Registration Dossier

Diss Factsheets

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015/2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

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

Constituent 1
Chemical structure
Reference substance name:
Fatty acids, C16-C18 and C18-unsatd., ME esters, epoxidized
EC Number:
605-143-8
Cas Number:
158318-67-3
Molecular formula:
Unspecified (UVCB)
IUPAC Name:
Fatty acids, C16-C18 and C18-unsatd., ME esters, epoxidized
Test material form:
liquid
Specific details on test material used for the study:
Batch identification: CE80580015
Content: 99.9% (100% - water content)
Storage stability: Expiry date: 01 May 2016
Physical state/appearance: Liquid/ colorless, clear

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: 10-12 weeks
- Weight at study initiation:
- Housing: single caging
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: six days

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

IN-LIFE DATES: 1st cohort From: 2015-07-09 To: 2015-07-29, 2nd cohort: from 2015-07-10 to 2015-07-30

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The oily test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature. For the test substance preparations, the specific amount of test substance was weighed, topped up with corn oil in a graduated flask and intensely mixed with a magnetic stirrer until it is dissolved. During administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): test item is insoluble in water.
- Concentration in vehicle: adjusted to amount of vehicle
- Amount of vehicle (if gavage): 4mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in corn oil at room temperature over a period of 7 days had been verified prior to the start of the study. Given that test substance is completely miscible with corn oil Ph. Eur./DAB, solutions are considered to be homogenous without further analysis.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: referred to as day 0 of pregnancy
Duration of treatment / exposure:
14 days
Frequency of treatment:
daily
Duration of test:
GD 6-19 / 14 days
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: outcome of 28d and 90d repeated dose studies (no adverse findings, NOAEL 1000 mg/kg bw)

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS:
Time schedule: Mortality/Morbidity, pertinent behavioral changes and/or signs of overt toxicity. were checked twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays (GO 0 to 20).

DETAILED CLINICAL OBSERVATIONS:
A cage-side examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occurred, the animals were examined several times daily (GD 0-20).

FOOD CONSUMPTION:
The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

BODY WEIGHT:
Time schedule for examinations: GO 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day: GD 20.
- On GD 20, the dams were sacrificed under isoflurane anesthesia by decapitation, in randomized order.
- After the dams had been sacrificed, they were necropsied and assessed for gross pathology, in randomized order. The uteri and the ovaries were removed and the following data were recorded: weight of the unopened uterus, number of corpora lutea, number and distribution of implantation sites classified as live fetuses and dead implantations.
Dead implantations include:
a) Early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single horn pregnancy)
b) Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c) Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)

- After the weight of the uterus had been determined, all subsequent evaluations of the dams and the gestational parameters were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose animal numbers were encoded.


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
- Other: Site of implantations in the uterus
Fetal examinations:
- External examinations: all per litter
- Soft tissue examinations: half per litter
- Skeletal examinations: half per litter

EXAMINATIONS OF THE FETUSES AFTER DISSECTION FROM THE UTERUS:
At necropsy each fetus was weighed, sexed, and external tissues and all orifices were examined macroscopically. The sex was determined by observing the distance between the anus and the base of the genitalia. Furthermore, the viability of the fetuses and the condition of placentae, umbilical cords, fetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded. Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital (Narcoren®; dose: 0.1 mL/fetus). After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and fixed in ethanol; the other half were placed in Harrison’s fluid for fixation.

SOFT TISSUE EXAMINATION OF THE FETUSES:
The fetuses fixed in Harrison’s fluid were examined for any visceral findings according to the method of BARROW and TAYLOR. After this examination these fetuses were discarded.

SKELETAL EXAMINATION OF THE FETUSES:
The skeletons of the fetuses fixed in ethanol were stained according to a modified method of KIMMEL and TRAMMELL. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After this examination the stained fetal skeletons were archived individually.

