Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 June 2008
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
Aug 1998
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3,7-dimethyloct-6-enenitrile
EC Number:
257-288-8
EC Name:
3,7-dimethyloct-6-enenitrile
Cas Number:
51566-62-2
Molecular formula:
C10H17N
IUPAC Name:
3,7-dimethyloct-6-enenitrile
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch number of test material: BASF SE, 19000329U0
- Purity: 95.6 area-%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability of the test material during storage: Expiry date: 01 May 2021
The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Homogeneity of the test material: given

OTHER
- Chemical name: 3,7-dimethyloct-6-enenitrile
- Physical state/appearance: Liquid/colorless, clear

Test animals

Species:
rabbit
Strain:
New Zealand White
Remarks:
Crl:KBL(NZW)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services,
Germany GmbH/ Charles River Laboratories, France
- Housing: Single housing
- Fasting period before study: Not applicable
- Diet (e.g. ad libitum): ad libitum; pelleted Kliba maintenance diet rabbit and guinea pig “GLP” (Granovit AG, Kaiseraugst/Switzerland)
- Water (e.g. ad libitum): Drinking water ad libitum
- Acclimation period: At least 5 d before artificial insemination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-21
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light (06:00 h - 18:00 h); 12 hours darkness (18:00 h - 06:00 h)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% CMC suspension in deionized water (with 10 mg/ 100 mL Cremophor EL)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The specific amount of test substance was weighed, topped up with 0.5% CMC suspension in deionized water (with 10 mg/ 100 mL Cremophor EL) in an Erlenmeyer flask and intensely mixed with a magnetic stirrer. Before and during administration, the preparations was kept homogeneous with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0, 0.25, 0.8, 2.50 g/100 mL
- Amount of vehicle (if gavage): 10 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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 a maximum of 7 days at room temperature had been verified prior to the start of the study with the same batch.
Samples of the test substance preparations were sent once (at the beginning of administration) to the analytical laboratory for verification of the concentrations. The samples taken for the concentration control analyses were also used to verify the homogeneity of the samples of the low- and high concentrations each (25 and 250 mg/kg bw/d). Three samples (one from the top, middle and bottom) were taken for each of these preparations from the preparation vessel with a magnetic stirrer running.
Details on mating procedure:
- Impregnation procedure: Artificial insemination (GD 0)
- 0.2 mL of a synthetic hormone (Receptal, Intervet Deutschland GmbH, Unterschleißheim, Germany), which releases LH and FSH from the anterior pituitary lobe, was injected intramuscularly into the female rabbits about 1 h before insemination
- The samples of the ejaculate used for the artificial insemination were derived from male New Zealand White rabbits of the same strain as the females. The male donors were maintained under the same conditions (air conditions, feed and water) as the females used in the study. The day of insemination was referred to as GD 0.
Duration of treatment / exposure:
GD 6-28
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
80 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In a three-week test study (BASF project No.: 01R0333/03R030), Citronellylnitril was administered to each three non-pregnant New Zealand White rabbits per test group at dose levels of 1000, 600, 300 and 0 (control) mg/kg bw/d. The highest test group was terminated at study day 6 due to severe effects on food consumption and body weights.

In a following maternal toxicity range-finding study (BASF project No.: 20R0333/03R031), Citronellylnitril was administered daily to each five pregnant New Zealand White rabbits per test group from implantation to one day prior to the expected day of parturition (GD 6-28). Dose levels of 300 and 600 mg/kg bw/d were used. At the high-dose level of 600 mg/kg bw/d, the following effects were observed: reduced feces in three out of five animals, no feces in four out of five animals, a pronounced reduction in mean food consumption at GD 6 onwards, a body weight loss, a decreased body weight change. One animal of this test group aborted and was sacrificed on GD 21, the other four animals of this test group were sacrificed premature. At the lower dose of 300 mg/kg bw/d, mean food consumption values were slightly reduced on GD 9-11. One out of five animals showed reduced feces, no feces was noted in two out of five animals. One animal of this test group aborted and was sacrificed on GD 21.

Therefore, based on the above-mentioned range-fining study, the following doses were chosen for the present prenatal developmental toxicity study in New Zealand White rabbits: 0, 25, 80 and 250 mg/kg bw/day.

