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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:
2016-05-30 to 2016-11-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted January 22, 2001
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
May 30, 2008
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
2,3-Epoxypropyl neodecanoate, oligomeric reaction products with toluene-4-sulfonic acid
EC Number:
500-281-4
EC Name:
2,3-Epoxypropyl neodecanoate, oligomeric reaction products with toluene-4-sulfonic acid
Cas Number:
98362-33-5
Molecular formula:
Exact identification is not feasible
IUPAC Name:
Copolymer of neodecanoic acid oxiranylmethyl ester and 4-methylbenzenesulfonic acid
Test material form:
liquid
Details on test material:
Characteristics: Slightly yellowish liquid
Content: ≥ 99.75%
Batch no.: MMS 15-15.13

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ORGANISMS: 
- Species: Rat
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Strain: CD / Crl: CD(SD)
- Age: 61 days
- body weight: 171.4 - 237.1 g
- Diet: ad libitum, Commercial diet ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water: ad libitum
- Acclimatisation period: 5 days
-Housing: Except during the mating period, the dams were kept singly in MAKROLON cages
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C +/- 3° C
- Humidity (%): 55% +/- 15 %
- Illumination: 12 hours artifical fluorescent light and 12 hours dark
- Ventilation rate: between fifteen to twenty air changes per hour.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
ADMINISTRATION: 
- Frequency: once daily, day 6 to 20 of gestation
- Dose volume: 2 ml/kg b.w.
- Dose: 0, 100, 300, 1000 mg/kg/bw
- Animals: 25 female rats/group
DOSAGE PREPARATION:
- The test item formulations were freshly prepared every day.
- The test item was suspended in the vehicle to the appropriate concentrations and was administered orally at a constant volume once daily from the 6th to the 20th day of gestation.
- The amount of the test item was daily adjusted to the current body weight of the animal.
- The control animals received the vehicle at the same administration volume daily in the same way.
- The male rats for mating remained untreated.
Analytical verification of doses or concentrations:
yes
Remarks:
The analysis was performed at LPT using a validated method.
Details on analytical verification of doses or concentrations:
For the analysis of the test item-vehicle formulations, samples of approximately 2 mL were taken at the following times and stored at -20°C or colder until analysis at LPT:
At start of dosing
- Analysis of stability and concentration: Immediately after preparation of the formulations as well as after 8 and 24 hours storage of formulations at room temperature. (3 samples/test item group), Number of samples: 2 x 3 x 3 = 18,
- Homogeneity: At the start of dosing, during (middle) administration and before dosing to the last animal of the test item group. (3 samples/test item group), Number of samples: 2 x 3 x 3 = 18
- Number of aliquots: 2 x 3 x 3 = 18

At the end of the dosing period (at a time when the majority of animals was dosed):
- Analysis of concentration: During treatment with the test item always before administration to the last animal of the dose level group. (1 sample/test item group), Number of samples: 2 x 1 x 3 = 6

Sum of all samples: 42
Details on mating procedure:
- Sexually mature ('proved') male rats of the same breed served as partners.
- The female breeding partners were randomly chosen.
- Mating was monogamous: 1 male and 1 female animal were placed together in one cage during the dark period.
- Each morning a vaginal smear was taken to check for the pres-ence of sperm. If findings were negative, mating was repeated with the same partner.
- The day on which sperm was found was considered as the day of conception (day 0 of pregnancy).
- This procedure was repeated until enough pregnant dams were available for all groups.
- Rats which did not become pregnant were excluded from the analysis of the results and replaced by other animals.
- A post-mortem negative staining according to SALEWSKI was carried out in the replaced animals in order to confirm the non-pregnancy status.