EVALUATION CRITERIA FOR ASSESSING THE FETUSES:
In the present study the glossary of WISE et al. (1997) and its updated version of MAKRIS et al. (2009) was essentially used to describe findings in fetal morphology. Classification of these findings was based on the terms and definitions proposed by CHAHOUD et al. (1999) and SOLECKI et al. (2001, 2003):
- Malformation: A permanent structural change that is likely to adversely affect the survival or health.
- Variation: A change that also occurs in the fetuses of control animals and/or is unlikely to adversely affect the survival or health. This includes delays in growth or morphogenesis that have otherwise followed a normal pattern of development. The term "unclassified observation" was used for those fetal findings, which could not be classified as malformations or variations. All fetal findings were listed in tables according to these classifications.
Statistics:
- DUNNETT's test: Food consumption, body weight, body weight change, corrected body weight gain, carcass weight, weight of the unopened uterus, weight of the placentas and fetuses, corpora lutea, implantations, pre- and postimplantation losses, resorptions and live fetuses
- FISHER's exact test: Number of pregnant animals at the end of the study, mortality rate (of the dams) and number of litters with fetal findings
- WILCOXON test: Proportion of fetuses with findings per litter
Indices:
- sex ratio
- The conception rate (in %) was calculated according to the following formula:
(number of pregnant animals / number of fertilized animals) x 100
- The preimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
((number of corpora lutea – number of implantations) / number of corpora lutea) x 100
- The postimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
((number of implantations – number of live fetuses) number of implantations) x 100
Historical control data:
yes, period over 5 years

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 100, 300 or 1000 mg/kg bw/d during the entire study period. Most of the females (18 out of 25) of the high-dose group (1000 mg/kg bw/d) and one female of the mid-dose group (300 mg/kg bw/d) showed transient salivation during the treatment period. Salivation occurred in the respective animals only within the 2-hour examination interval (i.e. < 2h after treatment). It is considered to be treatment-related, likely as a result of the bad taste of the test substance/vehicle preparation or due to local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no substance-related or spontaneous mortalities in any females of all test groups (0, 100, 300 or 1000 mg/kg bw/d).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights (BW) and the average body weight gains (BWC) of the low-, mid and high-dose dams (100, 300, 1000 mg/kg bw/d) were in general comparable to the controls throughout the entire study period. In addition, the corrected body weight gain of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) revealed no difference of any biological relevance to the concurrent control group. Moreover, mean carcass weights remained also unaffected by the treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption of the dams in test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) was generally comparable to the concurrent control throughout the entire study period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No necropsy findings which could be attributed to the test substance were seen in any dam. There occurred one spontaneous finding in test groups 0 and 1 (0 and 100 mg/kg bw/d), i.e. dilated renal pelvis. This finding was detected in one control and two low-dose animals and was therefore assessed as incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between the test groups 0-3 (0, 100, 300 and 1000 mg/kg bw/d) in the values calculated for the pre- and the postimplantation losses.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between the test groups 0-3 (0, 100, 300 and 1000 mg/kg bw/d) in conception rate, in the number of resorptions.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between the test groups 0-3 (0, 100, 300 and 1000 mg/kg bw/d) in number of resorptions.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between the test groups 0-3 (0, 100, 300 and 1000 mg/kg bw/d) in conception rate, in the number of viable fetuses.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The conception rate reached 96% in the mid-dose group (300 mg/kg bw/d) and 100% in the control, low- and high-dose groups (0, 100 and 1000 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of the study.
Other effects:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between the test groups 0-3 (0, 100, 300 and 1000 mg/kg bw/d) in the mean number of corpora lutea.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No toxic effects at the highest dose tested

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal weights of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) were not influenced by the test substance and did not show any biologically relevant differences in comparison to the concurrent control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) was comparable to the concurrent control fetuses.
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One fetus with two external malformations was recorded in test group 3 (1000 mg/kg bw/d). The total incidence of external malformations in treated animals did not differ significantly from the concurrent control group and was covered by the historical control data. No external variations were recorded. No external unclassified observations were recorded. No unclassified soft tissue observations were recorded.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A number of skeletal malformations were detected in fetuses of test groups 0, 1 and 2 (0, 100 and 300 mg/kg bw/d) affecting the skull and humerus. An association of these malformations to the treatment is not assumed. For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeleton and appeared without a relation to dosing. The overall incidences of skeletal variations were covered by the historical control data. Additionally, some isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the vertebral column, the sternum and ribs and did not show any relation to dosing.
Visceral malformations:
no effects observed
Description (incidence and severity):
No soft tissue malformations were recorded. Three soft tissue variations were detected, i.e. misaligned palatal rugae, dilated renal pelvis and dilated ureter. These variations were neither significantly different from the concurrent control nor dose-dependently altered. Therefore, they were not considered to be biologically relevant
Other effects:
no effects observed
Description (incidence and severity):
The mean placental weights of the low-, mid- and high-dose groups (100, 300 and 1000 mg/kg bw/d) were comparable to the concurrent control group.

