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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:
June to July 2017
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:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted January 22, 2001
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
May 30, 2008
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: pellets
Details on test material:
3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate homopolymer, isocyanurate type from Evonik Resource Efficiency GmbH, Batch no.: 16/149
Specific details on test material used for the study:
The test item was suspended in corn oil to the appropriate concentrations and was administered orally at a constant volume once daily from the 6th to the 20th day of gestation.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals 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: 197.7 - 261.9 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
- Humidity (%): 55 % +/- 15
- Illumination: 12 hours artifical fluorescent light and 12 hours dark
- Ventilation rate: between fifteen to twenty air changes per hour.
ENVIROMENTAL ENRICHMENT
- The animals received one piece of wood (certified for animal use) to gnaw on once weekly at change of the cages.
- Octagon-shaped red-tinted huts (polycarbonate) were placed in the cages to offer the animals a resting and hiding place.

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: 3 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. The following parameters were determined: Linearity, Accuracy, Precision, Sensitivity, Specificity, Stability
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: 3 x 3 = 9,
- 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: 3 x 3 = 9

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: 1 x 3 = 3

Sum of all samples: 21
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 25 mated dams were available for all groups.
- Those rats that did not litter were laparotomised on gestation day 24.
- The non-pregnant animals 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. 34637).
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 and no signs of toxicity were noted for the dams.
No embryotoxic properties (no dead fetuses, no malformations and no test item-related variations) were noted at any of the tested dose levels.
Therefore, for the present embryotoxicity study (LPT Report No. 34638), dose levels of 100, 300 or 1000 mg test item/kg b.w./day, administered orally, by gavage once daily (at a contact volume of 3 mL/kg b.w./day) from 6th to the 20th day of pregnancy, were selected.

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 relatingto 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.
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
- 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


Malformed fetuses
- individual data per fetus
- type of malformation
- number and incidence (%) per group and litter

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

Fetuses with variations
- type of variation
- 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 or abnormal appearance (e.g. size, colour, shape).
(b) The number of fetuses (alive and dead) and placentae (location in the uterus and the assignment of the fetuses) 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 test item-related changes in behaviour, the external appearance or the faeces that were considered to be of toxicological relevance were noted in any of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).
Changes in behaviour and external appearance that were noted in the intermediate and high dose group (300 or 1000 mg test item/kg b.w./day) and not considered to be test item-related are listed in the tables below.
At 300 mg test item/kg b.w./day, slight salivation was noted for dam no. 57 on GD 7. As only one dam was affected on one day, salivation was considered to be incidental and not adverse.
At 1000 mg test item/kg b.w./day, slight to moderate salivation (2 of 20 animals) and haemorrhagic canthus (1 of 20) were noted. As only 1 or 2 of 20 animals were noted for hemorrhagic canthus or salivation, these observations were considered to be incidental and not test item-related.

Start and duration of salivation
All observations of salivation for the intermediate and high dose groups (300 or 1000 mg test item/kg b.w./day) started immediately to 5 minutes post administration and disappeared within 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):
Body weight
No test item-related differences in body weight were noted between the dams of the control group and the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).
Body weight gain from GD 0 to 21
No test item-related difference for the body weight gain from GD 0 to 21 compared to the control group was noted for the animals treated with 100, 300 or 1000 mg test item/kg b.w./day.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related differences were noted between the control group and the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).
Statistically significant changes in food consumption that were considered to be spontaneous and not test item-related
Food efficiency:
no effects observed
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 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:
no effects observed
Description (incidence and severity):
Gravid uterus weight and Carcass weight
No test item-related differences were noted between the gravid uterus weight and the carcass weight of the control dams and the dams of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related observations were noted for the dams of the control group and 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.
The following findings were considered to be spontaneous and not test item-related as these findings were only single occurrences.
In the control, a change was noted for dam no. 4 in form of a dilated left uterine horn that was additionally filled with yellow liquid.
At 100 mg test item/kg b.w./day, dam no. 26 was noted with a mildly thickened and watery discolored uterus. In addition, dam no. 43 was noted with a cyst at the right kidney.
In the intermediate dose group (300 mg test item/kg b.w./day), dam no. 66 was noted with a yellow focus at the right kidney.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
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 pre- and post-implantation) were noted be-tween 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 pre- and post-implantation) were noted be-tween 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 pre- and post-implantation) were noted be-tween 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 pre- and post-implantation) were noted be-tween 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 pre- and post-implantation) were noted be-tween 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 pre- and post-implantation) were noted be-tween 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 pre- and post-implantation) were noted be-tween 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:
effects observed, non-treatment-related
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).
The slight but statistically significantly increased fetal weights that were noted at the high dose level were considered to be spontaneous as an increase in the fetal body weight is not an adverse.

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
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 (100, 300 or 1000 mg test item/kg b.w./day).
The slight but statistically significantly increased fetal weights that were noted at the high dose level were considered to be spontaneous as an increase in the fetal body weight is not an adverse.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No dead fetus was noted in the control group and in the test item treated groups (100, 300 or 1000 mg test item/kg b.w./day).
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No test item-related differences between the ratio of male and female fetuses (range: 0.88 - 1.06) were noted between the control group and the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
External inspection at laparotomy
No macroscopically visible external alterations (malformations or variations) were noted for the fetuses of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day) during the macroscopic inspection at laparotomy.
In the control group, one fetus (no. 4-1) was noted with a variation in form of a domed head and a malformation in form of a misshapen snout. These findings were within the normal range of spontaneous events.
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).
Skeletal malformations:
no effects observed
Description (incidence and severity):
Skeletal malformations
No skeletal malformations were noted for the fetuses of the control group and the test item-treated groups (100, 300 or 1000 mg test item/kg b.w./day) during the skeletal examination according to DAWSON.
Skeletal variations
Skeletal variations were noted for the ribs (less than 13 ribs ossified, short or wavy) and the sternebrae (bipartite or misaligned to a slight degree).
No test item-related increase in the incidence of the observed skeletal variations in comparison to the control group was noted for the fetuses of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).
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, dumbbell-shaped or reduced in size), the caudal vertebral bodies (only one body ossified), the lumbar vertebral bodies (bipartite) the os pubis (in-completely ossified) and the metacarpalia and metatarsalia (absence of ossification in metacarpalia 2 to 5 and metatarsalia 2 to 5). see table below
No test item-related increase in the incidence of skeletal retardations at 100, 300 or 1000 mg test item/kg b.w./day was noted during skeletal examination according to DAWSON.
Visceral malformations:
no effects observed
Description (incidence and severity):
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 kidneys (uni- or bilateral dilatation of the renal pelvis, hemorrhagic kidney or hemorrhagic adrenal), the liver (haemorrhagic focus/foci) or brain (dilatation of the cere-bral ventricle).
No test item-related differences and no statistically significant differences in the inci-dences 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).
Unclassified observations
A thoracic cavity filled with blood was noted for one fetus (no. 46-11) of the low dose group (100 mg test item/kg b.w./day). This observation is a preparation induced artefact and not considered as test item-related.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
One runt (2-06) was noted in the control group and one runt (43-14) was noted in the low dose group (300 mg test item/kg b.w./day). The single occurrence of one runt was with-in the normal range of variation and therefore considered to be not test item-related.
Details on embryotoxic / teratogenic effects:
no effects observed

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: prenatal developmental toxicity; highest dose tested

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Examination of the dams

test item

Group 1

Control

Group 2

100 mg/kg

Group 3

300 mg/kg

Group 4

1000 mg/kg

Treated dams

25

25

25

25

Not pregnant dams

1

2

5

1

Dams without viable fetuses

0

0

0

0

Dams with early delivery

0

0

0

0

Prematurely deceased animals

0

0

0

0

Not examined dams

(spare animals)

4

3

0

4

Evaluated litters

20

20

20

20

test item

Group 1

Control

Group 2

100 mg/kg

Group 3

300 mg/kg

Group 4

1000 mg/kg

Animal nos. of mated rats

1 - 25

26 - 50

51 - 75

76 - 100

Animal nos. with evaluable litters at laparotomy

1-18, 20, 21

27-38, 40‑47

51, 52, 54‑61, 63, 65-68, 71‑75

76-78, 80‑96

Dams not pregnant

(animal nos.)

19

26, 39

53, 62, 64, 69, 70

79

Dams with total implantation loss (animal nos.)

none

none

none

none

Prematurely deceased animals (animal nos.)

none

none

none

none

Reserve animals

(animal nos.)

22-25

48-50

none

97-100

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

275

13.8

289

14.5

280

14.0

268

13.4

Implantation sites

total

mean per dam

270

13.5

284

14.2

266

13.3

258

12.9

Resorptions

total

mean per dam

9

0.5

7

0.4

10

0.5

12

0.6

Early resorptions

total

mean per dam

6

0.3

7

0.4

10

0.5

11

0.6

Late resorptions

total

mean per dam

3

0.2

0

0.0

0

0.0

1

0.1

Live fetuses

total

mean per dam

261

13.1

277

13.9

256

12.8

246

12.3

Dead fetuses

total

0

0

0

0

Pre-implantation loss [%]

per group #1

mean per dam

1.8

4.4

1.7

1.7

5.0

5.2

3.7

5.7

Post-implantation loss [%]

per group #2

mean per dam

3.3

3.3

2.5

2.7

3.8

3.3

4.7

6.3

 

 

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 (*/**: p ≤ 0.05/p ≤ 0.01).

 

 

#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 (*/**: p ≤ 0.05/p ≤ 0.01).

 

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

% nominal concentration

Concentration

immediately after preparation

93.2 % - 95.8 %

before administration to the last animal on test day 21

95.0 % - 105.8 %

Stability

8h after preparation

90.3 % - 92.2 %

24h after preparation

91.7 % - 93.2 %

Homogeneity

before administration to the first animal

92.9 % - 94.3 %

during administration to the animals

90.9 % - 94.4 %

before administration to the last animal

96.2 % - 96.7 %

The measured actual concentrations of the test item in the test item vehicle-mixtures were between 90.3 % and 105.8 % of the nominal concentrations, indicating correctly prepared, stable and homogenous formulations.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, test item did not show any teratogenic potential.
Executive summary:

The aim of this prenatal developmental toxicity study (OECD 414, oral) was the examination of the influence of the 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.

Findings 

Examination of the dams:

 

Mortality

No premature deaths were noted.

 

 

Clinical signs

No test item-related signs of toxicity were noted.

 

 

Body weight and

body weight gain

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

 

 

Food consumption

No test item-related differences were noted for the food consumption.

 

 

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 were noted.

Reproduction data

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

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 malformations were noted during the macroscopic examinations at laparotomy (external inspection and inspection of the organs and tissues for gross lesions), the skeletal examination according to DAWSON and the soft tissue examination according to WILSON in the treatment groups.

Variations

The macroscopic examinations at laparotomy, the skeletal examination according to DAWSON and the soft tissue examination according to WILSON revealed no test item-related variations in the treatment groups.

Unclassified observations

No test item-related unclassified observations were noted.

 

 

Retardations

No test item-related retardations (delays in ossification) were noted.

Analysis of test item

formulations

 The measured actual concentrations of the test item in the test item vehicle mixtures were between 90.3 % and 105.8 % of the nominal concentrations, indicating correctly prepared, stable and homogenous formulations.

Conclusion

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

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

No prematurely deceased dams were noted during the study.

No test item-related changes in behaviour, external appearance or faeces were noted.

No test item-related change in food consumption, body weight or body weight gain was noted in any treatment group.

No changes were noted at the macroscopic examination during laparotomy.

The no-observed-adverse-effect level (NOAEL) for the fetal organism was also above 1000 mg test 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 test item-related differences for the placental and fetal weights were noted in the treatment groups.

No dead fetuses, no malformations and no test item-related variations or retardations were noted in the treatment groups.

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