Duration of treatment / exposure:
From gestation day 6 until gestation day 20
Frequency of treatment:
Once daily
Duration of test:
On gestation day 21, all rats were euthanized by carbon dioxide (CO2) inhalation and laparotomised.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle control
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Intermediate dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
HIgh dose
No. of animals per sex per dose:
25 female rats/dose , orally dosed with 0, 100, 300 or 1000 mg test item/kg b.w.
Evaluated litters: 20 litters per group
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were selected in agreement with the Sponsor based on the results of a dose-range-finding study for a prenatal developmental toxicity (LPT study no. 32703).
In this dose-range finding study, test item was administered to pregnant female rats at dose levels of 100, 300 or 1000 mg/kg b.w./day orally, by gavage, once daily from gestation day 6 to 20.
No premature deaths were noted. No test item-related influence was noted on behaviour or external appearance, body weight as well as intake of food of the dams at any tested dose level.
No embryotoxic properties (no dead fetuses, no malformations and no test item-related variations) were noted at any of the tested dose levels.
For the present rat embryotoxicity study (LPT Report No. 32963), dose levels of 100, 300 or 1000 mg test item/kg b.w./day, administered orally, by gavage once daily (at a conctant volume of 2 mL/kg b.w./day) from the 6th to the 20th day of pregnancy, were selected in agreement with the Sponsor.

Examinations

Maternal examinations:
Dated and signed records of all activities relating to the day to day running and maintenance of the study within the animal units, as well as to the
group observations and examinations outlined in the Study Plan, were recorded in the appropriate documentation. In addition, observations relating
to the individual animals made throughout the study were recorded.

The following observations were made during the course of the study:
Clinical signs
Individual animals were observed daily for any signs of behavioural changes, reaction to treatment, or illness.
Immediately after administration, any signs of illness or reaction to treatment were rec-orded. In case of changes, the animals were observed until thesymptoms disappeared. In addition, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m.
On Saturdays and Sundays, the animals were checked regularly starting from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately
3.30 p.m.
Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.


Viability
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This allowed
post mortem examinations to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed
except that the final check was carried out at approximately midday.
Animals showing signs of abortion or premature delivery would have been sacrificed on the same day. Fetuses obtained this way were examined for
abnormal development, whenever possible. No abortion occurred in the study.


Body weight
The weight of each rat was recorded on day 0 of gestation (the day of detection of a positive mating sign), followed by daily weighing - always at the same time of the day.
The body weight gain was calculated in intervals (i.e. day 0-3, 3 6, 6-9, 9-12, 12-15, 15-18 and 18-21), for the whole study (gestation day 0 - 21) and for the period after the start of dosing (gestation day 6 to gestation day 21). Furthermore the carcass weight and the net weight gain from day 6 is given.
These values are stated in the report.
These measurements were also used for calculating the daily amount of test item to be administered.

Food and drinking water consumption
The quantity of food consumed by each rat was recorded daily. Food intake per rat (g/rat/day) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment day.

The relative food consumption (g/kg b.w./day) was calculated using the following formula:
Daily food consumption [g/kg b.w./day]= Total food intake in g / Body weight in g x 1000

Daily monitoring by visual appraisal of the drinking water bottles was maintained throughout the study. Dehydration of the dams was avoided.

EXAMINATIONS (NECROPSY), Examination of the dams
Dissection technique and evaluation of the animals:
On gestation day 21, the rats were laparotomised under ether narcosis. The ovaries and the uteri of the dams were removed; the gravid uteri (in toto) were weighed. In order to check for possible test item effects, a dissection with macroscopic examination of the internal organs and placentae of the dams was carried out on the day of sacrifice or on the day on which the animals were found dead. In case of macroscopical findings, the affected maternal tissues were preserved in 7% buffered formalin for possible future histopathological examinations.
Organ weights
The following target organs of the dams were weighed: Heart, kidney (2) and liver.
The kidneys were weighed separately and identified as left and right.

