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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Name: EUCAROL AGE 91/S
CAS No.: 1228577-41-0
Batch No.: PIC 800
Chemical Name: reaction mass of D-Glucopyranose, oligomers, branched and linear C9-11-alkyl glycosides and Maleic Anhydride and sodium sulfite
Physical State: liquid
Colour: yellow
Density: 1.2 (20°C)
pH: 5.1 (2% in water)
Purity: 50% in water solution

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: males: 9-10 weeks old, females: 9-10 weeks old.
Body weight at the allocation of the animals to the experimental groups:
males: 236 - 267 g
(mean: 250.65 g, ± 20% = 200.52 – 300.78 g)
females: 160 - 188 g
(mean: 175.43 g, ± 20% = 140.34 – 210.51 g)

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 3°C
- Relative humidity: 55 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 1025)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept individually in IVC cages (except during the mating period when one female will be paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 011012)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: aqua ad injectionem
Details on exposure:
The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before dose administration.
The test item formulation was prepared freshly on each administration day before the administration procedure.
The vehicle was also used as control item.
Details on mating procedure:
Mating will be performed using a ratio of 1:1 (male to female). The vaginal smear of the females will be checked every morning after the start of the mating period to confirm the pregnancy. The day of the vaginal plug and/or sperm will be considered as day 0 of gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.6.01 software (p<0.05 was considered as statistically significant).
Duration of treatment / exposure:
The animals will be treated with the test item formulation or vehicle on 7 days per week for a period of 54 days, i.e. during 14 days of pre-mating and 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males will be dosed after the mating period until the minimum total dosing period of 28 days is completed.
Frequency of treatment:
daily
Details on study schedule:
According to the results of the dose range finding study (BSL project no. 130341) and in consultation with the sponsor the following doses (Table 1) were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C).
The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed.
The dosages were corrected regarding the purity of the test item which is 50%. Hence, the dosages of the active ingredient in the test item are 20 mg/kg BW, 100 mg/kg BW, and 500 mg/kg BW.
The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL. The doses were selected on the basis of data from a Dose Range Finding Study (BSL study no. 130343).
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume as used for the high dose group.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 40, 200, 1000 mg/kg bw
Basis:

No. of animals per sex per dose:
80 animals (40 males and 40 females) will be included in the study (10 male and 10 female animals per group).
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS: Yes
General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality were recorded.
Once before the first exposure, and at least once a week thereafter, DETAILED clinical observations were made in all animals outside the home cage in a standard arena.

Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals.
Haematological and clinical biochemistry evaluations were performed on blood samples collected at terminal sacrifice from five males and five randomly selected females from each group. Urinalysis was performed on samples collected at terminal sacrifice from five randomly selected males from each group.
Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behavior observations were performed in the week before the treatment and at the end of the study.

Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the pregnancy. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation.
The cages were arranged in such a way that possible effects due to cage placement were minimized. In case of unsuccessful mating, re-mating of females with proven males of the same group was considered.

Oestrous cyclicity (parental animals):
After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually.
Litter observations:
Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum.

Postmortem examinations (parental animals):
The males were sacrificed after completion of the mating period on treatment days 29 and 30 and the females along with their pups were sacrificed on post natal day 4. Non-pregnant females were sacrificed on day 26.
A full histopathological evaluation of the tissues was performed of 5 randomly selected male and female animals of the control and high dose groups. These examinations were extended to animals of all other dosage groups for treatment-related changes that were observed in the high dose group. Only organs and tissues of the other dosage groups showing changes in the high dose group were examined. Any gross lesion macroscopically identified will be examined microscopically in all animals.
Postmortem examinations (offspring):
Pups sacrificed on post natal day 4 and those found dead, were carefully examined for gross external abnormalities.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

