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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:
2021-2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3-[[3-[[(2-cyanoethyl)amino]methyl]-3,5,5-trimethylcyclohexyl]amino]propiononitrile
EC Number:
300-496-1
EC Name:
3-[[3-[[(2-cyanoethyl)amino]methyl]-3,5,5-trimethylcyclohexyl]amino]propiononitrile
Cas Number:
93940-97-7
Molecular formula:
C16H28N4
IUPAC Name:
3-[(3-{[(2-cyanoethyl)amino]methyl}-3,5,5-trimethylcyclohexyl)amino]propanenitrile
Test material form:
liquid
Specific details on test material used for the study:
Identity: 3-[[3-[[(2-cyanoethyl)amino]methyl]-3,5,5-trimethylcyclohexyl]amino]propiononitrile (13/0241-5)
Label name: Baxxodur(TM) PC 136
Alternative name: MIPDA
CAS number: 93940-97-7 (Batch no. 18315014)
Retest date: 21 July 2022
Appearance: clear colourless liquid (purity 97.0 area%)
Storage conditions: room temperature

Test animals

Species:
rat
Strain:
Wistar

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% CMC suspension in deionized water (with 10 mg/100 mL Cremophor EL)
Analytical verification of doses or concentrations:
yes
Remarks:
The stability of the test substance in 0.5% CMC suspension in deionized water (with 10 mg/100 mL Cremophor EL) over a period of 7 days at room temperature had been verified prior to the start of the study in a similar batch.
Details on analytical verification of doses or concentrations:
Analyses of the test substance preparations The analytical investigations of the test substance preparations were carried out as a separate study. The study was carried out in compliance with the Principles of Good Laboratory Practice. The stability of the test substance in 0.5% CMC suspension in deionized water (with 10 mg/100 mL Cremophor EL) over a period of 7 days at room temperature had been verified prior to the start of the study in a similar batch (Project No.: 01Y0241/13L005; Final report Study No. 18L00074).
Samples of the test substance preparations were sent thrice to the analytical laboratory for verification of the concentrations: once at the beginning of the administration period (sample Nos. 3-9) and, additionally due to technical issues (incomplete transfer from vial into flask), twice after the end of the administration period (reserve sample Nos. 6R-9R (sampling at the beginning of the administration period) and sample Nos. 12-18 (sampling towards the end of
the administration period)). The samples were also used to verify the omogeneity of the lowand high-concentrations (12 and 120 mg/kg bw/d). Three samples (one from the top, middle and bottom in each case) were taken from the beaker with a magnetic stirrer running. All test samples, plus a duplicate set of reserve samples, were withdrawn by staff of the Reproduction Toxicology. All reserve samples and further samples were stored at the Laboratory Reproduction Toxicology frozen (at -20 °C). Analysis of these samples were performed in case of equivocal analytical results with the original samples or after loss of/damage to original samples after agreement by the Study Director. Following finalization of the report, all analytical samples, including reserve samples, will be discarded.
Details on mating procedure:
The animals were paired by the breeder (“time-mated”); the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory. The following day was designated as “GD 1”. The animals were acclimated to the laboratory conditions between start of the study (beginning of the experimental phase) and first administration (GD 6).
Duration of treatment / exposure:
The test substance preparations were administered to the animals once a day orally by gavage, from implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always at approximately the same time in the morning. The animals of the control group were treated with the vehicle (0.5% CMC suspension in deionized water [with 10 mg/100 mL Cremophor EL]) in the same way. The volume administered each day was 10 mL/kg body weight. The
calculation of the administration volume was based on the most recent individual body weight.
Frequency of treatment:
once daily
Duration of test:
On GD 20, blood samples were obtained in a randomized order from all females by retrobulbar venous puncture. After blood sampling all surviving dams were sacrificed and examined. The fetuses were removed from the uterus and investigated.
Doses / concentrationsopen allclose all
Dose / conc.:
12 mg/kg bw/day (nominal)
Remarks:
low-dose level
Dose / conc.:
40 mg/kg bw/day (nominal)
Remarks:
mid-dose level
Dose / conc.:
120 mg/kg bw/day (nominal)
Remarks:
high-dose level
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
In a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats (OECD Guideline 422; Project No. 85R0241/13R184) signs of systemic toxicity at the highest dose of 150 mg/kg bw/d, such as a reduction in food consumption and a decrease in body weight (change), adverse changes in clinical pathology parameters (decreased eosinophil and increased monocyte cell counts) and histopathological findings (i.e. necrosis in liver and skeletal muscle cells; vacuolation in liver, kidney, spleen, lymph nodes) were seen.
Based on this results the following dose levels were chosen for the present prenatal developmental toxicity study in Wistar rats:
- 12 mg/kg body weight/day: as low-dose level
- 40 mg/kg body weight/day: as mid-dose level
- 120 mg/kg body weight/day: as high-dose level
The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.