- Rationale for animal assignment: During the acclimatization period the animals were assigned to the different test groups according to a randomization plan (NIJENHUIS and WILF) and on the basis of their body weights.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- At least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity
- A check for moribund and dead animals twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays (GD 0 to 29)
- During administration period (GD 6-28): all animals were checked daily for abnormal clinical signs before administration as well as within 5 hours after the administration. Abnormalities and changes are documented for each animal

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29

FOOD CONSUMPTION: Yes
- Time schedule for examination: daily during GD 0-29

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: Gross-pathological examination
The following weights were determined in all does sacrificed on schedule: adrenal glands, kidneys, liver, spleen
The following organs or tissues were fixed in 4% neutral buffered formaldehyde solution: all gross lesions, adrenal glands, kidneys, liver, spleen

CLINICAL PATHOLOGY
In the morning blood was taken from the ear vein from not-fasted animals without anesthesia. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence.

Hematology
Parameters examined:
- Leukocyte count (WBC)
- Erythrocyte count (RBC)
- Hemoglobin (HGB)
- Hematocrit (HCT)
- Mean corpuscular volume (MCV)
- Mean corpuscular hemoglobin (MCH)
- Mean corpuscular hemoglobin concentration (MCHC)
- Platelet count (PLT)
- Differential blood count
- Reticulocytes (RETA)

Clinical chemistry
Parameters examined:
- Alanine aminotransferase (ALT)
- Aspartate aminotransferase (AST)
- Alkaline phosphatase (ALP)
- gamma-Glutamyltransferase (GGT)
- Inorganic phosphate (INP)
- Calcium (CA)
- Urea (UREA)
- Creatinine (CREA)
- Glucose (GLUC)
- Total bilirubin (TBIL)
- Total protein (TPROT)
- Albumin (ALB)
- Globulins (GLOB)
- Triglycerides (TRIG)
- Cholesterol (CHOL)
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Other: Site of implantations in the uterus; Gross-pathological examination
Blood sampling:
Additional samples during venipuncture were collected for external research projects beyond the scope of this study and without GLP status. The results of the study were not influenced by this procedure because blood sampling occurred just prior to sacrifice/at necropsy. The last aliquot was taken for these projects, thus blood sampling for the regulatory investigations was not affected. Thus, the sampling procedures did not affect the outcome and compliance of the GLP study. The data from these research projects do not affect the outcome, assessment and compliance of this GLP study.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter (and the heads of any fetus which revealed severe findings during the external examination, e.g. anophthalmia, microphthalmia or hydrocephalus)
Statistics:
Means and standard deviations were calculated. In addition, the following statistical analyses were carried out:

- DUNNETT’s test: Food consumption, body weight, body weight change, corrected body weight gain, carcass weight, weight of the unopened uterus, weight of the placentas and fetuses, corpora lutea, implantations, pre- and postimplantation losses, resorptions and live fetuses
- FISHER's exact test: Number of pregnant animals at the end of the study, mortality rate (of the does) and number of litters with fetal findings
- WILCOXON test: Proportion of fetuses with findings per litter
- KRUSKAL-WALLIS and WILCOXON-test (two-sided) test: Blood parameters
- KRUSKAL-WALLIS H test (two-sided) and WILCOXON-test (two-sided): Weight parameters

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Only pregnant does were used for the calculations of mean maternal food consumption, body weight and body weight change. Only pregnant does with scheduled sacrifice (GD 29) were used for the calculation of mean gravid uterine weights, mean organ weights, corrected (net) body weight gain and summary of reproduction data.
The following females were excluded from the above-mentioned calculations:
Test group 0 (0 mg/kg bw/d): females Nos. 10, 15 - not pregnant
Test group 1 (25 mg/kg bw/d): female No. 50 - died after gavage error
Test group 2 (80 mg/kg bw/d): females Nos. 59, 64 - not pregnant
Test group 3 (250 mg/kg bw/d): females Nos. 86, 92 - not pregnant

One female, each, of the control and the high-dose groups had blood in bedding before and after treatment: No. 3 (0 mg/kg bw/d) on GD 19 and No. 91 (250 mg/kg bw/d) on GD 25.
Furthermore, low-dose doe No. 50 (25 mg/kg bw/d) showed, pre-terminally, labored respiration after treatment on GD 18 which was caused by a gavage error on this very day.
No defecation was observed in one low-dose, one mid-dose and three high-dose females. Reduced defecation was observed in one control, four low-dose, three mid-dose and three high-dose females (0, 25, 80 and 250 mg/kg bw/d). Incidence and distribution of these findings do not indicate a relationship to the test substance.
There were no further clinical findings in the other does in this study.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One low-dose female (No. 50 - 25 mg/kg bw/d) died after gavage error on GD 19.
There were no further substance-related or spontaneous mortalities in any of the does in the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No statistically significant differences were observed for the mean body weights and the average body weight gain of the low-, mid- and high-dose females (25, 80 or 250 mg/kg bw/d) when compared to the control group.
Nevertheless, corresponding to food consumption, the high-dose rabbits gained 35% less weight than the controls during the treatment period (GD 6-28) and also during the study period (GD 0-29, -24%), the latter difference attained statistical significance.

Mean carcass weights and the corrected body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) were not significantly different between all test groups including controls (0, 25, 80 or 250 mg/kg bw/d).
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
The average food consumption of the low-, mid- and high-dose rabbits (25, 80 and 250 mg/kg bw/d) was not significantly different to the concurrent control (0 mg/kg bw/d) throughout the entire study period.
However, the high-dose does consumed less food than the control does on each day of the treatment period, although the difference never gained statistical significance. The overall food consumption of the high-dose group was about 9% below control during the treatment period on GD 6 through 28.
A single food consumption value in the mid-dose group on GD 26-27 which was significantly below control was considered as a spurious finding.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At gestation day 29, red blood cell (RBC) counts, hemoglobin and hematocrit values were decreased
(hematocrit not statistically significantly) in does of test group 3 (250 mg/kg bw/d), whereas absolute reticulocyte counts and mean corpuscular volume (MCV) were significantly increased. These alterations were regarded as treatment-related and adverse.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At gestation day 29, in does of test group 3 (250 mg/kg bw/d) γ-glutamyl transferase (GGT) activities were significantly increased whereas creatinine values were significantly decreased. These alterations were regarded as treatment-related and adverse.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute weights
All 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:
Relative weights Females
Test group 1 (25 mg/kg bw/d): 97%
Test group 2 (80 mg/kg bw/d): 97%
Test group 3 (250 mg/kg bw/d): 107%*
*: p <= 0.05
All other mean relative weight parameters did not show significant differences when compared to the control group 0. The significantly increased relative liver weights in test group 3 animals represent a marginal change, since only the relative weights were significantly increased, the weight increase was
very mild and the absolute and relative liver weights lay within the range of historical control values. In combination with increased γ-glutamyl transferase (GGT) activities in this test group, a relation to the treatment cannot be completely ruled out. However, because the weight increase was very mild, it was regarded as not adverse, if treatment related at all.
Gross pathological findings:
no effects observed
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.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between the different test groups in the values calculated for pre- and post-implantation losses.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between the different test groups in the number of resorptions.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Female rabbits were placed into the study in four cohorts. Each dose group was represented in each cohort. The conception rate was 92% in the control, mid- and high-dose groups (0, 80 and 250 mg/kg bw/d) and 100% in the low-dose group (25 mg/kg bw/d). A sufficient number (approximately 20, but not fewer than 16 females with implantation sites) of pregnant females was available for the purpose of the study.
There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Uterus weights
The mean gravid uterus weight of the rabbits in test group 3 (250 mg/kg bw/d) was significantly reduced (-21% in comparison to the concurrent control). This decrease was coherent with a lower number of corpora lutea and implantation sites and a, consequently, lower number of viable fetuses in this test group (see section 4.2.2.2. Reproduction data). As the potential origin of these findings lies before the start of treatment, an association to the test substance is not assumed.
The mean gravid uterus weight of the rabbits of test groups 1 and 2 (25 or 80 mg/kg bw/d) was not influenced by the test substance. The differences between these groups and the control group showed no dose-dependency and were assessed to be without biological relevance.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
80 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
other: mild anemia

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
In test group 3 (250 mg/kg bw/d), the mean number of corpora lutea (8.9* (p ≤ 0.05) vs. 10.6 in control) and as a result of this, mean number of implantation sites (7.8 vs. 9.5 in control) were lower and below their historical control ranges (HCD corp.lut.: mean 10.2 (9.3 - 11.1); HCD impl. sites: mean 9.6 (8.7 - 10.9)). Subsequently, the number of live fetuses (both sexes combined) was also significantly lower (7.0* (p ≤ 0.05) vs. 9.1 in control; HCD viable fetuses
(litter size): mean 9.0 (7.9 - 10.2)). Furthermore, there seems to be a shift towards a lower percentage of female live fetuses (mean% 32.4** (p ≤ 0.01) vs. 50.8 in control) in this group. However, as ovulation, fertilization and formation of corpora lutea take place 6 days before the start of treatment, a relationship of these apparent findings to the test substance cannot be assumed.