Ovaries and uterine content:
Corpora lutea
- number per dam
- absolute number per group
- mean per group

Implantations
- number per dam
- distributions in the uterine horns
- absolute number per group
- mean per group

Resorptions
- number per dam
- distributions in the uterine horns
- absolute number per group
- mean per group
- mean % per group
- early resorptions < 2 mm
- Late resorptions > 2 mm

Weight of placentae
- individual data per fetus
- mean per litter
- mean per group
- mean per sex and group

Weight of fetuses
- individual data per fetus (alive and dead)
- mean per litter
- mean per group
- litter mean per sex and group

Fetuses
- number per dam (alive)
- number per dam (dead)
- number of fetuses (alive and dead) per sex and dam
- distribution in the uterine horns
- absolute number of fetuses alive per group
- mean number of fetuses alive per group
- mean % of fetuses alive per group
- male/female ratio (alive and dead)


Runts
- number per dam
- mean per group

Dead fetuses
- number per dam
- mean per group

Malformed fetuses
- individual data per fetus
- type of malformation: number and incidence (%) per group and litter
- number of affected fetuses per group (fetuses affected by several changes were counted as one fetal incidence)

Total malformation rate [%] = malformed fetuses per group / fetuses per group x 100

Fetuses with variations
- type of malformation
- individual data per fetus
- number and incidence (%) per group and litter

Total variation rate [%] = fetuses per group with variations / fetuses per group x 100

Fetuses with retardations
- type of retardation
- individual data per fetus
- number and incidence (%) per group and litter


Indices of pre-implantation loss and post-implantation loss:

Calculation of group indices
Pre implantation
loss [%] = [Corpora lutea (per group) - Implantations (per group)] / Corpora lutea (per group) x 100


Post implantation
loss [%] = [Implantations (per group) - living fetuses (per group)] / Implantations (per group) x 100


Calculation of mean indices per litter
Pre implantation
loss [%] = sum of pre-implantation losses per litter in a group [%] / number of litters in a group

Post implantation
loss [%] = sum of post-implantation losses per litter in a group [%] / number of litters in a group



Fetal examinations:
The fetuses were removed and the following examinations performed:
(a) Macroscopic inspection (gross evaluation) of the placentae for example for focal indurations.
(b) The number of fetuses (alive and dead) and placentae (location in uterus and as-signment to the fetus) was determined.
(c) Sex and viability of fetuses were determined. Animals are said to be viable when they are found alive (spontaneous breathing, spontaneous movement).
(d) Number and size of resorptions were determined.
(e) Corpora lutea in the ovaries, implantations and location of fetuses in the uterus were determined.
(f) Weights of fetuses and weights of the placentae were determined (fetuses were considered as runts if their weight was less than 70% of the mean litter weight).
(g) All fetuses (dead and alive) were inspected externally for damages, especially for malformations .
(h) The fetuses were sacrificed by an ether atmosphere.
(i) Examination of fetuses and determination of number and kind of retardations, variations or malformations:
1) 50% of the number of fetuses in each litter were examined for skeletal anomalies. The thorax and peritoneal cavity (without damage to ribs and sternum) were opened and the location, size and condition of the internal organs were determined.
Then the skeleton was double-stained with Alcian blue for the examination of cartilage and with Alizarin red to reveal ossifications (according to DAWSON). The skeletal system was examined (determination of the number and type of retardations, variations as well as malformations).
2) The remaining 50% of the number of fetuses in each litter were examined for soft tissue anomalies. Body sections were made and examined according to WILSON.
The fetuses were allocated to the evaluation of DAWSON or WILSON on an alternating basis.
Statistics:
Parametrical data:
The statistical evaluation of the parametrical values was done by Provantis using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), inter-group comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

Non-parametrical data
The statistical evaluation of non-parametrical values was done using the FISHER or Chi2 test:
FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01)
or
Chi2 test, n ≤ 0.01 (p ≤ 0.05 and p ≤ 0.01)

The respective calculations for the FISHER and Chi2 test were performed using Provantis (maternal macroscopic findings at necropsy or findings during the external macro-scopic examination of the fetuses). or an internal computer program (e.g. findings during the fetal skeletal or soft tissue examination).
Note:
The statistical evaluation of the pre- and post-implantation index (per group) using the number of corpora lutea, implantation sites and/ or fetuses per group (see table 7-1 Re-production Data - Summary - Values per Group) was done using StatXact 4.0.1 software, as such a calculation is not possible in Provantis.