Examinations

Maternal examinations:
Mortality
A check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20).

Clinical symptoms
A cage-side examination was conducted at least once daily before and after treatment period (GD 0-5 and 20). During treatment period (GD 6-19) all rats were checked daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity before administration
as well as within 2 hours and between 2 and 5 hours after administration.

Water consumption
Water consumption was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

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

Body weight data
All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. The body weight change of the animals was calculated based on the obtained results.

Corrected (net) body weight gain
Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).

Pathology
Necropsy
On GD20 all surviving animals were sacrificed by decapitation under isoflurane anesthesia in a randomized sequence. After the animals have been sacrificed, organs were assessed by gross pathology.

Organ weights
The following weights were determined in all dams sacrificed on schedule:
1. Thyroid glands (with parathyroid glands) (fixed)
2. Liver
3. Kidneys
All paired organs were weighed together (left and right). The carcass weights (GROSSE-System) were transferred to the ProLIMS-System to calculate the relative organ weights.

Organ/tissue fixation
The following organs or tissues were fixed in 4% neutral buffered formaldehyde solution or in modified Davidson’s solution from all animals, sacrificed on schedule:
1. All gross lesions
2. Adrenal glands
3. Brain
4. Eyes with optic nerve (modified Davidson’s solution)
5. Kidneys
6. Liver
7. Lungs
8. Lymph nodes (axillary and mesenteric)
9. Skeletal muscle
10. Spleen
11. Stomach (forestomach and glandular stomach)
12. Thyroid glands (with parathyroid glands)

Histopathology
Fixation of tissues from all animals was followed by histotechnical processing, examination by light microscopy and assessment of findings:
1. Thyroid glands
2. Liver
3. Kidneys
Special stains of organs of individual animals were prepared at the discretion of the study pathologist for the assessment of histopathological findings. In detail, an Oil-Red-O (ORO)-
stain for the detection of neutral lipids and a Sudan black stain for the detection of neutral lipids and phospholipids were performed exemplarily on the liver of animals nos. 7, 80, 81 and 82,
and on the kidneys of animals nos. 6, 93 and 98. Additionally, on the liver of animals nos. 80 and 82, a Periodic-Acid-Schiff (PAS)-stain for the detection of glycogen was done. Thyroid glands were processed according to the “Revised guides for organ sampling and trimming in rats and mice – Part 2” (Kittel et al., 2004). A correlation between gross lesions and histopathological findings was attempted. After completion of the histopathological assessment by the study pathologist, an internal peer review was also performed including the liver and kidneys of all animals of the control group and all treatment groups. Results presented in this report reflect the consensus opinion of the study pathologist and the peer review pathologist.
Ovaries and uterine content:
Cesarean section
On GD 20, the dams were sacrificed under isoflurane anesthesia by decapitation, in randomized order. After the dams had been sacrificed, they were necropsied and assessed for gross pathology. The uteri and the ovaries were removed and the following data were recorded:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
• Live fetuses
• Dead implantations:
a) Early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single horn pregnancy)
b) Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c) Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)
After the weight of the uterus had been determined, all subsequent evaluations of the dams and the gestational parameters (except of gross pathology including organ sampling and weights) were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose animal numbers were encoded.
Blood sampling:
Clinical pathology
Blood samples were taken from all females by puncturing the retrobulbar venous plexus following isoflurane anesthesia. Blood sampling and blood and serum examinations were carried out in a randomized sequence. The list of randomization instructions was compiled with a computer. The following parameters were measured in all pregnant females: The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in International System (SI) units.

Hematology
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter.

Clinical chemistry
An automatic analyzer was used to examine the clinicochemical parameters.

Thyroid hormones
The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter. T3 and T4 Elisa was measured with a Sunrise MTP-reader and evaluated with the Magellan-Software of the instrument producer.
Fetal examinations:
EXAMINATIONS OF THE FETUSES
All fetal analyses were conducted by technicians unaware of the treatment group, in order to minimize bias.

Examinations of the fetuses after dissection from the uterus
At necropsy each fetus was weighed, sexed, and external tissues and all orifices were examined macroscopically. The sex was determined by observing the distance between the anus and the base of the genitalia. Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded. Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital (Narcoren®; dose: 0.1 mL/fetus). The anogenital distance (defined as the distance from the center of the anal opening to the base of the genital tubercle) measurements were conducted, using a measuring ocular, on all liveborn fetuses. After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and fixed in ethanol; the other half was placed in Harrison’s fluid for fixation.