Two animals were found dead / were euthanized during this study. Both were not assumed to be related to the test item.
Some clinical symptoms were observed in MD and HD group which were assumed to be test item related. These symptoms display discomfort and no direct toxicological relevance.
No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups.
No toxicological relevant change in body weights of treatment groups were measured when compared to C animals.
No considerable effect of EUCAROL AGE 91/S on food consumption was found in any of the groups of male animals and in female animals.
No treatment-related effect was observed during the precoital interval or during the duration of gestation when compared with the control group.
The group mean numbers of corpora lutea, number of implantation sites, number of live pups born on PND 0, percentage of pre-implantation loss and post-implantation loss remained unaffected due to the treatment with test item when compared with the control group.
No test item related effect could be found regarding the reproductive indices if treated groups when compared with C group.
No significant effect on survival of the pups from PND 0 to PND 4 was observed in any treatment group when compared with controls.
No treatment-related gross external findings were observed in any of the treated groups.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
not specified
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified

Details on results (F1)

No treatment-related effect of the litter data was observed such as the total number of pups born, number of male and females, sex ratio, live pups on PND 0 and PND 4.
The statistical analysis of the litter weight data revealed no change in any parameter of treated groups when compared to C group.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Mortality

1 female animal of the LD group (#60) was euthanized due to animal welfare towards the end of the treatment period. Histopathology revealed a prominent urogenital inflammation (bilateral ureteral abscess, cystitis, unilateral pyelitis and advanced chronic progressive nephropathy) which was considered to be due to incidental infection and not to be treatment-related. A number of other histological changes noted in this animal were considered to be secondary to the inflammatory and agonal condition (diffuse acute pneumonia of the lung, moderate extramedullary hemopoiesis of the spleen, moderate atrophy/regression of the thymus).

1 female animal of the HD group (#70) was found dead during the early treatment period. In histopathology hemorrhages in the trachea, epithelial damage in the bronchi and multifocalintraalveolar eosinophilic material in the lung was found. Hence, this could be related to aspiration of the test material, possibly due to regurgitation, and is therefore not directly related to systemic adverse effects due to the test item.

Hence, both deaths are not considered to be a systemic test item related effect.

Clinical Observations

In male animals, clear test item related clinical symptoms could be observed in HD animals. The most relevant were slight piloerection (4/10 HD animals) and moving the bedding (5/10 HD animals).

In female animals, test item related clinical signs were observed in MD and HD animals. Again, the most relevant were slight piloerection (2/10 MD animals; 3/10 HD animals) and moving the bedding (2/10 MD animals; 9/10 HD animals).

These symptoms were signs of discomfort but do not display a general toxic effect. Hence, these symptoms alone are not of toxicological relevance.

Functional Observations

No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups.

Body Weight Development

In both males and females, the mean body weight increased with the progress of the study in the C, LD, MD, and HD group.

In male HD group, the mean body weight gain was significantly higher in HD group when compared to C group between pre-mating day 1 and terminal sacrifice (p<0.05; C: 39.1 g/week; HD: 53.4 g /week).

In female animals, no significant change was found in body weight and body weight development.

The increased weight gain in HD group is not considered to be of toxicological relevance.

Food Consumption

In correlation to the body weight and body weight change, the food consumption in both males and females increased with the progress of the study in all groups.

No considerable effect of EUCAROL AGE 91/S on food consumption was found in any of the groups of male animals and in female animals.

Litter Data

No treatment-related effect of the litter data was observed such as the total number of pups born, number of male and females, sex ratio, live pups on PND 0 and PND 4.

A slight tendency to a decreased sex ratio (m/f) in HD group (when compared to C group) is not assumed to be test item related.

Litter Weight Data

The statistical analysis of the litter weight data revealed no change in any parameter of treated groups when compared to C group.

Precoital Interval and Duration of Gestation

No treatment-related effect was observed during the precoital interval or during the duration of gestation when compared with the control group. The values were comparable between the groups.

Pre- and Post-Natal Data

The group mean numbers of corpora lutea, number of implantation sites, number of live pups born on PND 0, percentage of pre-implantation loss and post-implantation loss remained unaffected due to the treatment with test item when compared with the control group.

Although there was a slight increase in post-implantation loss in the MD group compared to the controls, due to the lack of statistical significance and dose-dependency, this effect cannot be attributed to the treatment.

Reproductive Indices

All animals were found sperm positive or – if not – were found to be pregnant. Hence, the copulation index was 100 % in all groups. All animals, except one of the HD group were found to be pregnant. Hence, C, LD, and MD groups had a fertility index of 100 %. HD group had a fertility index of 90 %. This is not assumed to be treatment related. All pregnant dams delivered viable pubs, if possible. Female animals, which died before littering or which were euthanized before littering were excluded from this calculation.

The viability index was slightly reduced in MD group when compared with the control group. This is not assumed to be test item related.

Pup Survival Data

No significant effect on survival of the pups from PND 0 to PND 4 was observed in any treatment group when compared with controls.

One pup (from animal No. 64; MD) was found dead within 24 hours after birth. This is not assumed to be test item related but incidental.

Pup External Findings

No treatment-related gross external findings were observed in any of the treated groups. Few incidences of external findings were observed in all groups which were considered to be spontaneous and not related to the test item.

Haematology and Coagulation

All haematological parameters of treated groups were unchanged when compared with C group.

Furthermore, blood coagulation was not affected by EUCAROL AGE 91/S.

Clinical Biochemistry

All parameters of clinical chemistry were within the normal range of variation for this strain and no biological relevant difference could be found between C and treatment groups.

Urinalysis

The urinalysis performed in male animals revealed a slight increase in leucocytes in MD and HD group. However, this increase was very slight and no dose response was found. Hence, this is assumed to be not test item related but incidental

In female animals no change was found in urine parameters of treated groups.

Pathology

Few specific gross pathological changes were recorded for the male and female animals which were not considered to be treatment-related but incidental.

Organ Weight

In male animals, absolute weights of adrenals were increased when compared to C animals. In particular, +25 % (LD), +18 % (MD), and +12 % (HD) were measured. A similar pattern could be found for the relative weights (to body weight). Due to the absence of statistical significance as well as missing dose dependency, a test item relation is not assumed.

In male animals, absolute weights of the pituitary gland were increased in LD (+14 %) and MD (+18 %) group. By tendency, this could be also found for relative weights (to body weight). Since no dose dependency could be found (missing increase in HD group) and since no statistical significance was calculated, this is not assumed to be test item related.

In male animals, absolute weights of thymi were increased in LD (+15 %), MD (+16 %), and HD (+27 %) group. In relation to body weights, the same tendencies could be found. However, no clear dose dependency is given and no histopathological findings display a test item related change of the thyme-weights. Hence, although it cannot be excluded a test item relation is not assumed and a toxicological relevance cannot be stated.

In female animals, absolute weights of spleens were increased in HD group (+23 %) when compared to C group. This could be confirmed by relative spleen weights (to body weight). Since no statistical significance could be calculated and since a dose dependency is missing (values of MD group were decreased in comparison to C group) a test item relation is not assumed.

Relative organ weights of the right ovaries was significantly decreased (p<0.05) in HD group when compared to C group. However, the total ovary values does not follow a clear dose response, show a high variability and are not statistical significantly different. Hence, this significant change is assumed to be incidental and not test item related.

In female animals, absolute weights of uteri were decreased in treatment groups. In particular, - 12% (LD), - 6% (MD), and -13 % (HD) were calculated. These tendencies could be confirmed in relation to body weight. No dose dependency could be found and no statistical significance was observed. Hence, no test item relation can be assumed.

In female animals, thyroid/parathyroid gland was increased in MD and HD groups. In particular, + 23 % (MD) and +56 % (HD) were observed. These weights could be confirmed in relation to body weight. Due to the high variation, no statistical significance could be calculated. Furthermore, no findings were revealed during the histological analysis. Hence, these weight changes are not assumed to be of toxicological relevance.

Histopathology

No test item-related macroscopic or histopathological findings were noted in reproductive and other organs under the conditions of this study. Two females treated at 40 mg/kg/day and one female treated at 1000 mg/kg/day were found not to be pregnant at terminal sacrifice, but without relationship to treatment.

Applicant's summary and conclusion

Conclusions:
On the basis of this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with EUCAROL AGE 91/S in male and female Wistar rats with dose levels of 40, 200, and 1000 mg/kg body weight day the following conclusions can be made:
No toxicological relevant effects of EUCAROL AGE 91/S were found at any dose level. Hence, NOAEL of EUCAROL AGE 91/S in this study is considered to be 1000 mg/kg body weight for P and F1 generation.