Cyclohexapentylose

Developmental toxicity / teratogenicity

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohexapentylose

EC Number:

233-007-4

EC Name:

Cyclohexapentylose

Cas Number:

10016-20-3

Molecular formula:

C36H60O30

IUPAC Name:

(1S,3R,5R,6S,8R,10R,11S,13R,15R,16S,18R,20R,21S,23R,25R,26S,28R,30R,31R,32R,33R,34R,35R,36R,37R,38R,39R,40R,41R,42R)-5,10,15,20,25,30-Hexakis(hydroxymethyl)-2,4,7,9,12,14,17,19,22,24,27,29-dodecaoxahe ptacyclo[26.2.2.23,6.28,11.213,16.218,21.223,26]dotetracontane-31,32,33,34,35,36,37,38,39,40,41,42-dodecol

Details on test material:

The test material .alpha.-cyclodextrin (.alpha.-CD) were received from the sponsor on February 11 and 12, 1991, respectively and stored at room temperature. Lactose was obtained form Borculo Whey Products, Borculo, the Netherlands.
Characteristics of the test substance as given by the sponsor were as follows:

Chemical and product name: .alpha.-cyclodextrin
Batch no.: 0030
CAS. reg. no.: 10016-20-3
Appearance: white crystalline powder
Solubility (in water): 6.0 %
Storage: at room temperature

Test animals

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 .alpha.-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.

Administration / exposure

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 % .alpha.-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

Doses / concentrations

Remarks:

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

No. of animals per sex per dose:

16 female animals per group

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.

Examinations

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 and 21.

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

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Test substance analyses:
At the start of the study the stability of the test substances, .alpha.-CD and lactose, in the diet was determined. All levels of either test substance appeared to be sufficiently stable in cereal-based diet when kept for 28 days at 4 °C and at room temperature. The distribution of .alpha.-CD and lactose in the basal diet was sufficiently homogeneous. The actual levels of .alpha.-CD and lactose in the various diets were slightly lower than, but sufficiently close to the nominal levels.

Clinical signs:
All animals survived according to schedule. In the animals belonging to the 5 % .alpha.-CD and the 20 % lactose group no clinical signs were observed.
In 2 animals of the 10 % .alpha.-CD group a subcutaneous abcess in the head was observed. Two animals suffered from nasal discharge, 1 of the 10 % .alpha.-CD. Alopecic areas were observed in 1 animal of the 10 % .alpha.-CD group. Mucous faeces were observed in 1 animal of the control group; diarrhoea in 1 animal of the 20 % .alpha.-CD group. In 2 cases blood on the excrement collecting tray was observed, once of an animal of the 10 % .alpha.-CD group. Both animals still were pregnant at caesarian section. Of all effects described above, only the diarrhoea was considered to be treatment-related.

Maternal body weight:
Mean maternal body weight during gestation did not differ between the test groups and the control group.

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 % .alpha.-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 1.4-2.2 g/kg bw/d for the 5 % .alpha.-CD group, 3.0-4.6 g/kg bw/d for the 10 % .alpha.-CD group, 5.9-7.5 g/kg bw/d for the 20 % .alpha.-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, a liver with a marked lobular pattern in 1 animal of the 5 % .alpha.-CD group. In 1 animal of the 10 % .alpha.-CD group the uterus was found to be filled with haemorrhagic fluid. In 2 animals, 1 of the control group and 1 of the 20 % .alpha.-CD group, the uterus was filled with pus. In 1 animal of the 20 % lactose group, the uterus had a red appearance.

Maternal performance: During gestation, 1 female of the control 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 but one (of the 20 % .alpha.-CD group) had live fetuses.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

10 other: % in the diet

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

> 20 other: % in the diet

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

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, except for the 5 % .alpha.-CD group, but this was considered to be a fortuitous finding. 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. Two fetuses with a dysmature appearance were observed, one in a litter of the 5 % .alpha.-CD group and 1 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 thefetal 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 % .alpha.-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, haemorrhagicurinary 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 20% .alpha.-CD group 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 1 fetus of the 20 % .alpha.-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. 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 crookedhind 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% 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 statisticaly 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.

Applicant's summary and conclusion

Conclusions:

Dietary administration of .alpha.-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.

Animal of the 20 % .alpha.-CD group group had diarrhoea, during a few days of the study only.

Food intake was not affected in most groups, apart from the 20 % .alpha.-cyclodextrin group 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 treatment-related 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 .alpha.-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 .alpha.-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 .alpha.-cyclodextrin in rabbits. In this study .alpha.-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.

Food intake was not affected in most groups, apart from the 20% .alpha.-cyclodextrin group 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 .alpha.-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 .alpha.-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 .alpha.-cyclodextrin for maternal effects is 10% in the diet.