Soft tissue examination of the fetuses
The fetuses fixed in Harrison’s fluid were examined for any visceral findings according to the method of BARROW and TAYLOR. After this examination these fetuses were discarded.

Skeletal examination of the fetuses
The skeletons of the fetuses fixed in ethanol were stained according to a modified method of KIMMEL and TRAMMELL. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After this examination the stained fetal skeletons were retained individually.

Evaluation criteria for assessing the fetuses
Classification and assessment of fetal findings is a matter of ongoing discussion (see e.g. BELTRAME and GIAVINI, CHAHOUD, SOLECKI). Despite considerable efforts to harmonize the nomenclature used to describe observations of fetal morphology, the terms still vary considerably between laboratories, investigators and textbooks in the fields of teratology and developmental toxicity. In the present study the internationally harmonized glossary of WISE et al. and the updated version of MAKRIS et al. was essentially used to describe findings in fetal morphology. Classification of these findings was based on the terms and definitions proposed by CHAHOUD and SOLECKI:

Malformation
A permanent structural change that is likely to adversely affect the survival or health.

Variation
A change that also occurs in the fetuses of control animals and/or is unlikely to adversely affect the survival or health. This includes delays in growth or morphogenesis that have otherwise followed a normal pattern of development. The term "unclassified observation" was used for those fetal findings, which could not be classified as malformations or variations. All fetal findings were listed in tables according to these classifications.

Anogenital index
The anogenital index was calculated accordingly.
Statistics:
Statistics of clinical pathology
Means, medians, standard deviations and deviation vs control of each test group were calculated for several parameters. In summary tables of part IB, mean values were rounded, but deviations of means versus control means were calculated with not rounded values. Therefore, slight differences may occur when changes were re-calculated with rounded means. In these tables “deviation vs control” means x-fold of controls expressed as percentages minus 100%.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
Only pregnant dams were used for the calculations of mean maternal food consumption, body weight and body weight change. Only pregnant dams with scheduled sacrifice (GD 20) were used for the calculation of mean gravid uterine weights, mean organ weights, corrected (net) body weight gain and summary of reproduction data. No females were excluded from the above-mentioned calculations.

No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 12, 40 or 120 mg/kg bw/d during the entire study period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any females of all test groups (0, 12, 40 or 120 mg/kg bw/d).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights of all substance-treated dams (12, 40 and 120 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period. The body weight change of the dams in test groups 2 and 3 was statistically significantly reduced during GD 6-8 (test group 2: 27%; test group 3: 53% below controls) but recovered afterwards, respectively. This effect was assessed as treatment related and adverse in the high dose group. In the mid dose group the slight temporarily decrease was regarded as possibly treatment related, but non adverse If calculated for the entire treatment period (GD 6-19), the mid- and high-dose dams gained comparable weight than the controls. The average body weight gain of the low-dose dams (12 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.

