Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Short description of key information:
According to Regulation (EC) No. 1907/2006, Annex VIII, 8.7.2, column 2, this screening test for reproduction does not need to be conducted if a reliable prenatal developmental toxicity study in one species is available.
A prenatal developmental toxicity study in one species has been performed and did not show any developmental effects when dosed up to 1000 mg/kg bw/d.

Effects on developmental toxicity

Description of key information

No reproductive/developmental alerts have been identified in Stages 1 or 2 of the Integrated Testing Strategy that would trigger the need for further testing. At greater than or equal to 100 tonnes/year (Annex IX) progression beyond Stage 1 and 2 triggers the standard data requirement of a prenatal developmental toxicity study (OECD TG 414). Rats are the preferred species for testing.

Such a study has been performed and did not reveal any maternal or teratogenic effects via oral application to rats during gestation days 6 to 20 when dosed up to 1000 mg/kg bw/d.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted January 22, 2001
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: CD / Crl:CD (SD)
Details on test animals and environmental conditions:
Mature female CD® rats were used for this experiment, a strain bred by Charles River Laboratories, Research Models and Services, Germany GmbH.
Each animal was given a thorough examination. Animals were judged to be healthy on the basis of general observations (physical examination). Health checks were performed on the day of delivery and at the beginning of mating. No replacements due to illness had to be carried out.
Species: Rat
Strain / Stock: CD® / Crl:CD (SD)
Breeder: Charles River Laboratories, Research, Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
Adaptation period: 5 days
Age: 60 to 69 days (on day 0 of pregnancy)
Body weight: 195.7 g - 258.8 g (on day 0 of pregnancy)
Selection of species: The rat is a commonly used rodent species for such embryotoxicity studies.
Number of animals: Treated animals in Groups 1 - 4: 25 mated females per group , Evaluated litters in Groups 1 - 4: 20 litters per group
Animal identification: For identification, each rat received a continuous number between 1 and 100: a pattern of points was set on paws and/or tail by tattoo. Additionally, the animal cages were labelled with the tattooed serial number, sex, study code number, type of study, route of administration, dose level, date of conception and dates of administration.
Route of administration:
oral: gavage
Vehicle:
other: 0.5% aqueous hydroxypropylmethylcellu-lose gel
Remarks:
Lot no.: 14G02-B11, Supplier: Fagron Services B.V., Molenwerf 13, 1911 DB Uitgeest, The Netherlands
Details on exposure:
Route of administration: Oral, via gavage
Frequency of administration: Once daily
Treatment period: Day 6 to 20 of gestation
Vehicle: 0.5% aqueous hydroxypropylmethylcellulose gel
Administration volume: 10 mL/kg b.w./day
Selection of route of administration: According to OECD guideline 414
Test item preparation: The test item formulations were freshly prepared every day.
The test item was suspended in the vehicle to the appropriate concentrations. In order to prevent bubble formation, the test item was first moistened with a small amount of the vehicle and stirred slowly. Then the suspension was filled up to the final volume and stirred thoroughly for approximately 15 minutes. The vehicle chosen is a commonly used vehicle for the preparation of homogeneous suspensions for water-insoluble test items.
The test item 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 test item formulation was continuously stirred throughout the entire administration procedure to ensure homogeneity. 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
Details on analytical verification of doses or concentrations:
For the analysis of the test item-vehicle formulations, samples of approximately 5 mL were taken at the following times and stored at ≤ -20 °C until analysis at LPT:
At start of dosing concentration and homogeneity was analysed on samples that were taken from the top, the middle and the bottom of the container of each dose formulation (3 samples/test item group = 3 x 3 = 9 samples
At the end of the dosing period, at a time when the majority of the animals was dosed concentration was analysed (during treatment always before administration to the last animal of the group with one sample per dose group (1 sample/test item group = 1 x 3 = 3 samples).
Thus, the total number of samples taken for analysis was 12.
The samples were labelled with the study number, species, type of sample, concentration, sampling time, date and test day.
The analytical method was validated in LPT Study No. 35348. The results of this validation revealed that the method employed was suitable for the determination and quantification of N,N'-(methylenedi-4,1-phenylene)bis(2-oxoazepane-1-carboxamide) in test item formulation samples and the samples were stable at room temperature for at least 6 hours.
The samples were analysed at LPT and the results of the test item formulation analysis are summarised in in section 'Analysis of test item formulations'.
The results of the test item-formulation analyses for the investigated parameters are listed below:
Concentration and Homogeneity (taken from the top, middle and bottom of the container of each test item formulation: 98.2% - 103.8% nominal concentration.
Concentration (before administration to the last animal of the group at the end of the study): 94.5% - 105.6% nominal concentration.
The measured actual concentrations of the test item in the test item vehicle-mixtures were between 94.5% and 105.6% of the nominal concentrations, indicating correctly prepared and homogenous formulations.
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 in that 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 presence 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 each group.
The non-pregnant rats 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 day of gestation 6 to day of gestation 20, once daily
Frequency of treatment:
Once daily by gavage (GD6 - GD20)
Duration of test:
15 days exposure time
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
low dose group
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
mid dose group
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
high dose group
No. of animals per sex per dose:
25 females per dose (including vehicle control); thus, in total 100 females
Control animals:
yes, concurrent vehicle
Details on study design:
Four (4) groups of pregnant rats were established, each obtained from matings which were carried out on a daily basis. Vaginal lavages were taken each morning. Day 0 of pregnancy was the day on which sperm was found in the vaginal lavage. When positive, the animals were assigned to the test groups by mating day using a Provantis (Provantis® Integrated preclinical software, version 9.4.0.1, Instem LSS Ltd.) generated randomization based on the body weight of the animals.
Dose group 1: 0 mg/kg b.w./day, p.o. (vehicle control), females # 1 – 25, 20 litters evaluated
Dose group 2: 100 mg/kg b.w./day, p.o. (low dose), females # 26 – 50, 20 litters evaluated
Dose group 3: 300 mg/kg b.w./day, p.o. (intermediate dose), females # 51 – 75, 20 litters evaluated
Dose group 4: 1000 mg/kg b.w./day, p.o. (high dose),females # 76 – 100, 20 litters evaluated
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. 35349).
In this dose-range finding study, N,N'-(methylenedi-4,1-phenylene)bis(2-oxoazepane-1-carboxamide) 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 changes in behaviour, external appearance or faeces as well as no test item-related influences on body weight or food consumption were noted for the treatment groups.
The reproduction parameters (number of implantations, number of resorptions and number of fetuses) were not influenced by the test item.
No embryotoxic properties (no dead fetuses, no malformations and no variations) were noted at any of the tested dose levels.
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 behavioural changes, reaction to treatment, or illness.
Immediately after administration, any signs of illness or reaction to treatment were recorded. In case of changes, the animals were observed until the symptoms 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 would have 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 or premature delivery occurred in the study.
Ovaries and uterine content:
Dissection technique and evaluation of the animals:
On gestation day 21, the rats were laparotomised under CO2 narcosis. The ovaries and the uteri of the dams were removed; the gravid uteri (in toto) were weighed. In order to check for possible drug 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.
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:
i 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).
I 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-WILK 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), intergroup 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 or internal macroscopic 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 was done using StatXact 4.0.1 software.
Indices:
Corpora lutea
- number/dam
- absolute number/group
- mean/group
Implantations
- number/dam
- distributions in the uterine horns
- absolute number/group
- mean/group
Resorptions
- number of early and late resorptions/dam
- distributions in the uterine horns
- absolute number/group
- mean/group
- early resorptions < 2 mm
- late resorptions > 2 mm
Weight of placentae
- individual data/fetus
- mean/litter
- mean/group
- mean/sex and group
Weight of fetuses
- individual data per fetus (alive and dead)
- mean/litter
- mean/group
- mean/sex and group
Fetuses
- number/dam (alive)
- number/dam (dead)
- number of fetuses (alive + dead)/sex and dam
- distribution in the uterine horns
- absolute number of fetuses alive per group
- mean number of fetuses alive per group
- male/female ratio (alive + dead)
Runts
- number/dam
- number/group
Dead fetuses
- number/dam
- number/group
Malformed fetuses
- type of malformation
- individual data/fetus
- number and incidence (%)/group and litter
- Total malformation rate [%] = malformed fetuses per group x 100/fetuses per group
Fetuses with variations
- type of variation
- individual data/fetus
- number and incidence (%)/group and litter
- Total variation rate [%] = fetuses per group with variations x 100/ fetuses per group
Fetuses with retardations
- type of retardation
- individual data/fetus
- number and incidence (%)/group and litter
- Total retardation rate [%] = fetuses per group with retardations x 100/fetuses per group
Indices of pre-implantation loss and post-implantation loss:
Calculation of group indices
- Pre-implantation loss [%] = (Corpora lutea - implantations) x 100/Corpora lutea
- Post-implantation loss [%] = (Implantations - living fetuses) x 100/Implantations
Calculation of mean indices per litter
- Pre-implantation loss [%] = Sum of pre-implantation losses per litter [%]/Number of litters
- Post-implantation loss [%] = Sum of post-implantation losses per litter [%]/Number of litters
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes in behaviour, the external appearance or the faeces were noted in the treatment groups (100, 300 or 1000 mg/kg b.w./day).
Observations that were considered to be spontaneous and not test item-related were noted for one animal of the low dose group and 2 animals of the high dose group as described below.
In the low dose group (100 mg/kg b.w./day), dam no. 42 was noted with slight salivation (on days 7 to 13), piloerection (on days 10 to 21) and breathing sounds (on days 7 to 21)
As dam no. 42 also had a low body weight, a negative net body weight gain was noted with an inflated stomach and an inflated small intestine the observations listed above were considered to be due to the poor health condition and not test item-related.