Significantly different data are indicated in the summary tables of the result sections of the report.
Indices:
Fertility Index, Viability Index, Resorption Index, Pre-Implantation Loss Index, Post-Implantation Loss Index, Runts Index, Variation Index,
Number of litters having abnormalities, Number of abnormalities per litter
Historical control data:
LPT Background Data, see Appendix 4
Summarized results of the 59 last embryotoxicity studies in Sprague-Dawley rats (Charles River Deutschland GmbH performed at LPT in the years 2000 to July 2016

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes in behaviour, the external appearance or the faeces were noted for the dams of the control group and the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).
Test item-related changes in behavior were noted in the intermediate and high dose groups (300 or 1000 mg test item/kg b.w./day). Due to the low incidence and/or short duration (salivation) these observations were considered to be not adverse and of no toxicological relevance.
Start and duration of salivation
In the intermediate and high dose group (300 and 1000 mg test item/kg b.w./day) salivation was noted immediately to 5 minutes after application and was observed until 20 to 60 minutes after administration

Mortality:
no mortality observed
Description (incidence):
No premature deaths were noted in the control group and in the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The comparison of the body weight of the dams of the control group to those of the test groups (100, 300 or 1000 mg test item/kg b.w./day) revealed no noteworthy difference.
Body weight gain
The comparison of the control groups to the treatment groups (100, 300 or 1000 mg test item/kg b.w./day) revealed no difference. Neither in in the treatment period (gestation day 6 - 21) nor during the entire experiment period (gestation day 0 - 21) a difference was noted.

Body weight gain from gestation day 6
No test item-related differences for the absolute and also the net body weight gain be-tween the control group and the treatment groups (100, 300 or 1000 mg test item/kg b.w./day) between gestation day 6 and gestation day 21 were noted.

Body weight at autopsy
No test item-related differences in the body weight at autopsy were noted between the dams of the control group and the dams of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).


Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item-related differences in food consumption were noted between the control group and the treat-ment groups.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
No noteworthy difference was noted between the dams of the control group and the dams of the low and intermediate dose groups (100 or 300 mg test item/kg b.w./day).
Compared to the control group, the high dose group (1000 mg test item/kg b.w./day) showed a transiently, slightly increased relative food consumption from gestation day 19 to 20 (+6.9%, statistically significant at p ≤ 0.05). This effect was considered to be spontaneous and not test item-related.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test item-related changes in drinking water consumption were noted between the dams of the control group and the dams of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day) by visual appraisal.
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The heart, the liver and the kidneys were weighed and no test item-related differences for the absolute and the relative organ weights were noted between the control group and the low dose group (100 mg test item/kg b.w./day).
Regarding the absolute organ weights, increased weights for the right kidney and the liver (+7.5% and +12.2%) were noted in the intermediate dose group (300 mg test item/kg b.w./day) in comparison to the control group.
In the high dose group (1000 mg test item/kg b.w./day) increased absolute weights of the left and right kidney (+15.2% and +15.4%) and of the liver (+28.9%) were noted.
The same organs of both groups were also statistically significantly increased investigating the relative organ weight. Compared to the control group, a statistically significant increase was noted for the right kidney and the liver (+8.3% and +12.9%) of the intermediate dose group (300 mg test item/kg b.w./day) and in the high dose group (1000 mg test item/kg b.w./day) for the left and right kidney (+14,7% and +14.9%) as well as for the liver (+28.4%).
The differences in the absolute and relative organ weights in the intermediate and high dose group (300 or 1000 mg test item/kg b.w./day) were due to an increased work load evoked by the application of the test item. Therefore, these observations were considered to be not adverse.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related observations were noted for the dams of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day) during the macroscopic inspection of the organs and tissues.
Findings have been noted in 2 dams (no. 2 and no. 6) of the control group and in 1 dam (no. 88) of the high dose group (1000 mg test item/kg b.w./day) that are listed in the table below. Since the noted effect in the high dose group was the only finding in the treatment groups, it was considered to be spontaneous and not test item-related. Maternal macroscopic findings
group finding
control uterus:
- filled with yellowish liquid (no. 2)
- partly brown discoloration (no. 2)
- filled with brownish-yellowish, clear liquid (no. 6)
liver:
- partly discoloration (no. 6)
high dose group
(1000 mg/kg) kidney (left and right):
- partly paleness (no. 88)
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Description (incidence and severity):
Gravid uterus and carcass weight
No test item-related differences were noted between the weights of the gravid uterus or carcass of the control dams and those of the dams in the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).