The corrected body weight gain (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6) was statistically significantly decreased in test group 3 (120 mg/kg bw/d - about 13% below control). This is assessed as treatment related and adverse. The corrected body weight gain of test groups 1 and 2 (12 and 40 mg/kg bw/d) revealed no difference of any biological relevance to the corresponding control group. Moreover, mean carcass weights of all test groups remained unaffected by the treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In test group 3 (120 mg/kg bw/d), the mean food consumption was statistically significantly reduced during GD 6-8 (about 8% below control) but recovered afterwards and was comparable to the control values from GD 8 onwards. If calculated for the entire treatment period (GD 6-19), the high-dose dams consumed a comparable amount of food than the controls. The mean food consumption of the dams in test groups 1 and 2 (12 and 40 mg/kg bw/d) was generally comparable to the concurrent control group throughout the entire study period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
The mean water consumption of the dams in test groups 2 and 3 (40 and 120 mg/kg bw/d) was statistically significantly increased on GD 15-17 (test group 2: +14%; test group 3: +13%). Nevertheless, except for this occasion, the mean water consumption values in these groups were comparable to the concurrent control throughout the entire treatment period and there was no dose-response relationship visible. If calculated for GD 6-19, all substance-treated dams (test groups 1-3) consumed a comparable amount of water than the controls.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At gestation day 20, in dams of test groups 2 and 3 (40 and 120 mg/kg bw/d) absolute reticulocyte counts were significantly decreased, but the values were minimally above the historical control range (dams, absolute reticulocytes 115.3-181.0 Giga/L). Therefore and as no other red blood cell parameters were altered, this finding was considered incidental and not treatment-related.
Additionally, in dams of test group 3 (120 mg/kg bw/d) absolute and relative eosinophil counts were significantly decreased. The values were at the low border (relative eosinophils) or below the historical control range (absolute eosinophils; dams, absolute eosinophils 0.03-0.09 Giga/L; relative eosinophil 0.6-1.7 %). This alteration in combination with a transient reduced food consumption and decreased body weight change may be an indication of subacute stress. In this context, the decreased absolute and relative eosinophil counts in dams of test group 3 may be regarded as treatment related and adverse.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At gestation day 20, in dams of test group 3 (120 mg/kg bw/d) alkaline phosphatase (ALP) activities were significantly decreased. The values were at the low border of the historical control range (dams, ALP 0.84-1.37 μkat/L). However, this was the only changed clinical chemistry parameter. Therefore, it was regarded as maybe treatment related, but non adverse (ECETOC Technical Report No. 85, 2002).
Endocrine findings:
no effects observed
Description (incidence and severity):
Thyroid hormones
In dams of test groups 1, 2 and 3 (12, 40 and 120 mg/kg bw/d), no treatment-related alterations of T3, T4 and TSH levels were observed.
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute weights
When compared to control group 0 (=100%), the mean absolute weights of thyroid glands were significantly decreased in test group 3. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights
When compared to control group 0 (=100%), the mean relative weights of the liver were significantly increased in test group 3 All other mean relative weight parameters did not show significant differences when compared to the control group 0. Although, the relative liver weights in test group 3 were only minimally increased, the increase was assumed to be treatment related. The significantly decreased thyroid gland weights in test group 3 were regarded as incidental, as there was no dose response-relationship.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in kidneys and livers of animals of test group 3.
Most animals of test group 3 showed a macro- and microvesicular vacuolation of the kidneys. Both types of vacuoles occurred predominantly in cortical tubular epithelial cells. Macrovesicular vacuoles appeared mostly in collecting ducts, whereas the microvesicular vacuoles seemed to affect more the convoluted proximal and distal tubules. However, a clear distinction was not always possible.
Microvesicles appeared as many small cytoplasmic and optically empty vacuoles, whereas macrovesicles were characterized by one large cytoplasmic and optically empty vacuole, displacing the nucleus and occasionally distorting the cell. Both types of vacuoles were negative in the ORO-stain. Microvesicles revealed a questionable result with the Sudan black stain, macrovesicles were clearly negative for Sudan black. The macro- and microvesicular change of cortical tubular epithelial cells in test group 3 was regarded as treatment related.

The liver of test group 3 animals showed a minimal up to moderate centrilobular, cytoplasmic, microvesicular vacuolation of hepatocytes. The vacuoles were distributed throughout the cytoplasm and appeared optically empty in the H&E-stain and were negative in the ORO-stain. The Sudan black stain revealed a questionable result. The microvesicular change of hepatocytes in test group 3 was regarded as treatment related and correlated to significantly increased liver weights. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
The conception rate was 100% in all test groups (0, 12, 40 and 120 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of this study. There were no test substance-related and/or biologically relevant differences between the test groups 0-3 in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The conception rate was 100% in all test groups (0, 12, 40 and 120 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of this study. There were no test substance-related and/or biologically relevant differences between the test groups 0-3 in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The conception rate was 100% in all test groups (0, 12, 40 and 120 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of this study. There were no test substance-related and/or biologically relevant differences between the test groups 0-3 in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The conception rate was 100% in all test groups (0, 12, 40 and 120 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of this study. There were no test substance-related and/or biologically relevant differences between the test groups 0-3 in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.
Dead fetuses:
no effects observed
Description (incidence and severity):
The conception rate was 100% in all test groups (0, 12, 40 and 120 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of this study. There were no test substance-related and/or biologically relevant differences between the test groups 0-3 in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The conception rate was 100% in all test groups (0, 12, 40 and 120 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of this study. There were no test substance-related and/or biologically relevant differences between the test groups 0-3 in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Uterus weight
The mean gravid uterus weights of the animals of test groups 1-3 (12, 40 and 120 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
LOAEL
Effect level:
120 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
haematology
Key result
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other:
Description (incidence and severity):
Dams
- Reduction in food consumption (GD 6-8: about 8% below control) but recovered
afterwards
- Decrease in body weight change (GD 6-8: about 53% below control) and corrected body weight gain (about -13%)
- Decreased absolute and relative eosinophil cell counts

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Weight of the placentae
The mean placental weights of the low-, mid- and high-dose groups (12, 40 and 120 mg/kg bw/d) were comparable to the corresponding control group.