Effect levels (fetuses)

Key result
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 toxic effects observed at the highest dose tested

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Total indcidence of external malformations

 test group  Dam No, Fetus No, Sex  Findings
 0 (0 mg/kg bw) none  
 1 (100 mg/kg bw)  none  
 2 (300 mg/kg bw)   none  
 3 (1000 mg/kg bw) 94 -10 M  anal atresia, thread-like tail

                                                                             

Total soft tissue variations

     group 0 - 0 mg/kg bw/d  group 1 -100 mg/kg bw/d  group 2 -300 mg/kg bw/d  group 3 -1000 mg/kg bw/d
 Litter Fetuses N N   25 136  24 121 23 108   24 119
 fetal incidence  N (%) 2 (1.5)  1 (0.8)  1 (0.9)  2 (1.7)
 litter incidence  N (%)  2 (8)  1 (4.2)  1 (4.3)  2 (8.3)
 affected fetuses / litter  mean % 1.6  1.0  1.1  1.9

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Individual fetal skeletal malformations

 test group  Dam No, Fetus No, Sex  Findings
 0 (0 mg/kg bw)  12-01 F misshapen tuberositas deltoidea
 1 (100 mg/kg bw)  41-07 M exoccipital fused with 1st cervical arch,misshapen basisphenoid 
 2 (300 mg/kg bw)   69-13 M71-05 M severely malformed skull bonesshortened humerus
 3 (1000 mg/kg bw)   none  

Total fetal skeletal malformations

     group 0 - 0 mg/kg bw/d  group 1 -100 mg/kg bw/d  group 2 -300 mg/kg bw/d  group 3 -1000 mg/kg bw/d
 Litter Fetuses N N   23 119  25 132 25 122   25 130
 fetal incidence  N (%)  1 (0.8) 0.0  2 (1.6) 2 (1.5)
 litter incidence  N (%) 1 (4.3) 0.0 1 (4.0) 2 (8.0) 
 affected fetuses / litter  mean % 0.9 0.0 1.3 1.6

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

Total fetal skeletal variations

     group 0 - 0 mg/kg bw/d  group 1 -100 mg/kg bw/d  group 2 -300 mg/kg bw/d  group 3 -1000 mg/kg bw/d
 Litter Fetuses N N   25 143  25 132 23 121  24 130
 fetal incidence  N (%)  1 (0.7) 1 (0.8)  2 (1.7) 0.0
 litter incidence  N (%) 1 (4.0) 1 (4.0) 2 (8.7) 0.0
 affected fetuses / litter  mean % 0.6 0.6 1.2 0.0

Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)

For a better overview, all skeletal variations with statistically significant differences between the control and the treated groups were compiled in the table below. All incidences were expressed on a fetus per litter basis and any statistically significant differences, which were outside historical control ranges were marked in bold and italics types.

 Finding  group 0 - 0 mg/kg bw/d  group 1 -100 mg/kg bw/d  group 2 -300 mg/kg bw/d  group 3 -1000 mg/kg bw/d HCD Mean %(range) 

Incomplete ossification

of parietal; unchanged cartilage

2.0 6.8 8.9*  9.0*  10.5(4.5 – 17.1)

Incomplete ossification

of thoracic centrum;

unchanged cartilage

0.0 2.2 0.7 1.7  1.0(0.0 – 4.8)

Dumbbell ossification of

thoracic centrum;

unchanged cartilage

0.8 5.5 4.3 5.0 5.2(0.0 – 14.5)
 Supernumerarythoracic vertebra 0.8   6.1  5.9*  1.3  4.1(0.8 - 11.0)

HCD = Historical control data; % = per cent * = p ≤ 0.05 (Wilcoxon-test [one-sided]) ** = p ≤ 0.01 (Wilcoxon-test [one-sided])

As can be seen from this table the increased incidences of skeletal variations were either not related to the dose or they were inside the historical control range. They are in any case not considered as adverse events.

Applicant's summary and conclusion