All other differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

One dead fetus was found at C-section of mid-dose doe No. 62 which is a rare finding but may occur spontaneously in this rabbit strain. As there was no evidence for potential developmental toxicity of the test item in this study, this is considered to be an incidental finding.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in test groups 1-3 (25, 80 and 250 mg/kg bw/d) was comparable to the control fetuses. Any observable differences were without biological relevance.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean placental weights in test groups 1, 2 and 3 were not influenced by the test substance and were comparable to the control value.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
External malformations were recorded for one fetus, each, in test groups 0, 2 and 3 (0, 80 and 250 mg/kg bw/d). In high-dose fetus No. 89-07, the multiple external malformations (short snout, absent nostrils, malpositioned eyes, flattened back of head) were associated with additional visceral and skeletal malformations. However, these findings were isolated events in a single fetus, thus, they are considered to be incidental. No statistically significant differences of overall incidences were noted between the groups.

Individual fetal external malformations:
0 (0 mg/kg bw/d), 23-14 F: umbilical hernia
1 (25 mg/kg bw/d): none
2 (80 mg/kg bw/d), 57-02 F: umbilical hernia
3 (250 mg/kg bw/d) 89-07 M a): multiple external malformations
a) fetus with additional visceral and skeletal alformations
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations were detected in single fetuses of test groups 1, 2 and 3 (25, 80 and 250 mg/kg bw/d). Two fetuses had additional external and/or soft
tissue malformations. All findings appeared without a dose-response relationship and nearly all of them can be found in the historical control data at comparable incidences, thus, they were assessed as not treatment-related.
In summary, these events added up to a slightly increased affected fetuses per litter-incidence of skeletal malformations in test group 3 which attained statistical significance. However, in all test groups, the overall incidences of skeletal malformations were covered by the historical control range (mean%: 0.8 [0.0 - 2.4]).

Individual fetal skeletal malformations
Test group, Doe No.-Fetus No., Sex: Finding
0 (0 mg/kg bw/d): none
1 (25 mg/kg bw/d), 30-08 F: malpositioned and bipartite sternebra; 49-05 F: additional vertebral arch and corresponding rib, branched rib
2 (80 mg/kg bw/d), 54-12 M b): sternebrae severely fused (bony plate); 69-01 F: sutures fused
3 (250 mg/kg bw/d), 84-01 F: malpositioned and bipartite sternebra; 84-07 F: thoracic hemivertebra; 85-01 F: misshapen lumbar vertebra
89-07 M a)b): multiple skeletal malformations
a) fetus with additional external malformations
b) fetus with additional soft tissue malformations
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- Fetal soft tissue malformations
Soft tissue malformations occurred in all test groups including control (0, 25, 80 and 250 mg/kg bw/d). Two fetuses in different test groups had additional external and/or skeletal malformations.
The distribution of the findings about the test groups does not indicate an association to the treatment and no statistically significant differences between the groups were noted. The total incidence of soft tissue malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data.
Individual fetal soft tissue malformations
Test group, Doe No.-Fetus No., Sex: Finding
0 (0 mg/kg bw/d), 6-09 M: absent subclavian
1 (25 mg/kg bw/d), 40-03 M: cor triloculare; 49-01 F: malpositioned kidney, short ureter; 49-03 F: absent kidney, absent ureter
2 (80 mg/kg bw/d), 54-12 M b): absent subclavian
3 (250 mg/kg bw/d), 85-03 F: hydronephrosis; 89-07 M a)b): misshapen brain; 97-03 M: absent subclavian
a) fetus with additional external malformations
b) fetus with additional skeletal malformations
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
External examination of the fetuses
- Fetal external variations:
No external variations were recorded.

- Fetal external unclassified observations:
One unclassified observation (‘amniotic fluid discolored’) was recorded in one fetus of test group 2 (80 mg/kg bw/d). This finding is considered not to be related to treatment.


Skeletal examination of the fetuses
- 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 without a relation to dosing. The statistically significantly increased incidences of ‘fused sternebra (unchanged cartilage)’ in test group 1 and 3 (affected fetuses/litter, mean%: 0.0/3.1*/1.1/1.9* [p ≤ 0.05]) were not related to the dose and, furthermore, were within the historical control range (HCD: mean% 0.9 [0.0 - 3.1]). Thus, this finding is assessed as not treatment-related.
The overall incidences of skeletal variations were neither statistically significantly different from control nor dose-dependent and, therefore, not considered biologically relevant.