Details on results:
no findings

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of post-implantation loss) were noted between the dams of the control group and the dams of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day). See table below.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of post-implantation loss) were noted between the dams of the control group and the dams of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day). See table below
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of post-implantation loss) were noted between the dams of the control group and the dams of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day). See table below
Early or late resorptions:
no effects observed
Description (incidence and severity):
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of post-implantation loss) were noted between the dams of the control group and the dams of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day). See table below
Dead fetuses:
no effects observed
Description (incidence and severity):
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of post-implantation loss) were noted between the dams of the control group and the dams of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day). See table below
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):
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of post-implantation loss) were noted between the dams of the control group and the dams of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day). See table below
Details on maternal toxic effects:
No test item-related influence on the reproductive parameters (number of implantation sites, fetuses, resorptions and the index of post-implantation loss) were noted between the dams of the control group and the dams of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day). See table below

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The placental and fetal weights showed no test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).


Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): The placental and fetal weights showed no test item-related differences between the control group and the treatment groups.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No dead fetuses were noted in the control group and in the test item-treated groups

Number of runts
No runts were noted in the control group and also in the treatment groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No test item-related differences between the ratio of male and female fetuses were noted between the control group and the treatment groups.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No macroscopically visible external observations (malformations or variations) were noted for the fetuses of the control and treatment groups (100, 300 or 1000 mg test item/kg b.w./day) during the macroscopic inspection at laparotomy.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No skeletal malformations were noted for the fetuses of the control group and the test item-treated groups during the skeletal examination according to DAWSON.

Skeletal variations
Skeletal variations were noted for the skull (distinct areas not ossified or fontanelle en-larged), the ribs (fused to a slight degree or wavy) and the sternebrae (bipartite or misa-ligned to a slight degree).
No test item-related increase in the incidence of the observed skeletal variations in comparison to the control group were noted for the fetuses of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).
A statistically significantly decreased total fetal incidence for skeletal variations was noted for the low and intermediate dose group (100 or 300 mg test item/kg b.w./day). Since these incidences were below the value of the control group, this was considered to be not test item-related.

Skeletal retardations
Retardations (delayed ossifications) were related to the skull (incomplete ossification of frontal, parietal, interparietal and/or supraoccipital areas), the hyoid (unossified), the sternum (sternebra(e) incompletely ossified, reduced in size or unossified), the thoracic vertebral bodies (bipartite or dumbbell-shaped, the caudal vertebral bodies (only one body ossified), the os ischii (incompletely ossified), the os pubis (incompletely ossified) and the metacarpalia and metatarsalia (absence of ossification in metacarpalia 2 to 5 and metatarsalia 2 to 5).
No test item-related differences in the incidences of skeletal retardations compared to the control group was noted in the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).
A statistically significantly increased incidence for total fetal skeletal retardations was noted for the low and intermediate dose groups (100 or 300 mg test item/kg b.w./day). As the value of the high dose group (1000 mg test utem/kg b.w./day) was below of those of the low and intermediate dose groups, a dose dependence-relationship was missing. Therefore, these observations were considered to be not test item-related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Gross inspection of the organs and tissues at laparotomy
The macroscopic inspection of the organs and tissues for gross alterations at laparotomy revealed no malformations or variations for the fetuses of the control group and the fetuses of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).