The poor health conditions were considered to be spontaneous and not test item-related as they were noted for only one animal.
At 1000 mg/kg b.w./day, dam no. 77 was noted with slight salivation on day 6 and dam no. 79 was noted with red eye discharge (left eye on days 9 to 13) and slightly reduced motility (on days 10 to 13).
As salivation was noted for only one dam (no. 77) on one test day, salivation was considered to be spontaneous and not test item-related.
Red eye discharge and reduced motility were noted only transiently for several test days for only one dam (no. 79). Therefore, red eye discharge and reduced motility were considered to be spontaneous and not test item-related. Result tables and background data please see below.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not relevant, as gavage study
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 substance/kg b.w./day).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item-related differences in body weight and body weight gain were noted between the dams of the control group and the treatment groups (100, 300 or 1000 mg/kg b.w./day). Result tables and background data as well as graphical view please see below.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item-related difference was noted between the control group and the treatment groups (100, 300 or 1000 mg/kg b.w./day).
A slight but statistically significantly (at p ≤ 0.05) increased food consumption was noted between GD 0 to GD 1 for the dams treated with 300 mg/kg b.w./day (9.6% above the value of the control group). As this was before start of dosing, this increase was considered to be spontaneous. Result tables and background data as well as graphical view please see below.
Food efficiency:
not examined
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 examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Result tables and background data as well as graphical view please see below.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test item-related differences were noted on uterus and carcass weights. Result tables and background data as well as graphical view please see below.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes were noted during the macroscopic inspection of the dams at necropsy.
Dilation of the renal pelvis (kidney(r.), approx. 5 mm in diameter), observed in one animal of low dose group only, was assessed as incidental. Slight reddish partly discolouration of lungs in two high dose group animals was considered spontaneous. Result tables and background data as well as graphical view please see below.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
No abortion or premature delivery occurred in the study. Result tables and background data please see below.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No test item-related influence on the reproductive parameters such as the index of pre- and post-implantation loss was noted during the study. Result tables and background data please see below.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No test item-related influence on the reproductive parameters such as resorptions were noted during the study. Result tables and background data please see below.
Early or late resorptions:
no effects observed
Description (incidence and severity):
No test item-related influence on the reproductive parameters such as early or late resorptions were noted during the study. Result tables and background data please see below.
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead fetus was noted in the control group and in the low and high dose group (100 or 1000 mg test item/kg b.w./day).
In the intermediate dose group (300 mg test item/kg b.w./day), dam no. 51 was noted with one dead fetus (no. 51-6). External examination of the dead fetus revealed no observations. The observation of only one dead fetus was considered to be spontaneous and not test item-related. Result tables and background data please see below.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Result tables and background data please see below.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
From 25 treated dams 23, 25, 25 and 24 became pregnant in des control, 100 mg/kg bw/d, 300 mg/kg bw/d and 1000 mg/kg bw/d groups, respectively. Thus, there was no significant effect observed. Result tables and background data please see below.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
In the low dose group (100 mg N,N'-(methylenedi-4,1-phenylene)bis(2-oxoazepane-1-carboxamide)/kg b.w./day), dam no. 42 was noted with slight salivation, piloerection and breathing sounds. As dam no. 42 also had a low body weight (see tables and background material below), a negative net body weight gain (see table Gravid Uterine, Carcass and Net Body Weight Gain) and was noted with an inflated stomach and an inflated small intestine these observations were considered to be due to the poor health condition and not test item-related. The poor health conditions were considered to be spontaneous and not test item-related as they were noted for only one animal in the low dose group.
At 1000 mg/kg b.w./day, dam no. 77 was noted with slight salivation and dam no. 79 was noted with red eye discharge (left eye) and slightly reduced motility. As salivation was noted for only one dam (no. 77) on one test day, salivation was considered to be spontaneous and not test item-related.
Red eye discharge and reduced motility were noted only transiently for several test days (5 and 4, respectively) for only one dam (no. 79) and thus, was considered not test-item related.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
No test item-related differences were noted between the control group and the treatment groups. The fetal weights showed no test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg/kg b.w./day). Result tables and background data please see below.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No test item-related deaths of fetuses were noted in any of the test groups.
No dead fetus was noted in the control group and in the low and high dose group (100 or 1000 mg/kg b.w./day).
In the intermediate dose group (300 mg/kg b.w./day), dam no. 51 was noted with one dead fetus (no. 51-6). External examination of the dead fetus revealed no observations. The observation of only one dead fetus was considered to be spontaneous and not test item-related. Result tables and background data please see below.
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 (100, 300 or 1000 mg/kg b.w./day).
The following table gives the ratio of male per female fetuses per group:
Control: Ratio 0.94 male/female fetuses
100 mg/kg b.w./d: Ratio 1.16 male/female fetuses
300 mg/kg b.w./d: Ratio 1.03 male/female fetuses
1000 mg/kg b.w./d: Ratio 1.00 male/female fetuses
Result tables and background data please see below.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
One runt was noted for the control group (no. 15-5), 2 runts (nos. 27-1 and 27-4) were noted for the low dose group (100 mg/kg b.w./day) and one runt (no. 67-15) was noted for the intermediate dose group (300 mg/kg b.w./day). The occurrence of up to two runts per dose group is within the normal range of biological variability and therefore was considered to be not test item-related. Result tables and background data please see below.
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related malformations were noted during the macroscopic examinations at laparotomy (external inspection and inspection of the organs and tissues for gross lesions),
No macroscopically visible external malformations were noted for the fetuses of the intermediate and high dose group (300 or 1000 mg/kg b.w./day) during the external inspection at laparotomy. In the control group, one fetus (no. 15-5) was noted with a malformation in form of a small head and a variation in form of subcutaneous oedema at the neck and thorax. At 100 mg/kg b.w./day, a malformation in form of an omphalocele (approx. 5 mm in diameter with a prolapse of organs) was noted for fetus no. 32-1. The single observation of one omphalocele in a dose group was considered to be spontaneous and not test item-related. Result tables and background data please see below.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No malformations were noted during the skeletal examination according to DAWSON and the soft tissue examination according to WILSON.
No skeletal malformations were noted for the fetuses of the control group and the test item-treated groups (100, 300 or 1000 mg/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 or ribs wavy), the skull (fontanelle enlarged) and the sternum (bipartite, dumbbell-shaped, fused or misaligned to a slight degree).
No test item-related difference 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/kg b.w./day).
Incidences of the test groups that were statistically significantly different from the control group but were considered to be not test item-related are listed in the table on the following page.
Less than 13 ribs ossified
Control: 0.0%; historical background data 0.0 - 2.9% (n = 52 controls)
Group 2: 0.7%; historical background data 0.0 – 5.6% (n = 156 controls)
Group 3: 0.7%; historical background data 0.0 – 5.6% (n = 156 controls)
Group 4: 2.8% *; historical background data 0.0 – 5.6% (n = 156 controls)
Total skeletal variations
Control: 2.0%; historical background data 0.0 – 28.8% (n = 52 controls)
Group 2: 10.7% ** #2 #3; historical background data 0.0 – 28.7% (n = 156 controls)
Group 3: 2.8%; historical background data 0.0 – 28.7% (n = 156 controls)
Group 4: 5.6%; historical background data 0.0 – 28.7% (n = 156 controls)
*/**: (p ≤ 0.05 / p ≤ 0.01) Fisher or Chi2 - test
#2: Considered to be spontaneous as within LPT background data range.
#3 The increased incidence of total skeletal variations was mainly due to a statistically not significantly increased incidence of fetuses with an enlarged fontanelle and/or bipartite sternebrae. However, the observations were still in the LPT background data range (sternebrae bipartite) or only noted for fetuses of one litter (fontanelle enlarged) and therefore were considered to be not test item-related. Result tables and background data please see below.
Visceral malformations:
no effects observed
Description (incidence and severity):
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.
No malformations were noted for the fetuses of the control group and the fetuses of the treatment groups (100, 300 or 1000 mg/kg b.w./day) during the soft tissue examination according to WILSON.
During the examination of the organs and tissues according to WILSON, variations were noted for the brain (dilatation of the 4th cerebral ventricle), the kidneys (uni- or bilateral dilatation of the renal pelvis or malpositioned) 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 treatment groups (100, 300 or 1000 mg/kg b.w./day). Result tables and background data please see below.
Other effects:
no effects observed
Description (incidence and severity):
No unclassified observations were noted for the control group and for the low and intermediate dose group (100 or 300 mg/kg b.w./day).
An unclassified observation in form of a thoracic cavity filled with blood was noted for one fetus (no. 86-10) of the high dose group (1000 mg/kg b.w./day). This observation was considered to be a preparation-induced artefact and not test item-related.
No test item-related malformations or variations were 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).
Furthermore, no test item-related retardations (delay in ossification) were noted in any of the treatment groups (100, 300 and 1000 mg/kg b.w./day). Result tables and background data please see below.
Details on embryotoxic / teratogenic effects:
No enbryotoxic or teratogenic effects were observed.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Result tables and background data for OECD 414 study with N,N'-(methylenedi-4,1-phenylene)bis(2-oxoazepane-1-carboxamide)

Summary of animals examined

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

2

0

0

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 evaluated dams

(spare animals)

3

5

5

4

Evaluated litters

20

20

20

20

 

Summary of animals evaluated

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 - 6, 8 -18, 20 - 22

26 - 45

51 ‑ 70

76 - 89,

91 - 96

Dams not pregnant

(animal nos.)

7, 19

none

none

90

Dams with total implantation loss (animal nos.)

none

none

none

none

Prematurely deceased animals (animal nos.)

none

none

none

none

Reserve animals

(animal nos.)

23 - 25

46 - 50

71 - 75

97 - 100

 

 

Maternal Clinical Signs - Summary

 

Observation Type: All Types Female

From Day 0 (Mating (A)) to 21 (Mating)

Gr.1: control

Gr.2: 100 mg/kg

Gr.3: 300 mg/kg

Gr.4: 1000 mg/kg

Number of Animals Examined:

20

20

20

20

Normal

Number of Animals Affected

20

20

20

20

First to Last seen

0 - 21

0 - 21

0 - 21

0 - 21

% of Affected Animals

100

100

100

100

Number of Times Recorded

440

425

440

434

Scheduled Removal (Terminal)

Number of Animals Affected

20

20

20

20

First to Last seen

21

21

21

21

% of Affected Animals

100

100

100

100

Number of Times Recorded

20

20

20

20

Piloerection

Number of Animals Affected

0

1

0

0

First to Last seen

-

10 - 21

-

-

% of Affected Animals

0

5

0

0

Number of Times Recorded

0

12

0

0

Salivation

Number of Animals Affected

0

1

0

1

First to Last seen

-

7 – 13

-

6

% of Affected Animals

0

5

0

5

Number of Times Recorded

0

7

0

1

Eye Discharge, Left

Number of Animals Affected

0

0

0

1

First to Last seen

-

-

-

9 – 13

% of Affected Animals

0

0

0

5

Number of Times Recorded

0

0

0

5

Motility reduced

Number of Animals Affected

0

0

0

1

First to Last seen

-

-

-

10 – 13

% of Affected Animals

0

0

0

5

Number of Times Recorded

0

0

0

4

Breathing sounds

Number of Animals Affected

0

1

0

0

First to Last seen

-

7 – 21

-

-

% of Affected Animals

0

5

0

0

Number of Times Recorded

0

15

0

0

Non pregnant females (NP) are not considered in the calculation.

 

 

Maternal Body Weights – Summary

 

Day(s) Relative to Mating

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

Gr.1:

Mean

223.33

229.82

237.76

245.80

253.64

255.82

262.23

264.54

270.00

274.22

280.60

286.44

292.33

296.86

300.90

307.74

318.93

331.22

347.71

364.13

377.98

398.87

SD

14.53

15.87

15.69

13.80

17.13

15.62

13.81

14.46

16.61

13.75

14.62

15.50

15.23

15.47

16.24

17.58

17.54

18.72

20.41

22.01

22.99

25.83

N

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

Gr.2:

Mean

222.01

230.06

237.81

246.49

252.54

254.66

260.21

262.89

267.10

273.61

280.71

287.59

293.73

300.19

303.25

311.93

321.85

337.10

352.96

368.67

385.19

406.19

SD

13.48

15.23

15.72

16.67

16.50

17.81

20.27

20.59

23.77

26.19

29.87

32.07

32.21

30.14

29.42

31.65

33.44

34.94

37.84

39.83

41.80

43.77

N

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

%Diff

-0.6

0.1

0.0

0.3

-0.4

-0.5

-0.8

-0.6

-1.1

-0.2

0.0

0.4

0.5

1.1

0.8

1.4

0.9

1.8

1.5

1.2

1.9

1.8

Gr.3:

Mean

221.31

229.98

237.13

243.92

249.11

254.52

258.99

262.10

267.52

275.34

280.06

286.15

292.43

297.39

302.15

309.12

320.98

333.22

349.35

367.05

379.82

401.44

SD

13.14

13.04

14.56

15.25

15.49

12.91

15.48

14.70

15.06

20.13

20.41

20.58

20.47

19.72

19.51

20.25

21.21

22.18

21.91

23.56

23.58

25.27

N

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

%Diff

-0.9

0.1

-0.3

-0.8

-1.8

-0.5

-1.2

-0.9

-0.9

0.4

-0.2

-0.1

0.0

0.2

0.4

0.4

0.6

0.6

0.5

0.8

0.5

0.6

Gr.4:

Mean

223.08

229.58

239.05

246.75

252.08

257.28

260.54

265.59

269.04

275.56

283.47

289.65

297.36

301.25

306.64

314.21

324.51

336.53

355.46

370.17

387.32

407.03

SD

17.12

18.23

17.92

16.16

15.67

14.83

15.93

16.25

16.51

16.10

17.45

16.94

15.82

17.53

18.19

17.74

19.87

19.78

21.34

22.32

22.74

27.86

N

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

%Diff

-0.1

-0.1

0.5

0.4

-0.6

0.6

-0.6

0.4

-0.4

0.5

1.0

1.1

1.7

1.5

1.9

2.1

1.7

1.6

2.2

1.7

2.5

2.0

Anova & Dunnett(Rank)

 

 

Maternal Body Weight Gain – Summary (Day(s) Relative to Mating) [g]

 

Period:

0 – 3

3 – 6

6 – 9

9 – 12

12 – 15

15 – 18

18 – 21

6 – 21

0 – 21

6 – 21 (%)

0 – 21 (%)

Gr.1:

Mean

22.47

16.43

11.99

18.12

15.41

39.97

51.16

136.64

175.54

52.12

78.84

SD

5.92

6.42

6.83

5.61

6.77

5.89

7.31

16.85

19.43

5.82

9.37

N

20

20

20

20

20

20

20

20

20

20

20

Gr.2:

Mean

24.48

13.72

13.41

20.12

18.21

41.03

53.23

145.98

184.18

56.11

82.89

SD

5.27

7.36

18.51

8.45

4.17

7.53

11.46

32.44

36.64

12.10

15.29

N

20

20

20

20

20

20

20

20

20

20

20

%Diff

8.9

-16.5

11.8

11.0

18.1

2.6

4.1

6.8

4.9

-

-

Gr.3:

Mean

22.61

15.07

16.36

17.09

16.69

40.23

52.10

142.46

180.13

55.16

81.70

SD

4.65

3.84

8.10

10.97

3.97

6.03

8.22

19.22

22.67

7.89

11.71

N

20

20

20

20

20

20

20

20

20

20

20

%Diff

0.6

-8.3

36.4

-5.7

8.3

0.7

1.8

4.3

2.6

-

-

Gr.4:

Mean

23.67

13.79

15.02

21.81

16.85

41.25

51.58

146.50

183.95

56.27

82.81

SD

6.91

4.46

5.16

5.23

5.29

7.35

12.44

17.18

19.55

6.14

9.95

N

20

20

20

20

20

20

20

20

20

20

20

%Diff

5.3

-16.1

25.3

20.4

9.3

3.2

0.8

7.2

4.8

-

-

 

 

Group

Time interval

Gestation day 0 - 21

(whole study period)

 

Gain

in g#

Gain

in %#

Difference to control %#

Control

175.5

78.8

n.a.

Group 2 100 mg/kg

184.2

82.9

+4.9

Group 3 300 mg/kg

180.1

81.7

+2.6

Group 4 1000 mg/kg

184.0

82.8

+4.8

* Statistically significant at p ≤ 0.05 (ANOVA/DUNNETT test)

n.a. not applicable

# The values are taken from table 'Maternal Body Weight Gain - Summary'

 

 

Gravid Uterine Weight, Carcass Weight and Net Body Weight Gain, Summary [g]

 

Gravid Uterus

Carcass

Net Gain from GD 6

Gr.1:

Mean

105.59

293.28

31.06

SD

10.34

20.07

12.02

N

20

20

20

Gr.2:

Mean

106.79

299.40

39.20*

SD

17.05

31.44

21.55

N

20

20

20

%Diff

1.1

2.1

-

Gr.3:

Mean

100.76

300.69

41.70

SD

17.37

21.12

16.33

N

20

20

20

%Diff

-4.6

2.5

-

Gr.4:

Mean

105.22

301.81

41.28

SD

11.35

25.79

15.67

N

20

20

20

%Diff

-0.3

2.9

-

Anova & Dunnett(Rank): * = p ≤ 0.05

The statistical significance in group 2 noted for the net body weight gain from day 6 was due to the negative net body weight gain of no. 42 and considered to be spontaneous.

Comments and Markers

Day

Group

Sex

Measurement

Marker

Comment

21

2

Female

Net Gain from GD 6

*

Anova & Dunnett(Rank): * = p ≤ 0.05

 

 

Maternal Food Consumption – Summary Relative Food Consumption [g/kg bw/day], Day(s) Relative to Mating (Litter: A)

 

0 – 1

1 – 2

2 – 3

3 – 4

4 – 5

5 – 6

6 – 7

7 – 8

8 – 9

9 – 10

10 – 11

11 – 12

12 – 13

13 – 14

14 – 15

15 – 16

16 – 17

17 – 18

18 – 19

19 – 20

20 - 21

Gr.1:

Mean

86.22

97.82

99.05

100.50

99.05

94.64

93.73

96.22

97.07

88.30

87.79

92.73

88.83

87.78

84.67

86.02

85.54

84.94

81.60

75.79

73.69

SD

10.85

7.70

9.07

10.29

10.87

10.87

9.75

15.38

9.38

8.26

8.95

9.34

8.26

6.99

5.20

5.71

6.44

7.06

5.78

6.78

6.68

N

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

Gr.2:

Mean

92.20

96.00

99.56

101.07

96.86

97.29

94.36

100.33

95.58

87.78

87.11

91.77

91.11

88.54

87.43

89.90

87.77

86.68

83.02

80.95

73.44

SD

9.12

8.61

9.69

11.41

12.92

7.98

12.30

19.89

13.94

19.94

21.19

17.32

16.84

7.24

6.41

8.23

7.47

6.69

7.91

8.51

11.35

N

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

%Diff

6.9

-1.9

0.5

0.6

-2.2

2.8

0.7

4.3

-1.5

-0.6

-0.8

-1.0

2.6

0.9

3.3

4.5

2.6

2.1

1.7

6.8

-0.3

Gr.3:

Mean

94.47*

96.51

98.56

98.86

98.93

96.05

93.01

102.85

92.26

87.62

87.60

94.85

96.20

90.25

86.73

89.70

88.41

86.08

84.74

75.81

77.71

SD

7.84

10.74

10.20

17.53

13.04

7.54

8.36

12.56

21.13

24.75

15.14

11.52

8.07

8.59

7.22

8.22

8.57

10.40

9.51

9.68

10.14

N

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

%Diff

9.6

-1.3

-0.5

-1.6

-0.1

1.5

-0.8

6.9

-5.0

-0.8

-0.2

2.3

8.3

2.8

2.4

4.3

3.4

1.3

3.8

0.0

5.5

Gr.4:

Mean

88.17

93.78

91.26

102.36

97.08

97.30

91.83

94.90

95.84

90.20

91.58

91.68

91.37

90.16

84.41

89.50

86.43

85.28

83.86

77.20

78.26

SD

21.88

15.90

19.87

16.03

13.01

10.77

9.14

10.84

14.79

11.24

7.21

8.90

9.49

6.43

7.99

7.03

6.86

7.72

7.04

10.22

11.10

N

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

%Diff

2.3

-4.1

-7.9

1.9

-2.0

2.8

-2.0

-1.4

-1.3

2.1

4.3

-1.1

2.9

2.7

-0.3

4.0

1.0

0.4

2.8

1.9

6.2

Anova & Dunnett(Rank): * = p ≤ 0.05

Comments and Markers

Measurement

Group

Sex

Marker

Comment

Relative Food Consumption

3

Female 0 – 1

*

Anova & Dunnett(Rank): * = p ≤ 0.05

 

 

Maternal Macroscopic Findings at Necropsy, Placentae-Inspection, Summary

 

Gr.1: control

Gr.2: 100 mg/kg

Gr.3: 300 mg/kg

Gr.4: 1000 mg/kg

Number of Animals:

20

20

20

20

LUNGS

 

 

 

 

Submitted

20

20

20

20

Normal

20

20

20

18

Discoloration; dark, red

0

0

0

2

STOMACH

 

 

 

 

Submitted

20

20

20

20

Normal

20

19

20

20

Inflated

0

1

0

0

KIDNEY, right

 

 

 

 

Submitted

20

20

20

20

Normal

20

19

20

20

pelvis; Dilation

0

1

0

0

INTESTINES

 

 

 

 

Submitted

20

20

20

20

Normal

20

19

20

20

Inflated

0

1

0

0

 

 

Reproduction Data - Summary - Values per Group

 

Gr.1: control

Gr.2: 100 mg/kg

Gr.3: 300 mg/kg

Gr.4: 1000 mg/kg

Evaluated dams with:

20

20

20

20

 - viable fetuses

20

20

20

20

 - only dead fetuses

0

0

0

0

 - resorptions

7

9

3

6

 - resorptions (%)

35

45

15

30

 - complete resorption (NVF)

0

0

0

0

Corpora Lutea per group

306

320

286

294

Implantation sites per group

302

315

285

293

No. of resorptions per group

9

15

3

7

No. of early resorpt. Per gr.

9

12

2

7

No. of late resorpt. Per gr.

0

3

1

0

Evaluated dams with 1 Resorpt. (Yes)

5

5

3

5

Evaluated dams with 2 Resorpt. (Yes)

2

3

0

1

Evaluated dams with >2 Resorpt (Yes)

0

1

0

0

Fetuses (alive + dead)/group

293

300

282

286

M. fetuses (alive+dead)/group

142

161

143

143

F. fetuses (alive+dead)/group

151

139

139

143

M/F-Ratio (alive+dead)/group

0.94

1.16

1.03

1.00

Fetuses (dead) per group

0

0

1

0

Fetuses (alive) per group

293

300

281

286

Pre-implant. Loss (%)/group

1.3

1.6

0.3

0.3

Post-implant. Loss (%)/group

3.0

4.8

1.4

2.4

Runts per Group

1

2

1

0

Non pregnant females (NP) are not considered in the calculation. No statistically significant differences between the control and the test groups were noted for the pre- and the post-implantation loss.

 

 

Reproduction Data - Summary - Mean Values per Dam - Resorptions and Post-implantation loss -

Day(s): 21 Relative to Mating

Gr.1: control

Gr.2: 100 mg/kg

Gr.3: 300 mg/kg

Gr.4: 1000 mg/kg

Dams Aborted

 

0

0

0

0

Viab Fet Yes

 

20

20

20

20

Viab Fet No

 

0

0

0

0

Considered

N

20

20

20

20

Corpora Lutea

Mean

15.3

16.0

14.3

14.7

SD

1.6

2.1

2.6

1.8

Sum

306

320

286

294

Implantation sites

Mean

15.1

15.8

14.3

14.7

SD

1.6

1.9

2.6

1.8

Sum

302

315

285

293

Total Number Resorptions

Mean

0.5

0.8

0.2

0.4

SD

0.7

1.1

0.4

0.6

Sum

9

15

3

7

Early Resorptions

Mean

0.5

0.6

0.1

0.4

SD

0.7

0.9

0.3

0.6

Sum

9

12

2

7

Late Resorptions

Mean

0.0

0.2

0.1

0.0

SD

0.0

0.4

0.2

0.0

Sum

0

3

1

0

Pre-implant. loss (%)

Mean

1.24

1.47

0.31

0.31

SD

3.25

3.15

1.40

1.40

Post-implant loss (%)

Mean

2.88

4.65

1.23

2.41

SD

4.36

6.53

2.53

4.11

Non pregnant females (NP) are not considered in the calculation.

 

 

Reproduction Data - Summary - Mean Values per Dam - Live and Dead Fetuses -

Day(s): 21 Relative to Mating

Gr.1: control

Gr.2: 100 mg/kg

Gr.3: 300 mg/kg

Gr.4: 1000 mg/kg

Viab Fet Yes

 

20

20

20

20

Fetuses alive + dead

Mean

14.7

15.0

14.1

14.3

SD

1.5

2.0

2.5

1.9

Sum

293

300

282

286

Dead Fetuses

Mean

0.0

0.0

0.1

0.0

SD

0.0

0.0

0.2

0.0

Sum

0

0

1

0

Fetuses alive Mean

14.7

15.0

14.1

14.3

 

SD

1.5

2.0

2.5

1.9

Sum

293

300

281

286

Live Fetuses % of total

Mean

100.00

100.00

99.71

100.00

SD

0.00

0.00

1.32

0.00

N

20

20

20

20

Only dams with viable fetuses are considered.

 

 

Fetal and Placental Weights - Mean Values per Group

Day(s): 21 Relative to Mating

Gr.1: control

Gr.2: 100 mg/kg

Gr.3: 300 mg/kg

Gr.4: 1000 mg/kg

Mean Fetal Weight (M) [g]

Mean

5.54

5.39

5.57

5.62

SD

0.33

0.45

0.31

0.26

N

20

20

20

20

%Diff

.

-2.6

0.6

1.5

Mean Fetal Weight (F) [g]

Mean

5.26

5.15

5.25

5.30

SD

0.32

0.41

0.29

0.24

N

20

20

19

20

%Diff

.

-2.1

-0.1

0.8

Mean Fetal Wt. (both) [g]

Mean

5.40

5.28

5.43

5.45

SD

0.31

0.42

0.32

0.24

N

20

20

20

20

%Diff

.

-2.1

0.5

1.0

Mean Plac. Weight (M) [g]

Mean

0.512

0.527

0.553

0.534

SD

0.079

0.048

0.081

0.064

N

20

20

20

20

%Diff

.

3.1

8.1

4.3

Mean Plac. Weight (F) [g]

Mean

0.487

0.521

0.524

0.532

SD

0.062

0.077

0.088

0.060

N

20

20

19

20

%Diff

.

7.0

7.6

9.3

Mean Plac. Wt. (Both) [g]

Mean

0.498

0.525

0.541

0.531

SD

0.067

0.060

0.083

0.059

N

20

20

20

20

%Diff

.

5.3

8.5

6.5

 

 

 

Summary of reproductive parameters



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

306

15.3

320

16.0

286

14.3

294

14.7

Implantation sites

total

mean per dam

302

15.1

315

15.8

285

14.3

293

14.7

Resorptions

total

mean per dam

9

0.5

15

0.8

3

0.2

7

0.4

Early resorptions

total

mean per dam

9

0.5

12

0.6

2

0.1

7

0.4

Late resorptions

total

mean per dam

0

0.0

3

0.2

1

0.1

0

0.0

Live fetuses

total

mean per dam

293

14.7

300

15.0

281

14.1

286

14.3

Dead fetuses

total

0

0

1

0

Pre-implantation loss [%]

per group #1

1.3

1.6

0.3

0.3

Post-implantation loss [%]

per group #2

3.0

4.8

1.4

2.4

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

#1The 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).

#2The 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 live fetuses/implantation sites of the control group using the Chi2test (*/**: p ≤ 0.05/p ≤ 0.01).

 

 

The following table gives the ratio of male per female fetuses per group:

Ratio

[male/female fetuses]

Control

0.94

100 mg/kg

1.16

300 mg/kg

1.03

1000 mg/kg

1.00

 

 

Summary of All Classified and Unclass. Fetal External Observations

Exam Type: All

Gr.1: control

Gr.2:100 mg/kg

Gr.3: 300 mg/kg

Gr.4:1000 mg/kg

Dams with Viable Fetuses:

20

20

20

20

Total Number of Fetuses in Group:

293

300

282

286

Number of Fetuses Examined:

293

300

282

286

All classifications

 

 

 

 

 

Number of Fetuses [f]

 

1

1

0

0

Group % of Fetuses

 

0.3

0.3

0.0

0.0

Number of Litters [f]

 

1

1

0

0

 

%

5.0

5.0

0.0

0.0

Litter % of Fetuses

Mean

0.38

0.45

0.00

0.00

 

Max

7.7

9.1

0.0

0.0

 

Min

0.0

0.0

0.0

0.0

Malformation

 

 

 

 

 

Number of Fetuses [f]

 

1

1

0

0

Group % of Fetuses

 

0.3

0.3

0.0

0.0

Number of Litters [f]

 

1

1

0

0

 

%

5.0

5.0

0.0

0.0

Litter % of Fetuses

Mean

0.38

0.45

0.00

0.00

 

Max

7.7

9.1

0.0

0.0

 

Min

0.0

0.0

0.0

0.0

Variation

 

 

 

 

 

Number of Fetuses [f]

 

1

0

0

0

Group % of Fetuses

 

0.3

0.0

0.0

0.0

Number of Litters [f]

 

1

0

0

0

 

%

5.0

0.0

0.0

0.0

Litter % of Fetuses

Mean

0.38

0.00

0.00

0.00

 

Max

7.7

0.0

0.0

0.0

 

Min

0.0

0.0

0.0

0.0

Head/Neck

 

 

 

 

 

Head, Small - Malformation

Fetuses N(%)

1(0.3)

0(0.0)

0(0.0)

0(0.0)

 

Litters N(%)

1(5.0)

0(0.0)

0(0.0)

0(0.0)

Neck, Subcutaneous oedema - Variation

Fetuses N(%)

1(0.3)

0(0.0)

0(0.0)

0(0.0)

 

Litters N(%)

1(5.0)

0(0.0)

0(0.0)

0(0.0)

Trunk

 

 

 

 

 

Abdomen, Omphalocele - Malformation

Fetuses N(%)

0(0.0)

1(0.3)

0(0.0)

0(0.0)

 

Litters N(%)

0(0.0)

1(5.0)

0(0.0)

0(0.0)

Thorax, Subcutaneous oedema - Variation

Fetuses N(%)

1(0.3)

0(0.0)

0(0.0)

0(0.0)

 

Litters N(%)

1(5.0)

0(0.0)

0(0.0)

0(0.0)

[f] - Chi-Squared & Chi-Squared - Pearson

 

 

Summary of All Classified and Unclassified Fetal Internal Observations, No. Fetuses/Group

 

No. Fetuses (alive+dead)

Intern Exam. Body

With Intern Malformation

With Intern Variation

Gr.1: control

293

147

0

0

Gr.2: 100 mg/kg

300

150

0

0

Gr.3: 300 mg/kg

282

142

0

0

Gr.4: 1000 mg/kg

286

143

0

0

 

 

Summary of all classified fetal skeletal observations

 

Test Group 1

Control

Test Group 2

100 mg/kg

Test Group 3

300 mg/kg

Test Group 4

1000 mg/kg

Litters Evaluated

N

20

20

20

20

Fetuses Evaluated

N

147

150

142

143

 Live

N

147

150

142

143

 Dead

N

0

0

0

0

TOTAL MALFORMATIONS

 

 

 

 

 Fetal Incidence

N

0

0

0

0

 

%

0.0

0.0

0.0

0.0

 Litter Incidence

N

0

0

0

0

 

%

0.0

0.0

0.0

0.0

 Affected Fetuses/Litter

MEAN%

0.0

0.0

0.0

0.0

 

S.D.

0.0

0.0

0.0

0.0

TOTAL VARIATIONS

 

 

 

 

 Fetal Incidence ##

N

3

16**

4

8

 

%

2.0

10.7

2.8

5.6

 Litter Incidence

N

2

9*

3

5

 

%

10.0

45.0

15.0

25.0

 Affected Fetuses/Litter

MEAN%

2.1

10.5

2.6

6.0

 

S.D.

7.0

16.4

6.5

12.3

TOTAL RETARDATIONS

 

 

 

 

 Fetal Incidence ##

N

126

133

130

126

 

%

85.7

88.7

91.5

88.1

 Litter Incidence

N

20

20

20

20

 

%

100.0

100.0

100.0

100.0

  Affected Fetuses/Litter

MEAN%

85.2

87.4

92.6

87.4

 

S.D.

18.1

21.2

12.1

19.9

TOTAL FETAL SKELETAL MALFORMATIONS

 

 

 

 

 Fetal Incidence

N

0

0

0

0

 

%

0.0

0.0

0.0

0.0

 Litter Incidence

N

0

0

0

0

 

%

0.0

0.0

0.0

0.0

FONTANELLE ENLARGED

 

 

 

 

 Fetal Incidence

N

2

5

0

2

 

%

1.4

3.3

0.0

1.4

 Litter Incidence

N

1

1

0

1

 

%

5.0

5.0

0.0

5.0

LESS THAN 13 RIB(S) OSSIFIED

 

 

 

 

 Fetal Incidence

N

0

1

1

4*

 

%

0.0

0.7

0.7

2.8

 Litter Incidence

N

0

1

1

2

 

%

0.0

5.0

5.0

10.0

RIB(S) WAVY

 

 

 

 

 Fetal Incidence

N

0

1

1

1

 

%

0.0

0.7

0.7

0.7

 Litter Incidence

N

0

1

1

1

 

%

0.0

5.0

5.0

5.0

STERNEBRA(E) BIPARTITE

 

 

 

 

 Fetal Incidence

N

1

5

0

1

 

%

0.7

3.3

0.0

0.7

 Litter Incidence

N

1

5

0

1

 

%

5.0

25.0

0.0

5.0

STERNEBRA(E) DUMBBELL-SHAPED

 

 

 

 

 Fetal Incidence

N

0

3

2

0

 

%

0.0

2.0

1.4

0.0

 Litter Incidence

N

0

1

1

0

 

%

0.0

5.0

5.0

0.0

STERNEBRA(E) FUSED (SEVERITY: SLIGHT)

 

 

 

 

 Fetal Incidence

N

1

0

0

0

 

%

0.7

0.0

0.0

0.0

 Litter Incidence

N

1

0

0

0

 

%

5.0

0.0

0.0

0.0

STERNEBRA(E) MISALIGNED (SEVERITY: SLIGHT)

 

 

 

 

 Fetal Incidence

N

1

2

0

0

 

%

0.7

1.3

0.0

0.0

 Litter Incidence

N

1

2

0

0

 

%

5.0

10.0

0.0

0.0

TOTAL FETAL SKELETAL VARIATIONS

 

 

 

 

 Fetal Incidence ##

N

3

16**

4

8

 

%

2.0

10.7

2.8

5.6

 Litter Incidence

N

2

9*

3

5

 

%

10.0

45.0

15.0

25.0

ABSENCE OF OSSIFICATION IN METACARPALIA 2 TO 5

 

 

 

 

 Fetal Incidence

N

1

1

1

0

 

%

0.7

0.7

0.7

0.0

 Litter Incidence

N

1

1

1

0

 

%

5.0

5.0

5.0

0.0

ABSENCE OF OSSIFICATION IN METATARSALIA 2 TO 5

 

 

 

 

 Fetal Incidence

N

1

0

0

 

 

%

0.7

0.0

0.0

0.0

 Litter Incidence

N

1

0

0

0

 

%

5.0

0.0

0.0

0.0

CAUDAL VERTEBRAL BODIES, ONLY ONE BODY OSSIFIED

 

 

 

 

 Fetal Incidence

N

0

1

0

0

 

%

0.0

0.7

0.0

0.0

 Litter Incidence

N

0

1

0

0

 

%

0.0

5.0

0.0

0.0

HYOID UNOSSIFIED

 

 

 

 

 Fetal Incidence

N

77

84

84

84

 

%

52.4

56.0

59.2

58.7

 Litter Incidence

N

19

17

20

18

 

%

95.0

85.0

100.0

90.0

OS ISCHII UNOSSIFIED

 

 

 

 

 Fetal Incidence

N

0

0

1

0

 

%

0.0

0.0

0.7

0.0

 Litter Incidence

N

0

0

1

0

 

%

0.0

0.0

5.0

0.0

OS PUBIS INCOMPLETELY OSSIFIED

 

 

 

 

 Fetal Incidence

N

0

0

1

3

 

%

0.0

0.0

0.7

2.1

 Litter Incidence

N

0

0

1

2

 

%

0.0

0.0

5.0

10.0

SKULL INCOMPLETE OSSIFICATION

 

 

 

 

 Fetal Incidence

N

42

47

55*

48

 

%

28.6

31.3

38.7

33.6

 Litter Incidence

N

12

16

18*

16

 

%

60.0

80.0

90.0

80.0

STERNEBRA(E) INCOMPLETELY OSSIFIED

 

 

 

 

 Fetal Incidence

N

7

2

4

8

 

%

4.8

1.3

2.8

5.6

 Litter Incidence

N

3

2

4

4

 

%

15.0

10.0

20.0

20.0

STERNEBRA(E) REDUCED IN SIZE

 

 

 

 

 Fetal Incidence

N

87

77

89

71

 

%

59.2

51.3

62.7

49.7

 Litter Incidence

N

20

17

20

17

 

%

100.0

85.0

100.0

85.0

STERNEBRA(E) UNOSSIFIED

 

 

 

 

 Fetal Incidence

N

6

8

8

8

 

%

4.1

5.3

5.6

5.6

 Litter Incidence

N

4

6

7

4

 

%

20.0

30.0

35.0

20.0

THORACIC VERTEBRAL BODY/BODIES BIPARTITE

 

 

 

 

 Fetal Incidence

N

9

10

5

6

 

%

6.1

6.7

3.5

4.2

 Litter Incidence

N

5

8

4

5

 

%

25.0

40.0

20.0

25.0

THORACIC VERTEBRAL BODY/BODIES DUMBBELL-SHAPED

 

 

 

 

 Fetal Incidence

N

10

5

8

5

 

%

6.8

3.3

5.6

3.5

 Litter Incidence

N

9

4

5

3*

 

%

45.0

20.0

25.0

15.0

TOTAL FETAL SKELETAL RETARDATIONS

 

 

 

 

 Fetal Incidence ##

N

126

133

130

126

 

%

85.7

88.7

91.5

88.1

 Litter Incidence

N

20

20

20

20

 

%

100.0

100.0

100.0

100.0

Significantly different from controls:  * = p≤0.05  ** = p≤0.01 (Fisher or Chi-square test)

## Fetuses affected by several changes will be counted as one fetal incidence

 

 

Summary of all classified fetal soft tissue observations

 

Test Group 1

Control

Test Group 2

100 mg/kg

Test Group 3

300 mg/kg

Test Group 4

1000 mg/kg

Litters Evaluated

N

20

20

20

20

Fetuses Evaluated

N

146

150

140

143

 Live

N

146

150

139

143

 Dead

N

0

0

1

0

TOTAL MALFORMATIONS

 

 

 

 

  Fetal Incidence

N

0

0

0

0

 

%

0.0

0.0

0.0

0.0

 Litter Incidence

N

0

0

0

0

 

%

0.0

0.0

0.0

0.0

  Affected Fetuses/Litter

MEAN%

0.0

0.0

0.0

0.0

 

S.D.

0.0

0.0

0.0

0.0

TOTAL VARIATIONS

 

 

 

 

 Fetal Incidence ##

N

7

8

10

8

 

%

4.8

5.3

7.1

5.6

  Litter Incidence

N

5

5

8

5

 

%

25.0

25.0

40.0

25.0

 Affected Fetuses/Litter

MEAN%

5.2

5.7

7.0

5.5

 

S.D.

12.0

10.5

9.8

12.2

TOTAL FETAL SOFT TISSUE MALFORMATIONS

 

 

 

 

 Fetal Incidence

N

0

0

0

0

 

%

0.0

0.0

0.0

0.0

 Litter Incidence

N

0

0

0

0

 

%

0.0

0.0

0.0

0.0

DILATATION OF CEREBRAL VENTRICLE

 

 

 

 

 Fetal Incidence

N

0

0

2

0

 

%

0.0

0.0

1.4

0.0

 Litter Incidence

N

0

0

2

0

 

%

0.0

0.0

10.0

0.0

DILATATION OF RENAL PELVIS

 

 

 

 

 Fetal Incidence

N

6

6

5

7

 

%

4.1

4.0

3.6

4.9

 Litter Incidence

N

4

4

3

5

 

%

20.0

20.0

15.0

25.0

KIDNEY(S): MALPOSITIONED

 

 

 

 

 Fetal Incidence

N

0

0

0

1

 

%

0.0

0.0

0.0

0.7

 Litter Incidence

N

0

0

0

1

 

%

0.0

0.0

0.0

5.0

LIVER: HAEMORRHAGIC FOCUS/FOCI

 

 

 

 

 Fetal Incidence

N

1

2

3

0

 

%

0.7

1.3

2.1

0.0

 Litter Incidence

N

1

2

3

0

 

%

5.0

10.0

15.0

0.0

TOTAL FETAL SOFT TISSUE VARIATIONS

 

 

 

 

 Fetal Incidence ##

N

7

8

10

8

 

%

4.8

5.3

7.1

5.6

 Litter Incidence

N

5

5

8

5

 

%

25.0

25.0

40.0

25.0

THORACIC CAVITY: FILLED WITH BLOOD

 

 

 

 

 Fetal Incidence

N

0

0

0

1

 

%

0.0

0.0

0.0

0.7

 Litter Incidence

N

0

0

0

1

 

%

0.0

0.0

0.0

5.0

TOTAL FETAL SOFT TISSUE UNCLASSIFIED OBSERVATIONS

 

 

 

 

 Fetal Incidence

N

0

0

0

1

 

%

0.0

0.0

0.0

0.7

 Litter Incidence

N

0

0

0

1

 

%

0.0

0.0

0.0

5.0

Significantly different from controls:  * = p≤0.05  ** = p≤0.01 (Fisher or Chi-square test)

## Fetuses affected by several changes will be counted as one fetal incidence

 

 

External Malformations

Number of

fetuses with malformations

Group 1

Control

Group 2

100 mg test item/kg

Group 3

300 mg test item/kg

Group 4

1000 mg test item/kg

fetal incidence N

 %

1

0.3

1

0.3

0

0.0

0

0.0

litter incidence N

 %

1

5.0

1

5.0

0

0.0

0

0.0

 

 

External Variations

Number of

fetuses with variations

Group 1

Control

Group 2

100 mg test item/kg

Group 3

300 mg test item/kg

Group 4

1000 mg test item/kg

fetal incidence N

 %

1

0.3

0

0.0

0

0.0

0

0.0

 litter incidence N

 %

1

5.0

0

0.0

0

0.0

0

0.0

 

 

Malformations - Fresh Visceral #1

Number of

fetuses with malformations

Group 1

Control

Group 2

100 mg test item/kg

Group 3

300 mg test item/kg

Group 4

1000 mg test item/kg

fetal incidence N

 %

0

0.0

0

0.0

0

0.0

0

0.0

litter incidence N

 %

0

0.0

0

0.0

0

0.0

0

0.0

#1:

Macroscopic gross inspection of internal organs and tissues at laparotomy

 

 

 

Variations - FreshVisceral#1

Number of

fetuses with variations

Group 1

Control

Group 2

100 mg test item/kg

Group 3

300 mg test item/kg

Group 4

1000 mg test item/kg

 fetal incidence N

 %

0

0.0

0

0.0

0

0.0

0

0.0

 litter incidence N

 %

0

0.0

0

0.0

0

0.0

0

0.0

#1:

Macroscopic gross inspection of internal organs and tissues at laparotomy

 

 

 

Visceral - Malformations #1

 

Number of

fetuses with malformations

Group 1

Control

Group 2

100 mg test item/kg

Group 3

300 mg test item/kg

Group 4

1000 mg test item/kg

 

 fetal incidence N

 %

0

0.0

0

0.0

0

0.0

0

0.0

 

 litter incidence N

 %

0

0.0

0

0.0

0

0.0

0

0.0

 

#1:

Soft tissue examination according to WILSON

 

Visceral - Variations #1

Number of

fetuses with variations

Group 1

Control

Group 2

100 mg test item/kg

Group 3

300 mg test item/kg

Group 4

1000 mg test item/kg

 fetal incidence N

 %

7

4.8

8

5.3

10

7.1

8

5.6

 litter incidence N

 %

5

25.0

5

25.0

8

40.0

5

25.0

#1:

Soft tissue examination according to WILSON

 

 

 

 

 

 

Skeletal Malformations #1

 

Number of

fetuses with malformations

Group 1

Control

Group 2

100 mg test item/kg

Group 3

300 mg test item/kg

Group 4

1000 mg test item/kg

 

 fetal incidence N

 %

0

0.0

0

0.0

0

0.0

0

0.0

 

 litter incidence N

 %

0

0.0

0

0.0

0

0.0

0

0.0

 

#1:

Skeletal examination according to DAWSON

 

Skeletal Variations #1

Number of

fetuses with variations

Group 1

Control

Group 2

100 mg test item/kg

Group 3

300 mg test item/kg

Group 4

1000 mg test item/kg

 fetal incidence N

 %

3

2.0

16

10.7 **

4

2.8

8

5.6

 litter incidence N

 %

2

10.0

9

45.0 *

3

15.0

5

25.0

#:1

Skeletal examination according to DAWSON

*/**

Statistically significant at p < 0.05 / 0.01 (Fisher test)

 

Skeletal Retardations #1

 

Number of

fetuses with retardations

Group 1

Control

Group 2

100 mg test item/kg

Group 3

300 mg test item/kg

Group 4

1000 mg test item/kg

 

 fetal incidence N

 %

126

85.7

133

88.7

130

91.5

126

88.1

 

 litter incidence N

 %

20

100.0

20

100.0

20

100.0

20

100.0

 

#1:

Skeletal examination according to DAWSON

 

Conclusions:
Under the conditions of the study, the test substance did not show any significant maternal toxicity and no teratogenic potential. Thus, the maternal and the developmental NOAEL were set to 1000 mg/kg bw/d, both.
Executive summary:

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.

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 premature death was noted for any test group.

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

No influence on the body weight, body weight gain or the net body weight gain was noted for the treatment groups.

No test item-related 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 deaths of fetuses and no test item-related malformations, variations or retardations were noted.

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

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the outcome of an OEC 414 developmental toxicity study, showing no developmental effects when dosed up to 1000 mg/kg bw/d to rats, a classification for reproductive toxicity according to CLP (Regulation EC 1272/2008) is not required.