- Fetal skeletal unclassified cartilage observations
Some isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the vertebral column, sternum and the ribs. The overall incidences of skeletal unclassified cartilage observations in the substancetreated
groups did not differ significantly from the concurrent control group.
The incidence of ‘bipartite processus xiphoideus’ was statistically significantly increased in test group 2 (affected fetuses/litter, mean%: 2.5/3.5/9.8**/2.4 [p ≤ 0.01]). However, as the value of test group 2 was within the historical control range (mean% 4.8 [0.5 - 22.1]) and there is no dose-response, no association to the treatment is assumed.


Soft tissue examination of the fetuses
- Fetal soft tissue variations
The examinations of the soft tissues revealed malpositioned carotid branches in the test groups 0, 1 and 2. An absent lung lobe (Lobus inferior medialis) was noted in all test groups including the control (0, 25, 80 and 250 mg/kg bw/d). Other variations, such as cystic dilatation of the brain (test group 2), supernumerary branch from aortic arch (test groups 0 and 3), dilated aortic
arch and narrowed pulmonary trunk (test group 1, respectively) and dilated ureter (test groups 1 and 3) occurred in individual fetuses of the different test groups.
No statistically significant or toxicologically relevant differences between the groups were noted, and the overall incidences were within the historical control range.

- Fetal soft tissue unclassified observations
Two unclassified soft tissue observations were recorded. A blood coagulum around urinary bladder was seen in 14 control, six low-dose, five mid-dose and eight high-dose fetuses. This finding can be found in the historical control data at a comparable incidence, therefore, it was neither assessed as treatment-related nor as adverse. Furthermore, a fluid-filled pericardium was recorded in one fetus of test group 0.
Details on embryotoxic / teratogenic effects:
Assessment of all fetal external, soft tissue and skeletal observations
There were noted external (Tab. ID-003), soft tissue (Tabs. ID-007 - ID-009) and skeletal malformations in all test groups (0, 25, 80 or 250 mg/kg bw/d). The distribution of total malformations about the groups was not related to dose.
Three fetuses of the same litter in test group 1 (25 mg/kg bw/d), one fetus in test group 2 (80 mg/kg bw/d) and one fetus in test group 3 (250 mg/kg bw/d) had more than one malformation. Female low-dose fetus No. 49-01 had a malpositioned kidney and a short ureter, while female fetus 49-03 showed an absent kidney and absent ureter; furthermore, for female fetus No. 49-05 an additional vertebral arch with corresponding rib and a branched rib were
recorded. Male mid-dose fetus No. 54-12 had an absent subclavian in combination with severely fused sternebrae (bony plate) during skeletal examination. Finally, male high-dose fetus No. 89-07 was multiple-malformed across the different examination areas. The findings consisted of multiple external malformations, such as short snout, absent nostrils,
malpositioned eyes, flattened back of head, a misshapen brain (small cerebellum, no separation of cerebral hemisphere) and multiple skeletal malformations affecting the skull, cervical and lumbar vertebrae. No ontogenetic pattern is recognizable for the individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of these test groups.
The finding ‘absent subclavian’ which was seen in a multiple malformed mid-dose fetus, occurred also in one individual fetus, each, of test groups 0 and 3. Other malformations, such as umbilical hernia, cor triloculare, hydronephrosis, sutures fused, thoracic hemivertebra, misshapen lumbar vertebra, as well as malpositioned and bipartite sternebra were scattered observations in individual fetuses of all test groups including the control. They all were not dose-related and, except ‘hydronephrosis’ which occurred in one single high-dose fetus, all of
them can be found in the historical control data at comparable frequency. An association of these findings to the treatment is not assumed.

External variations did not occur in any fetus in this study. A spontaneous origin is assumed for the soft tissue variations and the range of skeletal variations which were noted in all test groups including controls. If all different types of
variations are summarized, none of the incidences showed a relation to dosing
and can be found in the historical control data at comparable frequency.

A spontaneous origin is assumed for the unclassified external, unclassified soft tissue and unclassified skeletal cartilage observations which were observed in several fetuses of all test groups (0, 25, 80 and 250 mg/kg bw/d). The
distribution and type of these findings do not suggest any relation to treatment.
Finally, fetal examinations revealed that there is no adverse effect of the compound on the respective morphological structures up to the highest dose tested (250 mg/kg bw/d).

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects up to the highest dose tested

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion