Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
reproductive toxicity, other
Remarks:
enhanced combined repeated dose toxicity study with reproduction/developmental toxicity screening test
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
In order to attain statistical power equivalent to that of a full-scale Generation study (e.g. OECD 416), this study enhanced the guideline recommendations by increasing the group sizes to 22 - 25 (pregnant females).
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
9-Octadecenoic acid (Z)-, reaction products with 2-[(2-aminoethyl)amino]ethanol
EC Number:
272-379-2
EC Name:
9-Octadecenoic acid (Z)-, reaction products with 2-[(2-aminoethyl)amino]ethanol
Cas Number:
68815-51-0
Molecular formula:
C18 H34 O2 . C4 H12 N2 O
IUPAC Name:
2-(2-aminoethylamino)ethanol; (Z)-octadec-9-enoic acid
Details on test material:
- Name of test material (as cited in study report): Kerocom F 100
- Physical state: solid (melt)/brown
- Analytical purity: ca. 100%
- Lot/batch No.: 09001529U0
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the manufacturer, and the manufacturer holds this responsibility
- Storage condition of test material: Room temperature, avoid temperatures >50 °C

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: About 11-12 weeks
- Diet (e.g. ad libitum): Ground Kliba maintenance diet mouse/rat
- Water (e.g. ad libitum): Drinking water


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was liquefied at a temperature of about 40°C in a drying chamber. Thereafter, the specific amount of test substance was weighed, topped up with olive oil in a beaker and intensely mixed with a magnetic stirrer. During administration, the preparations was kept homogeneous with a magnetic stirrer. The test substance preparations were prepared at intervals which guarantee that the test substance concentrations in the vehicle will remain stable. The test substance preparations were stored at room temperature.


VEHICLE
- Amount of vehicle (if gavage): 4 ml/kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
28 days in males; 48 days in females
Frequency of treatment:
Once daily
Details on study schedule:
- Age at mating of the mated animals in the study: 13-14 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
10, 100, 300 and 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- After the end of the administration period (at least 28 days) all surviving parental males were sacrificed and examined.
- The parental females were allowed to deliver and rear their pups until post natal day (PND) 4. On PND 4, all pups were sacrificed and examined.
- On PND 4 of the female, which delivers last or on one of the following days, all parental females were sacrificed and examined.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations checked: any signs of morbidity, pertinent behavioral changes and signs of overt toxicity


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed clinical observations (DCO) were performed in 10 parental males and females per group once before the first administration and thereafter at weekly intervals


BODY WEIGHT: Yes
- Time schedule for examinations: once a week


FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes, once a week
Sperm parameters (parental animals):
Parameters examined in P male parental generations: testis weight, epididymis weight, weight of Prostate and Seminal vesicles including coagulation glands.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities


GROSS EXAMINATION OF PUPS:
On post natal day (PND) 4, all pups were sacrificed with CO2 and examined externally, eviscerated and their organs were assessed macroscopically, paying particular attention to potential pericardial blood vessel effects. Pups that died or were sacrificed in a moribund state were eviscerated and examined for possible defects and/or the cause of death, paying particular attention to potential pericardial blood vessel effects. All pups were preserved in 4% neutral buffered formaldehyde after performed gross examination.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [28 days after dosing]
- Maternal animals: All surviving animals [PND 4]


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera with particular attention on the reproductive organs