Weight of the fetuses
The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group. The statistically significantly increased mean fetal weight of all viable fetuses and especially of male fetuses, in test group 1 is considered to be biologically irrelevant due to the lack of dose response relationship.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in test groups 1-3 (12, 40 and 120 mg/kg bw/d) was comparable to the control fetuses.
Changes in litter size and weights:
no effects observed
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
The anogenital distance and anogenital index of all male and female fetuses in the test groups 1-3 were comparable to the concurrent control values.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
Fetal external malformations
External malformations were recorded in one fetus, each, of all test groups including the controls. All malformations were associated with visceral or skeletal malformations. The distribution of the findings about the test groups does not indicate an association to the treatment. The overall incidences of external malformations were comparable to those found in the historical control data.

Fetal external variations
No external variations were recorded.

Fetal external unclassified observations
No external unclassified observations were recorded.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Skeletal examination of the fetuses
Fetal skeletal malformations
Skeletal malformations were detected in three fetuses of the control and one fetus, each, of the mid- and high-dose groups. Female control fetus No. 12-04 had multiple skeletal malformations affecting the whole fetal body compared with an additional external malformation. Two further fetuses had associated external malformations. All findings are assessed as not treatment-related since they occurred in single fetuses without a relation to dose. The total incidences of skeletal malformations did not differ significantly from control and were comparable to the historical control data.

Fetal skeletal variations
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared in the majority of cases without a relation to dose. The overall incidences of skeletal variations were comparable to the historical control data.
All skeletal variations with statistically significant differences between the control and any treated group were compiled in the table below. All incidences were expressed on a fetus per litter basis and any statistically significant differences, which were outside the historical control range were marked in italicized bold types.

The finding ‘incomplete ossification of parietal (unchanged cartilage)’ was statistically significantly increased and outside the historical control range in test groups 2 and 3 (40 and 120 mg/kg bw/d).
The findings ‘incomplete ossification of interparietal (unchanged cartilage)’, ‘incomplete ossification of skull (unchanged cartilage)’, ‘basioccipital hole(s)’ and ‘wavy rib’ were statistically significantly increased in test group 3 (120 mg/kg bw/d). All values were outside the historical control range. These skeletal variations were assessed as treatment-related.

Fetal skeletal unclassified cartilage observations
Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the vertebral column, ribs, sternum and forelimbs and did not show any relation to dosing.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Fetal soft tissue malformations
One fetus in test group 1 (40 mg/kg bw/d) had a visceral malformation, which was associated with an external malformation. Since this was a single event,
and the finding can be found in the historical control data at a comparable incidence, it is assessed as not treatment-related. The total incidence of soft tissue malformations in treated animals did not differ significantly from the concurrent control group and was covered by the historical control data.

Fetal soft tissue variations
Three soft tissue variations were detected, i.e. short innominate in test groups 0 and 3, dilated renal pelvis in all test groups and dilated ureter in test groups 0-2. The incidence of ‘dilated renal pelvis’ (0.8/6.5*/1.6/2.4 mean% affected fetuses/litter) was statistically significantly increased in test group 1. Since this low-dose value was well within the historical control range (HCD: mean% 4.2 [0.9-8.7]) and there is no dose-response relationship visible, it is assessed as incidental. The total incidences of these variations were neither statistically significantly nor dose dependently increased in the treated groups. All of them can be found in the historical control data at comparable incidences. Therefore, they are assessed as not treatment-related.

Fetal soft tissue unclassified observations
No soft tissue unclassified observations were recorded.
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Assessment of all fetal external, soft tissue and skeletal observations
External, soft tissue and skeletal malformations were noted in three control, one low-dose, one mid-dose and one high-dose fetuses (0, 12, 40 and 120 mg/kg bw/d).
Four fetuses carried more than one malformation: female control fetus No. 12-04 had anasarca and multiple skeletal malformations concerning the whole fetal body, such as misshapen basisphenoid, a big hole in basioccipital, absent lumbar vertebra, sternebrae malpositioned and bipartite, shortened femur, shortened and bent tibia and shortened fibula. Female lowdose fetus No. 36-09 (12 mg/kg bw/d) showed an umbilical hernia and situs inversus, while female mid-dose fetus No. 58-05 (40 mg/kg bw/d) had a curled tail and a small thoracic arch. Furthermore, for female high-dose fetus No. 85-11 (120 mg/kg bw/d) malpositioned digits (confirmed as supernumerary phalanges), a generalized disturbance of ossification (concerning skull, sternum, cervical and sacral vertebrae, pelvic girdle and forelimbs) and a misshapen cervical vertebra were recorded. Further malformations, i.e. split basisphenoid and cleft sternum, were observed in individual fetuses of the control group. All these findings were single cases without relation to treatment, no ontogenetic pattern is recognizable for all these individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of these test groups. They also do neither form a pattern or syndrome with other minor anomalies which may raise toxicological concern nor do they influence the overall rate of malformations in this study. There is no evidence for any association of these scattered findings with the treatment.

External variations did not occur in any of the fetuses in this study. Three soft tissue variations and a range of skeletal variations were noted in all test groups including the controls. None of the total incidences showed a relation to dosing. The majority of individual variations are equally distributed about the different test groups, if normal biological variation is taken into account, and can be found in the historical control data at a comparable frequency. The finding ‘incomplete ossification of parietal (unchanged cartilage)’ was statistically significantly increased and outside the historical control range in test groups 2 and 3 (40 and 120 mg/kg bw/d). The findings ‘incomplete ossification of interparietal unchanged cartilage)’, ‘incomplete ossification of skull (unchanged cartilage)’, ‘basioccipital hole(s)’ and ‘wavy rib’ were statistically significantly increased in test group 3 (120 mg/kg bw/d). All values were outside the historical control range. These findings indicate a delay in ossification. They are known to be reversible. Particularly, no changes in the underlying cartilages and no abnormalities in the respective bone structures were observed. No unusual pattern of ossification was otherwise observable in the treated fetuses and the observed skeletal variations did not influence the overall rate of fetal variations.
Ossification of skull bones and ribs occurs at the end of gestation and the fetal ossification status is highly influenced by many factors, such as difference in mating time, time of cesarean section or by stress due to maternal toxicity (Carney & Kimmel, 2007). Overall, the above-mentioned findings were assessed as minor effects indicating a developmental delay and were, therefore, assessed as treatment-related.
Unclassified external and unclassified soft tissue observations did not occur in any of the fetuses in this study. A spontaneous origin is assumed for the unclassified skeletal cartilage observations which were observed in several fetuses of all test groups (0, 12, 40 and 120 mg/kg bw/d). The distribution and type of these findings do not suggest any relation to treatment.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
120 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
no

Any other information on results incl. tables

Pathology (* : p <= 0.05, **: p <= 0.01)





















Absolute weightsFemales
Test group (mg/kg)1
(12)
2
(40)
3
(120)
Thyroid glands+2.6%+6.4%-9.6%*




















Relative weightsFemales
Test group (mg/kg)1
(12)
2
(40)
3
(120)
Liver+2.3%+1.5%+6.8%**

 


Histopathology


















































































Kidneys
 Female animals
Test group (mg/kg)0
(0)
1
(12)
2
(40)
3
(120)
No. of animals25252525
Vacuolation, macrovesicular00013
·     Grade 1   10
·     Grade 2   2
·     Grade 3   1
Vacuolation, microvesicular00019
·     Grade 1   10
·     Grade 2   7
·     Grade 3   2





















































Liver
 Female animals
Test group (mg/kg)0
(0)
1
(12)
2
(40)
3
(120)
No. of animals25252525
Vacuolation, microvesicular00015
·     Grade 1   12
·     Grade 2   2
·     Grade 3   1

 


Individual fetal external malformations

































Test groupDam No.-Fetus No., SexFinding
0 (0 mg/kg bw/d)12-04 F a)anasarca
1 (12 mg/kg bw/d)36-09 F b)umbilical hernia
2 (40 mg/kg bw/d)58-05 F a)curled tail
3 (120 mg/kg bw/d)85-11 F a)malpositioned digit
mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female
a)  fetus with additional skeletal malformation
b)  fetus with additional visceral malformation

 


Total external malformations
















































  Test group 0
0 mg/kg bw/d
Test group 1
12 mg/kg bw/d
Test group 2
40 mg/kg bw/d
Test group 3
120 mg/kg bw/d
Litter
Fetuses
N
N
25
255
25
263
25
264
25
271
Fetal incidenceN (%)1 (0.4)1 (0.4)1 (0.4)1 (0.4)
Litter incidenceN (%)1 (4.0)1 (4.0)1 (4.0)1 (4.0)
Affected fetuses/litterMean%0.50.30.40.3
per kilogram body weight per day; N = number; % = per cent

 


Individual fetal soft tissue malformations

































Test groupDam No.-Fetus No., SexFinding
0 (0 mg/kg bw/d)none 
1 (12 mg/kg bw/d)36-09 F a)situs inversus
2 (40 mg/kg bw/d)none 
3 (120 mg/kg bw/d)none 
mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female
a)  fetus with additional external malformation

 


Total soft tissue malformations
















































  Test group 0
0 mg/kg bw/d
Test group 1
12 mg/kg bw/d
Test group 2
40 mg/kg bw/d
Test group 3
120 mg/kg bw/d
Litter FetusesN N25
121
25
127
25
126
25
129
Fetal incidenceN (%)0.01 (0.8)0.00.0
Litter incidenceN (%)0.01 (4.0)0.00.0
Affected
fetuses/litter
Mean%0.00.60.00.0
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 


Total soft tissue variations
















































  Test group 0
0 mg/kg bw/d
Test group 1
12 mg/kg bw/d
Test group 2
40 mg/kg bw/d
Test group 3
120 mg/kg bw/d
Litter FetusesN N25
121
25
127
25
126
25
129
Fetal incidenceN (%)3 (2.5)7 (5.5)2 (1.6)4 (3.1)
Litter incidenceN (%)2 (8.0)6 (24)1 (4.0)4 (16)
Affected
fetuses/litter
Mean%3.56.51.63.2
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 


Individual fetal skeletal malformations

































Test groupDam No.-Fetus No., SexFinding
0 (0 mg/kg bw/d)3-03 M
8-03 M
12-04 F a)
cleft sternum
split basisphenoid
multiple skeletal malformations
1 (12 mg/kg bw/d)none 
2 (40 mg/kg bw/d)58-05 F a)small thoracic arch
3 (120 mg/kg bw/d)85-11 F a)generalized disturbance of ossification,
misshapen cervical vertebra, supernumerary phalanx
mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female
a)  fetus with additional external malformation

 


Total skeletal malformations
















































  Test group 0
0 mg/kg bw/d
Test group 1
12 mg/kg bw/d
Test group 2
40 mg/kg bw/d
Test group 3
120 mg/kg bw/d
Litter FetusesN N25
134
25
136
25
138
25
142
Fetal incidenceN (%)3 (2.2)0.01 (0.7)1 (0.7)
Litter incidenceN (%)3 (12)0.01 (4.0)1 (4.0)
Affected
fetuses/litter
Mean%2.30.00.70.7
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 


Total fetal skeletal variations
















































  Test group 0
0 mg/kg bw/d
Test group 1
12 mg/kg bw/d
Test group 2
40 mg/kg bw/d
Test group 3
120 mg/kg bw/d
Litter FetusesN N25
134
25
136
25
138
25
142
Fetal incidenceN (%)130 (97)129 (95)127 (92)138 (97)
Litter incidenceN (%)25 (100)25 (100)25 (100)25 (100)
Affected
fetuses/litter
Mean%97.094.891.397.2
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 


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
























































FindingTest group 0
0 mg/kg bw/d
Test group 1
12 mg/kg bw/d
Test group 2
40 mg/kg bw/d
Test group 3
120 mg/kg bw/d
HCD
Mean % (range)
Incomplete ossification of parietal;
unchanged cartilage
9.76.620.9**25.4**8.9
(1.9 - 19.8)
Incomplete ossification of interparietal;
unchanged cartilage
29.815.032.339.0*19.4
(11.8 - 31.8)
Incomplete ossification of skull; unchanged cartilage10.77.712.820.3*8.7
(2.1 - 17.1)
Basioccipital hole(s)0.00.80.02.1*0.3
(0.0 - 1.5)
Wavy rib3.12.33.816.2**3.7
(0.0 - 13.1)
mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = per cent
* = p £ 0.05 (Wilcoxon-test [one-sided])
** = p
£ 0.01 (Wilcoxon-test [one-sided])

 


Total unclassified cartilage observations
















































  Test group 0
0 mg/kg bw/d
Test group 1
12 mg/kg bw/d
Test group 2
40 mg/kg bw/d
Test group 3
120 mg/kg bw/d
Litter FetusesN N25
134
25
136
25
138
25
142
Fetal incidenceN (%)97 (72)105 (77)93 (67)102 (72)
Litter incidenceN (%)25 (100)24 (96)24 (96)25 (100)
Affected
fetuses/litter
Mean%72.475.866.972.0
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 


Total fetal malformations
















































  Test group 0
0 mg/kg bw/d
Test group 1
12 mg/kg bw/d
Test group 2
40 mg/kg bw/d
Test group 3
120 mg/kg bw/d
Litter FetusesN N25
255
25
263
25
264
25
271
Fetal
incidence
N (%)3 (1.2)1 (0.4)1 (0.4)1 (0.4)
Litter incidenceN (%)3 (12)1 (4.0)1 (4.0)1 (4.0)
Affected fetuses/litterMean%1.20.30.40.3
mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 


