used in this study had no effect on the clinical condition, food consumption or weight gain of the dams and there were no significant effects on intra-uterine mortality, viable fetuses or on the incidence or type of congenital malformation. The anomalies recorded in this study were regarded as sporadic and unrelated to treatment, and had been seen previously among fetuses from over 600 control dams. Under the conditions of this study, there was no evidence of fetotoxicity or teratogenicity

A teratogenicity study was conducted with beta-Cyclodextrin in CFY rats in which groups of 40 mated females (30 in control group) with positive vaginal smears were given beta-cyclodextrin by gavage in 1% methyl cellulose suspension at dose levels of 0, 200, 400 and 600 mg/kg bw/dy on days 7-11 post coitus. The animals were killed on day 21 and the number of implants, resorptions, live and dead fetuses, fetal weights and rates of congenital anomalies recorded (no details were given of the methodology of the teratological examinations). The conception rate was poor (about 30%) giving only 11-12 pregnant animals in each group. Five animals from the top and middose groups and 1 from the low-dose group died during the study, the deaths being attributed to bronchopneumonia due to misdosing. Maternal weight gain was reduced in a dose-related manner but there were no changes in mean number of implants (12.7 -14.1), resorptions (1.3-7.3%) fetal viability (93-99%) or fetal weight (3.5-3.9 g). There were 5 congenital anomalies reported out of 628 fetuses, 2 in the low-dose group (hydronephrosis, cardiac anomaly), 1 in the middose group (cardiac anomaly) and 2 in the top-dose group (both absence of right kidney); no anomalies were seen in the control group. No details were given of any examination for skeletal anomalies.

Justification for classification or non-classification

The criteria for classifcation and labelling for reproductive and developmental toxicity according CLP are not fulfilled.