HISTOPATHOLOGY / ORGAN WEIGHTS
- The tissues prepared for microscopic examination: All gross lesions, Brain, Spinal cord (cervical, thoracic and lumbar cord), Sciatic nerve, Pituitary gland, Salivary glands (glandula mandibularis and glandula sublingualis), Thyroid glands (with parathyroid glands), Adrenal glands, Prostate gland, seminal vesicles, coagulation glands, Ovaries (fixed in modified Davidson's solution), Uterus, oviducts, vagina, Testes (fixed in modified Davidson's solution), Epididymides (fixed in modified Davidson's solution), Female mammary gland, Thymus, Lymph nodes (axillary and mesenteric), Spleen, Trachea, Lungs, heart, Aorta, Liver, Pancreas, Kidneys, Esophagus, Stomach (forestomach and glandular stomach), Duodenum, jejunum (with Peyer’s patches), ileum, Cecum, colon, rectum, Urinary bladder, Sternum with marrow, Larynx, Pharynx, Nose (nasal cavity), Bone marrow (femur), Eyes with optic nerve, Femur with knee joint, Skin, Skeletal muscle.
- The weights of following organs were determined: Anesthetized animals, Liver, Kidneys, Adrenal glands, Testes, Epididymides, Prostate, Seminal vesicles including coagulation glands, Ovaries, Uterus, Thymus, Spleen, Brain, Heart, Thyroid glands (with parathyroid glands).
Statistics:
Dunnett test (two-sided) was performed for food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of pups delivered per litter. Fisher's exact test was used for number of live and dead pups and different indices (e.g. mating index, fertility index, gestation index) and number of litters with necropsy findings in pups. Wilcoxon test (one-sided) was used for proportion of pups with necropsy findings per litter. Kruskal-Wallis test (two-sided) and Wilcoxon test was used for feces, rearing, grip strength forelimbs, grip strength hind-limbs, landing foot-splay test, motor activity.
Reproductive indices:
- Male reproduction data: The pairing partners, the number of mating days until vaginal sperm was detected in female animals, and the gestational status of the females were recorded for F0 breeding pairs. For the males, mating and fertility indices were calculated.
- Female reproduction and delivery data: The pairing partners, the number of mating days until vaginal sperm were detected, and gestational status were recorded. For the females, mating, fertility and gestation indices were calculated. The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters. The implantations were counted and the postimplantation loss (in %) was calculated ((To determine the number of implantation sites, the apparently non-pregnant uteri were stained for about 5 minutes in 10% ammonium sulfide solution according to the method of SALEWSKI (Salewski, E.; 1964). Then the uteri were rinsed carefully under running water. Thereafter the implantation sites were recorded for calculation of the postimplantation loss.).
Offspring viability indices:
- Pup viability/mortality: The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters. In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturday, Sunday or public holidays. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4 were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on PND 0 and on PND 4. The viability index was calculated according to the following formula: Viability index (%) = [(number of live pups on day 4 after birth)/(number of live pups on the day of birth)] x 100.

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no test substance-related or spontaneous mortalities in any of the groups.
Clinical observations for males and females (except gestation and lactation periods): All male and 17 female F0 parental animals of the 1000 mg/kg bw/d-group, 24 male and 2 female animals of the 300 mg/kg bw/d-dose group and one F0 male animal of the 100 mg/kg bw/d-group showed transient salivation during major parts of the treatment period. Salivation persisted in the respective animals only for some minutes after daily gavage dosing (i.e. up to 10 minutes). This symptom was initially observed during study week 0. The temporary salivation is considered to be test substance-induced. From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse toxicologically relevant finding. No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any male or female F0 generation parental animals at the 10 mg/kg bw/d dose level. One F0 parental female of the high-dose group (1000 mg/kg bw/d) showed a skin lesion on its left flank during study week 7. This finding is considered to be incidental.
Clinical observations for females during gestation of F1 litter: At a dose level of 1000 mg/kg bw/d) 22 out of 25 F0 females and at a dose level of 300 mg/kg bw/d) 4 out of 25 F0 females (300 mg/kg bw/d) showed transient salivation during major parts of gestation period (GD 0 – 21 in test group 4, GD 4 – 9 in test group 3). Two low-dose F0 females (10 mg/kg bw/d) were not sperm-positive. However, one of these females had pups, giving evidence for successful copulation. Furthermore, one sperm-positive F0 female of test group 1 (10 mg/kg bw/d), three of test group 3 (300 mg/kg bw/d) and one of test group 4 (1000 mg/kg bw/d) did not deliver any F1 pups. There were no clinical findings in F0 females during the gestation period at the 10 and 100 mg/kg bw/d dose levels.
Clinical observations for females during lactation of F1 litter: In the high-dose group (1000 mg/kg bw/d), 12 out of 24 F0 females showed transient salivation during PND 0 – 4. There were no test substance-related clinical findings in F0 females at the 10, 100 and 300 mg/kg bw/d dose levels. One low-dose F0 female (10 mg/kg bw/d) nursed its pups not properly on PND 1 and 2. As a consequence, all pups of this litter showed hypothermia and were cannibalized on PND 3 at the latest. This single finding is not considered to be related to the test substance.
Detailed clinical observations: The detailed clinical observations, which took place in study weeks 0-4 in all parental animals and, additionally, in weeks 5-7 just in females, did not reveal any abnormalities in the animals of all test groups (0, 10, 100, 300 and 1000 mg/kg bw/d).

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Mean body weights and mean body weight gain of the F0 males in test groups 1-4 (10, 100, 300 and 1000 mg/kg bw/d) were comparable to the concurrent control throughout the entire study. Mean body weights of the F0 females were comparable to the controls throughout the entire study at all dose levels (10, 100, 300 and 1000 mg/kg bw/d). Females at the 300 and 1000 mg/kg bw/d dose levels gained more weight than the controls during gestation, although the increase was significant only at 1000 mg/kg bw/d during GD 7-14. On the other hand, these females lost previously gained surplus weight within days after parturition (statistically significant), which resulted in comparable body weights of control, 300 and 1000 mg/kg bw/d dose groups on PND 4. Thus, an association of this up and down of body weight change to treatment is not assumed. Mean body weight gain of the 100 and 1000 mg/kg bw/d females was temporarily increased during premating week 1-2, which are also considered to be spontaneous findings. Mean body weight gain of the 10 mg/kg bw/d and 100 mg/kg bw/d (apart from the incidental increase week 1-2) groups was comparable to the control group throughout the entire premating, gestation and lactation periods.
Food consumption of F0 males was statistically significantly increased during premating week 1-2 at the 300 and 1000 mg/kg bw/d dose levels while it was comparable to the controls during the entire study at the 10 and 100 mg/kg bw/d dose levels. The slight and temporary increase in test groups 3 and 4 is not considered to be treatment-related. Food consumption of F0 females was statistically significantly increased during premating week 1-2 at the 1000 mg/kg bw/d dose level, while it was comparable to the controls during remaining premating, gestation and lactation. Food consumption of F0 females was comparable to the controls during the entire study at the 10, 100 and 300 mg/kg bw/d dose levels. The slight and temporary increase in test group 4 is not considered to be treatment-related.

REPRODUCTIVE FUNCTION (PARENTAL ANIMALS)
One male of test group 1 (10 mg/kg bw/d), 3 males of test group 3 (300 mg/kg bw/d, and 1 male of test group 4 (1000 mg/kg bw/d) showed an impaired fertility. Accordingly, one female of test group 1 (10 mg/kg bw/d), 3 females of test group 3 (300 mg/kg bw/d, and 1 female of test group 4 (1000 mg/kg bw/d) were not pregnant. All females that were not pregnant and the male mating partners did not show gross lesions. The male with impaired fertility in test group 1 (10 mg/kg bw/d) showed unilaterally a spermatogenic granuloma in the epididymis. In one male with impaired fertility of test group 3, a minimal unilateral focal tubular degeneration was observed unilaterally in the testis. In addition, this male showed a severe multifocal lymphoid cell infiltration in the prostate but these findings cannot explain the infertility. All females that were not pregnant and the other male mating partners did not show relevant histopathological findings.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Male reproduction data: For nearly all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index varied between 96% (test group 1 - 10 mg/kg bw/d) and 100% (test groups 0, 2, 3, and 4 – 0, 100, 300, and 1000 mg/kg bw/d). Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter. One low-dose male, three high mid-dose males and one high-dose male did not generate F1 pups. Thus, the male fertility index ranged between 88% (test group 3 - 300 mg/kg bw/d), 96% (test groups 1 and 4 - 10 and 1000 mg/kg bw/d) and 100% (control and test group 2 - 0 and 100 mg/kg bw/d) without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study. All respective values are within the range of the historical control data of the test facility. The apparently infertile male rats did not show histopathological findings that could explain infertility. Only the apparently infertile low-dose male showed unilaterally a spermatogenic granuloma in the epididymis.

- Female reproduction and delivery data: The female mating index calculated after the mating period for F1 litter varied between 96% (test group 1 – 10 mg/kg bw/d) and 100% (test groups 0, 2, 3, and 4 – 0, 100, 300, and 1000 mg/kg bw/d). The mean duration until sperm was detected (GD 0) amounted to 2.5, 2.0, 2.2, 2.2, and 2.4 days (0, 10, 100, 300, and 1000 mg/kg bw/d, respectively). All sperm positive rats delivered pups or had implants in utero with the following exceptions: three high mid-dose females and one high-dose female did not become pregnant. Although no sperm was detected in vaginal smears of one low-dose rat, it was pregnant and had pups. The female fertility index varied between 88% (test group 3 - 300 mg/kg bw/d), 96% (test group 4 - 1000 mg/kg bw/d), and 100% (test groups 0, 1, 2 - 0, 10, 100 mg/kg bw7d). These values reflect the normal range of biological variation inherent in the strain of rats used for this study. All respective values are within the range of the historical control data of the test facility and do not show any relation to dosing. There were no corroborative histopathological findings in the sexual organs of the non-pregnant females. The mean duration of gestation values varied between 21.8 and 22.1 days without any relation to dosing. Implantation, prenatal development and delivery were not affected by the treatment since neither the mean number of implantation sites nor the postimplantation loss or the average litter size showed any statistically significant differences between the groups. The gestation index varied between 96% (test group 1 - 10 mg/kg bw/d) and 100% (test groups 0, 2, 3, and 4 - 0, 100, 300, and 1000 mg/kg bw/d). The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 99% (test groups 0, 1, 2 and 3 - 0, 10, 100 and 300 mg/kg bw/d) and 100% (test group 4 - 1000 mg/kg bw/d). Moreover, the number of stillborn pups was comparable between the groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
The relative thymus weights were decreased in females of test groups 3 (300 mg/kg) and 4 (1000 mg/kg). Because there were no histopathological findings, a substance-related effect seems rather unlikely. Because there was no dose-response relationship and because there were no histopathological correlates, the increased kidney weights in females of test groups 2, 3 (absolute and relative) and 4 (relative) are considered incidental. The decreased spleen weights in males of test groups 1 (absolute and relative) and 3 (relative) as well as the increased absolute and relative weights of adrenal glands in males of test group 1 are considered incidental.

GROSS PATHOLOGY (PARENTAL ANIMALS)
All gross lesions occurred either singly or were biologically equally distributed over the control group and the treatment groups. They are considered to be incidental.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Mesenteric lymph nodes: Sinus histiocytosis was observed in the mesenteric lymph nodes of some treated males and females. The sinusoids were filled with a various number of macrophages containing foamy cytoplasm. The occurrence of sinus histiocytosis in the mesenteric lymph node might be caused by phagocytosis of the test substance and due to the lack of cytotoxicity is considered as non adverse.
Lungs: Two different findings were seen in the lungs, a granulomatous inflammation considered as treatment-related and spontaneous focal alveolar histiocytosis. A minimal to moderate granulomatous inflammation was observed in the lungs of some treated males. In all affected animals, the granulomatous inflammation was noted only in one of two investigated lobes (left lobe and right caudal lobe). The affected males showed few to multiple granulomatous foci in the left lobe (except of one animal: right caudal lobe), mainly near the bronchi and their branches. This distribution pattern indicates that after retraction of the gavage tube during gavage, small amounts of the test substance/ olive oil preparation might have been regurgitated, aspirated and finally caused the granulomatous inflammation. The granulomatous foci were characterized by a large number of intraalveolar macrophages, granulocytes, lymphocytes, desquamated epithelial cells and single multinucleated giant cells. The alveolar walls were lined by large cuboidal cells, partly with prominent nuclei. The cytoplasm of these alveolar epithelial cells was vacuolated or basophilic. Taken together, the occurrence of the granulomatous inflammation is considered as a direct and local effect of the test substance caused by gavage procedure. As consequence of a systemic toxic effect not only one lung lobe should be affected as seen in this study.
The granulomatous inflammation had to be differentiated from focal alveolar histiocytosis. The alveolar histiocytosis was characterized by variable degrees of intra-alveolar aggregation of macrophages containing foamy cytoplasm. In few males, the alveolar histiocytosis was associated with few inflammatory infiltrates. The incidence of alveolar histiocytosis was slightly increased in males of test group 4 (1000 mg/kg bw/d). Because there was no dose-response relationship and because the foci of alveolar histiocytosis occurred only singly (grade 1), a treatment-related effect seems rather unlikely.
All additional findings occurred either individually or were biologically equally distributed over the control group and the treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects on reproductive performance and fertility

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
The mean number of delivered pups per dam and the rate of liveborn and stillborn pups were evenly distributed among treated groups and control. The respective values reflect the normal range of biological variation inherent in the strain used in this study. The viability index as indicator for pup mortality between PND 0-4 varied between 96% (test group 1 - 10 mg/kg bw/d), 99% (test groups 2, 3, and 4 - 100, 300, and 1000 mg/kg bw/d) and 100% (control). The lower viability index in the 10 mg/kg bw/d group was caused by one dam, which nursed its pups not properly and thus lost all pups until PND3. This is considered to be an incidental finding. Sex distribution and sex ratios of live F1 pups on the day of birth and on PND 4 did not
show biologically relevant differences between treated groups and control.

CLINICAL SIGNS (OFFSPRING)
The F1 pups did not show any test substance-induced adverse clinical signs. Several pups of one low-dose dam showed hypothermia during lactation, because they were not properly attended to by their mother. One pup from one dam of test group 4 (1000 mg/kg bw/d) had a short tail caused by an accidental lesion.

BODY WEIGHT (OFFSPRING)
Mean pup body weights and pup body weight gain of all test substance-treated groups were comparable to the concurrent control group. One male runt was seen in test group 0 (control), 3 male and 3 female runts were seen in test group 1 (10 mg/kg bw/d), one male and 2 female runts were seen in test group 3 (300 mg/kg bw/d) and 3 male and 3 female runts were seen in test group 4 (1000 mg/kg bw/d). Distribution of runts among groups does not suggest a relationship to treatment.

GROSS PATHOLOGY (OFFSPRING)
Some F1 pups showed spontaneous findings at necropsy, such as post mortem autolysis, anasarca, situs inversus, abnormal liver lobation, small liver lobe, empty stomach, hydronephrosis, distended bladder, hydroureter, hemorrhagic testis and paw hyperflexion. The overall number of affected pups/litter with necropsy findings in the test substance-treated groups was comparable to the concurrent control group (0.9% / 1.9% / 2.7% / 2.1% / 3.1%). These pup necropsy findings occurred without relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Therefore, these findings were not considered to be associated to the test substance. Particular attention was paid to the pericardial blood vessels, where a number of F1 pups in the substance-treated groups showed findings (see table below). Macroscopical examination revealed aneurysms of 2 major pericardial blood vessels in pups of all dose groups (10, 100, 300, and 1000 mg/kg bw/d). The ductus arteriosus was the most frequently affected site where aneurysms occurred, thus, this seems to be the most sensitive area to develop aneurysms in this study.

Effect levels (F1)

Dose descriptor:
LOAEL
Generation:
F1
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity (aneurysm)

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Female reproduction and delivery - Mean number of implantation sites, postimplantation loss and average litter size: 

 

Control

10 mg/kg bw/d

100 mg/kg bw/d

300 mg/kg bw/d

1000 mg/kg bw/d

Implantations/dam

11.4

11.3

11.2

12.4

12.5

Postimplantation loss (%)

6.6

11.0

4.3

5.2

5.7

Mean litter size

10.6

10.7

10.8

11.7

11.8

 

Occurrence of pup necropsy observations concerning pericardial blood vessels (expressed as pup incidence, litter incidence and mean percentage of affected pups/litter): 

Finding

Test group 0
Control

Test group 1
10 mg/kg

Test group 2
100 mg/kg

Test group 3
300 mg/kg

Test group 4
1000 mg/kg

aneurysm of descending aorta

0
0
0.0

0
0
0.0

1
1
0.4

0
0
0.0

1
1
0.3

aneurysm of ductus arteriosus

0
0
0.0

1
1
0.3

0
0
0.0

2
2
0.8

6
5*
2.1**

Total aneurysms

0
0
0.0

1
1
0.3

1
1
0.4

2
2
0.8

7
5
2.4

*: p ≤ 0.05, **: p ≤0.01

 

Applicant's summary and conclusion

Conclusions:
The test substance had no adverse effects on fertility of the F0 parental animals of both sexes at 10, 100, 300 and 1000 mg/kg bw/d. The NOAEL for reproductive performance and fertility is 1000 mg/kg body weight/day for the F0 parental rats. The NOAEL for general, systemic toxicity of the test substance is 1000 mg/kg body weight/day for the F0 parental animals, the highest dose tested. The NOEL (no observed effect level) is 10 mg/kg bw/d for the F0 parental rats based on treatment-related, local and non-adverse effects such as sinus histiocytosis in the mesenteric lymph nodes at 300 and 1000 mg/kg bw/d and granulomatous inflammation of one lung lobe with no corroborative clinical or pathological findings indicative of systemic toxicity at 100, 300 and 1000 mg/kg bw/d.
Executive summary:

In order to attain statistical power equivalent to that of a full-scale Generation study (e.g. OECD 416), this study enhanced the guideline recommendations of OECD TG 422 by increasing the group sizes to 22 - 25 (pregnant females). The study was performed in compliance with GLP.

Kerocom F 100 was administered orally via gavage to groups of 25 male and 25 female Wistar rats (F0 animals) at doses of 10, 100, 300 and 1000 mg/kg body weight/day. Control animals were dosed daily with the vehicle (Olive oil Ph. Eur./DAB). The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females.

Kerocom F 100 had no adverse effects on fertility of the F0 parental animals of both sexes at 10, 100, 300 and 1000 mg/kg bw/d. Almost all of the F0 parental animals proved to be fertile. The occurrence of a few infertile pairs in test groups 1, 3 and 4 (10, 300 and 1000 mg/kg bw/d) did not suggest any relation to treatment. Gross and histopathological examinations of the respective animals of both genders did not reveal test substance-induced findings, which may have accounted for the observed infertility. The test substance caused also no impairments of the reproductive performance. Mating behaviour, conception, gestation, parturition, as well as sexual organ weights and gross and histopathological findings of these organs were not influenced.

The following test substance-related adverse effects/findings were noted:

 

Test group 4 (1000 mg/kg bw/d):

F0 parental animals:

- Minimal to slight sinus histiocytosis in the mesenteric lymph nodes of 13 males and minimal to severe sinus histiocytosis in the mesenteric lymph nodes of all females

- Minimal to moderate granulomatous inflammation in the lungs of 10 males

F1 pups:

- Macroscopic changes related to the major pericardial blood vessels, specifically 7/282 pups from 5 litters with aneurysms in the descending aorta (1) or ductus arteriosus (6), compared to 0/248 in control pups

 

Test group 3 (300 mg/kg bw/d):

F0 parental animals:

- Minimal to moderate sinus histiocytosis in the mesenteric lymph nodes of 12 females

- Minimal to slight granulomatous inflammation in the lungs of 3 males

F1 pups:

- Macroscopic changes related to the major pericardial blood vessels, specifically 2/245 pups from 2 litters with aneurysms in the ductus arteriosus, compared to 0/248 in control pups

 

Test group 2 (100 mg/kg bw/d):

F0 parental animals:

- Minimal granulomatous inflammation in the lung of 1 male

F1 pups:

- Macroscopic changes related to the major pericardial blood vessels, specifically 1/260 pup with an aneurysm in the aorta descendens, compared to 0/248 in control pups

 

Test group 1 (10 mg/kg bw/d):

F0 parental animals:

- No test substance-related adverse findings

F1 pups:

- Macroscopic changes related to the major pericardial blood vessels, specifically 1/228 pup with an aneurysm in the ductus arteriosus, compared to 0/248 in control pups

 

Conclusion: The NOAEL for reproductive performance and fertility is 1000 mg/kg body weight/day for the F0 parental rats. The NOAEL for general, systemic toxicity of the test substance is 1000 mg/kg body weight/day for the F0 parental animals, the highest dose tested. The NOEL (no observed effect level) is 10 mg/kg bw/d for the F0 parental rats based on treatment-related, local and non-adverse effects such as sinus histiocytosis in the mesenteric lymph nodes at 300 and 1000 mg/kg bw/d and granulomatous inflammation of one lung lobe with no corroborative clinical or pathological findings indicative of systemic toxicity at 100, 300 and 1000 mg/kg bw/d.