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Toxicological information

Developmental toxicity / teratogenicity

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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: 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:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
The study was performed according to OECD guideline 422. After a 14-day premating period, the male and female parental animals were mated overnight in a 1:1 ratio. 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.
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
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
Analytical verification of doses or concentrations:
yes
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
Duration of treatment / exposure:
28 days in males; 48 days in females
Frequency of treatment:
Once daily
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:
- 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

Maternal 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


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on PND 4
- Organs examined: external and internal examinations including the cervical, thoracic, and abdominal viscera with particular attention on the reproductive organs
Ovaries and uterine content:
Examinations included:
- Ovaries and uterus were weighed and subjected to histological examination
Fetal examinations:
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.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
The following test substance-related adverse effects/findings were noted in F0 female parental animals: 
- Test group 4 (1000 mg/kg bw/d): Decreased hemoglobin values and red blood cell counts, increased ALT activities and globulin values 
- Test group 3 (300 mg/kg bw/d): No test substance-related adverse findings
- Test group 2 (100 mg/kg bw/d): No test substance-related adverse findings 
- Test group 1 (10 mg/kg bw/d): No test substance-related adverse findings

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
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), giving some evidence that the test compound caused a rather specific type of developmental toxicity. 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. Aneurysms were observed in pups from the lower dose groups (10 and 100 mg/kg bw/d), but at a lower incidence compared to the high dose group. Additionally, it must not be overlooked that there was no apparent dose-related increase in the incidence of this lesion between the two lower Kerocom F 100 treatment groups (0, 1, 1, 2, 7), representing a 10-fold range in administered dose. This lack of internal consistency in the dose response at dose levels below 300 mg/kg bw/day must be taken into consideration when evaluating these findings. In other words, although the incidence of the lesion is higher in treatment groups than control, the ‘flat’ dose-response is contraindicative of a treatment-related finding below 300 mg/kg bw/day. On the other hand, aneurysms were not observed in any control animal. In such cases, it is usual to refer to historical control data to determine if treatment groups are different to a ‘normal population’, or whether the disparity is due to an aberrant response in the concurrent control. Macroscopically visible aneurysms have so far not been observed as spontaneous findings in the Wistar rat strain. However, some of these lesions were observed in a special control study in PND 4 pups, under conditions (i.e. microscopic evaluation of the great vessels) not routinely applied to control animals in OECD 421/422 or OECD 415/416 study designs. The incidence in this control study was 1 dissecting aneurysm and 2 focal hemorrhages within the vessel wall, which are thought to be precursor lesions of aneurysm. This shows that such lesions can also develop in untreated animals, although rarely.
There were no other findings in the offspring which might indicate a selective influence of the test compound on pre- and postnatal development.

Effect levels (fetuses)

Dose descriptor:
LOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: Macroscopical examination revealed aneurysms of 2 major pericardial blood vessels in pups of all dose groups.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

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 NOAEL for maternal toxicity of the test substance is 300 mg/kg body weight/day based on adverse clinical pathological findings at next higher dose. A NOAEL for developmental toxicity in the F1 progeny could not be determined based on the finding of aneurysms of 2 major pericardial blood vessels in pups of all dose groups. The LOAEL for developmental toxicity was determined to be 10 mg/kg bw/day based on the finding of one aneurysm in the ductus arteriosus in 1/228 pups in this dose group.
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.

 

The NOAEL for general, systemic maternal toxicity of the test substance was determined to be 300 mg/kg body weight/day based on adverse clinical pathological findings at next higher dose (decreased hemoglobin values and red blood cell counts, increased ALT activities and globulin values).

 

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.

Aneurysms were observed in pups from the lower dose groups (10 and 100 mg/kg bw/d), but at a lower incidence compared to the high dose group. Additionally, it must not be overlooked that there was no apparent dose-related increase in the incidence of this lesion between the two lower Kerocom F100 treatment groups (0, 1, 1, 2, 7), representing a 10-fold range in administered dose. This lack of internal consistency in the dose response at dose levels below 300 mg/kg bw/day must be taken into consideration when evaluating these findings. In other words, although the incidence of the lesion is higher in treatment groups than control, the ‘flat’ dose-response is contraindicative of a treatment-related finding below 300 mg/kg bw/day.

On the other hand, aneurysms were not observed in any control animal. In such cases, it is usual to refer to historical control data to determine if treatment groups are different to a ‘normal population’, or whether the disparity is due to an aberrant response in the concurrent control. Macroscopically visible aneurysms have so far not been observed as spontaneous findings in the Wistar rat strain. However, some of these lesions were observed in a special control study in PND 4 pups, under conditions (i.e. microscopic evaluation of the great vessels) not routinely applied to control animals in OECD 421/422 or OECD 415/416 study designs. The incidence in this control study was 1 dissecting aneurysm and 2 focal hemorrhages within the vessel wall, which are thought to be precursor lesions of aneurysm. This shows that such lesions can also develop in untreated animals, although rarely.

 

Conclusion: The NOAEL for maternal toxicity of the test substance was determined to be 300 mg/kg body weight/day based on adverse clinical pathological findings at next higher dose. Based on the current limited knowledge, a NOAEL for developmental toxicity in the F1 progeny could not be determined, and the LOAEL for developmental toxicity was set to be 10 mg/kg bodyweight/day based on the finding of aneurysms of 2 major pericardial blood vessels in pups of all dose groups (10, 100, 300, and 1000 mg/kg bw/d). There was no apparent dose-related increase in the incidence of this lesion between the two lower Kerocom F100 treatment groups (0, 1, 1, 2, 7), representing a 10-fold range in administered dose.