Soft tissue examination according to WILSON
Malformations
No malformations were noted for the fetuses of the control group and the fetuses of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day) during the soft tissue examination according to WILSON.

Variations
During the examination of the organs and tissues according to WILSON variations were noted for the cerebral ventricle (dilatation of the 4th ventricle), the kidneys (uni- or bilateral dilatation of the renal pelvis, malpositioned, hydroureter) and the liver (haemorrhagic focus/foci).
No test item-related differences and no statistically significant differences in the incidences of the observed variations were noted between the control group and the test item-treated groups (100, 300 or 1000 mg test item/kg b.w./day).

Details on embryotoxic / teratogenic effects:
No malformation was noted during the macroscopic inspection at laparotomy (including an external inspection and a gross inspection of the organs), the skeletal examination according to DAWSON and the soft tissue examination according to WILSON.
No test item-related variation was noted during the macroscopic inspection at laparotomy and the soft tissue examination according to WILSON and also during the skeletal examination according to DAWSON in all treatment groups group (100, 300 and 1000 mg test item/kg b.w./day).
No test item-related retardations were noted during the skeletal examination of the fetuses according to DAWSON in all treatment groups (100, 300 or 1000 mg test item/kg b.w./day).

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Reproduction data of the dams

Parameter

Group 1

Control

(n=20)

Group 2

100

mg/kg

(n=20)

Group 3

300 mg/kg

(n=20)

Group 4

1000 mg/kg

(n=20)

 

Corpora lutea

total

mean per dam

266

13.3

266

13.3

254

12.7

270

13.5

 

Implantation sites

total

mean per dam

263

13.2

263

13.2

248

12.4

261

13.1

 

Resorptions

total

mean per dam

6

0.3

8

0.4

6

0.3

4

0.2

 

Early resorptions

total

mean per dam

5

0.3

7

0.4

5

0.3

2

0.1

 

Late resorptions

total

mean per dam

1

0.1

1

0.1

1

0.1

2

0.1

 

Live fetuses

total

mean per dam

257

12.9

255

12.8

242

12.1

257

12.9

 

Dead fetuses

total

0

0

0

0

 

Pre-implantation loss [%]

per group ##1

mean per dam

1.1

1.1

1.1

1.1

2.4

2.4

3.3

2.8

 

Post-implantation loss [%]

per group ##2

mean per dam

2.3

2.4

3.0

3.0

2.4

2.5

1.5

1.6

 

 

 

Statistical analyses were performed for the mean values per dam using an ANOVA/DUNNETT test.

 

##1

The statistical comparison of the pre-implantation loss per group was done by comparing the values of implantation sites/corpora lutea of the test group with the ratio of implantation sites/corpora lutea of the control group using the Chi2test.

 

##2

The statistical comparison of the post-implantation loss per group was done by comparing the values of live fetuses/implantation sites of the test group with the ratio of fetuses/implantation sites of the control group using the Chi2test.

Analysis of test item formulations

The results of the test item-formulation analyses for the investigated parameters are listed in the table below:

Parameter

Sampling / dealing

Range of accuracy

(% of nominal concentration)

Concentration

before administration to the last animal on test day 6

101.1% - 103.5%

Stability

immediately after test item-vehicle mixture preparation

100.8% - 103.8%

Stability

8h after test item-vehicle mixture preparation

101.7% - 103.4%

Stability

24h after test item-vehicle mixture preparation

100.7% - 102.6%

Homogeneity

before administration to the first animal on test day 6

98.6% - 103.1%

Homogeneity

during administration to the animals on test day 6

99.7% - 102.3%

Homogeneity

before administration to the last animal on test day 6

101.1% - 103.5%

Concentration

before administration to the last animal on test day 21

99.0% - 103.5%

Applicant's summary and conclusion

Conclusions:
Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was above 1000 test item/kg b.w./day for the dams.
None of the dams died prematurely.
No differences in body weight and food consumption were noted in the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).
Increases in relative and absolute organ weights of the liver and kidneys that were not considered to be adverse were noted for the intermediate and high dose group (300 or 1000 test item/kg b.w./day).
The no-observed-adverse-effect level (NOAEL) for the fetal organism was above 1000 mgtest item/kg b.w./day.
The reproductive parameters (number of implantation sites, number of resorptions and number of fetuses) were not influenced by the test item.
No dead fetuses, no malformations, no test item-related variations or retardations were noted.
Under the conditions of the study, test item did not show any teratogenic potential.
Executive summary:

The aim of this dose-range-finding study was the examination of the influence of test item administered orally during the critical period of organogenesis and the fetal development (6th to 20th day of gestation) on the pregnant rat and the fetus.

 

In this prenatal developmental toxicity study, the test item was administered orally to female rats at dose levels of 100, 300 or 1000 mg/kg b.w./day from the 6th to 20th day of pregnancy.

Findings

Examination of the dams:

 

Mortality

No premature death was noted in the control group and in the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).

 

 

Clinical signs

No observations that were considered to be adverse or of toxicological relevance were noted in the treatment groups.

 

 

Body weight and

body weight gain

No test item-related differences in body weight and body weight gain were noted between the control group and the treatment groups.

 

 

Food consumption

 

No test item-related differences in food consumption were noted between the control group and the treatment groups.

 

 

Drinking water consumption

No differences were noted.

 

 

Necropsy findings

No test item-related changes were noted during the macroscopic inspection of the dams at necropsy.

 

 

Uterus and carcass weights

 

No test item-related differences in uterus or carcass weight were noted between the control group and the treatment groups.

 

 

Organ weights

Increased relative and absolute organ weights were noted in the intermediate and high dose group (300 or1000 mg test item/kg b.w./day) for the liver and/or the kidneys. However, these observations were due to an increased work load of these organs and are not considered to be adverse.

 

 

Reproduction data

No influence on the reproductive parameter (number of implantation sites, resorptions and fetuses) was noted.

 

Examination of the fetus

 

Mortality

No dead fetuses were noted in any of the test groups.

 

 

Body weight of the fetuses

and the placentae

No test item-related differences were noted between the control group and the treatment groups.

 

 

Fetal alterations

 

Malformations

No test item-related malformations were noted during the macroscopic examination at laparotomy(external inspection and inspection of the organs and tissues for gross lesions), the skeletal examination according toand thesoft tissueexamination according to.

 

 

Variations

No test item-related external variation was noted during macroscopic examination at laparotomy, the skeletal examination according to DAWSON and the soft tissue examination according to WILSON.

 

 

Retardations

No test item related retardation was noted during the skeletal examination according toin the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).

 

 

Analysis of test item

formulations

The measured actual concentrations of the test item in the test item vehicle mixtures were between 98.6% and 103.8% of the nominal concentrations, indicating correctly prepared and homogenized formulations which were stable at room temperature for at least 24h.

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was above 1000 test item/kg b.w./day for the dams.

None of the dams died prematurely.

No differences in body weight and food consumption were noted in the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).

Increases in relative and absolute organ weights of the liver and kidneys that were not considered to be adverse were noted for the intermediate and high dose group (300 or 1000 test item/kg b.w./day).

The no-observed-adverse-effect level (NOAEL) for the fetal organism was above 1000 mgtest item/kg b.w./day.

The reproductive parameters (number of implantation sites, number of resorptions and number of fetuses) were not influenced by the test item.

No dead fetuses, no malformations, no test item-related variations or retardations were noted.

Under the conditions of the study,test item did not show any teratogenic potential.