Total fetal variations
















































  Test group 0
0 mg/kg bw/d
Test group 1
12 mg/kg bw/d
Test group 2
40 mg/kg bw/d
Test group 3
120 mg/kg bw/d
Litter
Fetuses
N
N
25
255
25
263
25
264
25
271
Fetal incidenceN (%)133 (52)136 (52)129 (49)142 (52)
Litter
incidence
N (%)25 (100)25 (100)25 (100)25 (100)
Affected fetuses/litterMean%53.152.348.552.5
mg/kg bw/d = milligram per kilogram body weight per day; N= number; % = per cent

Applicant's summary and conclusion

Conclusions:
Under the conditions of this prenatal developmental toxicity study, the oral administration of 3-[[3-[[(2-cyanoethyl)amino]methyl]-3,5,5-trimethylcyclohexyl]amino]propiononitrile to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused signs of maternal toxicity (reduced food consumption and decrease in body weight change, decreased corrected body weight gain, decreased eosinophil cell counts) at 120 mg/kg bw/d. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity was 40 mg/kg bw/d. Single variations – indicating a slight delay in ossification in skull bones and ribs - were, at the top dose (120 mg/kg bw/d), present at incidences outside the historical control range. Those minor changes in connection with maternal toxicity in the high dose group are assessed as a possible effect of the treatment, but not as adverse. The no observed effect level (NOEL) for prenatal developmental toxicity was therefore 40 mg/kg bw/d; the NOAEL was 120 mg/kg bw/d, the highest dose tested. The test item is not teratogenic in rats.
Executive summary:

In a prenatal developmental toxicity study, the test substance 3-[[3-[[(2-cyanoethyl)amino]methyl]-3,5,5-trimethylcyclohexyl]amino]propiononitrile was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the
expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity.
Analyses confirmed the correctness of the prepared concentrations, the homogeneous distribution and the stability of the test substance in the vehicle. Regarding clinical examinations, at the high-dose level (120 mg/kg bw/d), signs of maternal toxicity were observed. In the beginning of the treatment period (GD 6-10), high-dose dams showed a slight reduction in mean food consumption (up to 8% below control) and a decrease in body weight change (up to 53% below control). Both parameters recovered to control values towards the end of the study. Corrected body weight gain of high-dose dams was statistically significantly decreased (13% below control). Due to the variety of affected parameters, the above-mentioned findings were assessed as treatment-related and adverse. At 40 and 12 mg/kg bw/d, no treatment-related, adverse effects were observed. Concerning clinical pathology, in dams of test group 3 (120 mg/kg bw/d) decreased absolute and relative eosinophils counts in combination with transient reduced food consumption and decreased body weight change may indicate subacute stress which is regarded as treatmentrelated. Regarding pathology, a minimal up to moderate macro- and microvesicular change of cortical tubular epithelial cells in the kidneys, and a minimal up to moderate microvesicular vacuolation of hepatocytes in the liver of test group 3 was noted. In the liver, the microvesicular vacuolation correlated to significantly increased relative liver weights. These findings were regarded as treatment related. However, since no additional signs of degeneration or necrosis were noted, findings were assessed as not adverse. The morphological appearance and distribution of vacuoles, the negative Oil-Red-O stain and existing knowledge of the test substance suggest a phospholipidosis. The Sudan black stain – which should have been positive in case of phospholipidosis – only revealed a questionable result. This was assigned to a poor staining quality and did not have an impact on the assessment of results, as the presence of phospholipids had already been shown for the test substance. A phospholipidosis is characterized by a lysosomal accumulation of phospholipids (Nonoyama and Fukuda, 2008; Lenz et al., 2018). Vacuoles were negative in the Oil-Red-O (ORO)-stain. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. No differences of toxicological relevance between the control and the treated groups (12, 40 or 120 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no toxicologically relevant influence of the test substance on sex distribution and anogenital distance/index of the fetuses was noted at any dose. In the high- and mid-dose fetuses, a slight delay in ossification was indicated by significantlyincreased incidences of five variations - ‘incomplete ossification of interparietal (unchanged cartilage)’, ‘incomplete ossification of skull (unchanged cartilage)’, ‘basioccipital hole(s)’ and ‘wavy ribs’ in test group 3 and ‘incomplete ossification of parietal (unchanged cartilage)’ in test groups 3 and 2. These findings are known to be reversible. Ossification of skull bones and ribs occurs at the end of gestation and the fetal ossification status is highly influenced by many factors, such as difference in mating time, time of cesarean section or by stress due to maternal toxicity (Carney & Kimmel, 2007). The isolated finding in mid-dose fetuses was assessed as of low toxicological relevance. The above-mentioned findings were considered to be minor, reversible effects indicating a developmental delay. They were assessed as possibly treatment-related but not as adverse. Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose.