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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
Study period:
09 Jul 2015 - 24 Feb 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-12 weeks
- 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
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: outcome of 28d studies (no adverse findings, NOAEL 1000 mg/kg bw)

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- 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 AND COMPOUND INTAKE (if feeding study): Yes
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: Yes
- 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 randomizedorder.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
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
Dead implantations:
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)

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: Yes: [all per litter ]
- Soft tissue examinations: Yes: [ half per litter ]
- Skeletal examinations: Yes: [ half per litter ]

Statistics:
DUNNETT's test: Food consumption, body weight, body weight change, DUNNETT's test corrected body weight gain, carcass weight, weight ofthe unopened uterus, weight of the placentas andfetuses, corpora lutea, implantations, pre- andpostimplantation losses, resorptions and live fetuses
FISHER's exact test: Number of pregnant animals at the end of the study, FISHER's exact test mortality rate (of the dams) and number of litters with fetal findings
WILCOXON test: Proportion of fetuses with findings per litter
Indices:
The conception rate (in %): (number of pregnant animals / number of fertilized animals) x 100
The preimplantation loss (in %): ((number of corpora lutea – number of implantations) / number of corpora lutea) x 100
The postimplantation loss (in %): ((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:
no effects observed
Description (incidence and severity):
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.
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. 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.
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
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
no changes of general behavior were detected in any female of all test groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The mean gravid uterus weights of the animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were not influenced by the test substance.
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:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
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). All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age
Other effects:
no effects observed
Details on maternal toxic effects:
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/day) in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age

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: 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/day) were not influenced by the test substance and did not show any biologically relevant differences in comparison to the concurrent control group.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
The sex distribution of the fetuses in test groups 1-3 (100, 300 and 1000 mg/kg bw/day) was comparable to the concurrent control fetuses.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
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/day, anal atresia, thread-like tail). 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.
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/day) 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.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
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
Details on embryotoxic / teratogenic effects:
Fetal examinations revealed that there is no effect of the compound on the respective morphological structures up to the highest dose tested (1000 mg/kg bw/d).

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: Highest dose tested

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
The administration of "Fatty acids, C16-C18 and C18-unsatd., ME esters, epoxidized" by gavage to male and female Wistar rats for 4 weeks did not cause test substance-related adverse signs of maternal or developmental or reproductive toxicity. Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 1000 mg/kg bw/day in Wistar rats and the substance does not need to be classified for developmental toxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The developmental toxicity of “Fatty acids, C16-C18 and C18-unsatd., ME esters, epoxidized” was examined in a OECD TG 414 study under GLP conditions. The test substance was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity. Generally, clinical observations including water consumption, food consumption and body weight gain revealed no toxicologically relevant difference between the animals receiving the test substance at 100, 300 or 1000 mg/kg bw/day and controls.

Most of the females (18 out of 25) of the high-dose group (1000 mg/kg bw/day) and one female of the mid-dose group (300 mg/kg bw/day) showed transient salivation during the treatment period. Salivation occurred in the respective animals only within the 2-hour examination interval. 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.

No differences of toxicological relevance between the control and the treated groups (100, 300 or 1000 mg/kg bw/day) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no influence of the test substance on fetal weight and sex distribution of the fetuses was noted at any dose. Finally, fetal examinations revealed that there is no developmental effect of the compound on the examined morphological structures up to the highest dose tested (NOAEL = 1000 mg/kg bw/day), the substance therefore does not need to be classified for developmental toxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).