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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
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)
Version / remarks:
25 Jun 2018
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3,7-dimethyloctan-3-ol
EC Number:
201-133-9
EC Name:
3,7-dimethyloctan-3-ol
Cas Number:
78-69-3
Molecular formula:
C10H22O
IUPAC Name:
3,7-dimethyloctan-3-ol

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 (Breeder: Charles River Laboratories, France).
- Age at study initiation: 16-17 weeks
- Weight at study initiation: 3233 - 4296 g at GD0
- Fasting period before study: no
- Housing: singly in Type 4X03B700CP cages supplied by TECNIPLAST Deutschland GmbH, Hohenpeißenberg, Germany (floor space 4264 mm², internal height 450 mm). For enrichment, wooden gnawing blocks were added (Typ SAFE® gnawing block), supplied by J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: min. 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-21
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% Sodium carboxymethyl cellulose (CMC) suspension in deionized water (with 5 mg/ 100 mL Cremophor EL)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature.
For the test substance preparation, the specific amount of the test substance was weighed, topped up with 0.5% CMC suspension in deionized water (with 5 mg/100 mL Cremophor EL) in an Erlenmeyer flask and intensely mixed with a magnetic stirrer. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0.5, 1.5 and 5 g/100 ml in the low, mid and high dose group, respectively
- Amount of vehicle (if gavage): 10 ml/kg bw/d
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 5 mg/100 mL Cremophor EL) over a period of a maximum of 7 days at room temperature was demonstrated prior to the start of the study in a similar batch.

Samples were taken for concentration control analyses and to verify the homogeneity of the samples of the low- and high-concentrations each. 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. The homogeneous distribution of the test substance in the vehicle was demonstrated.

The results of the analyses of the test substance preparations confirmed the correctness of the prepared concentrations. The measured concentrations of the retain samples of treatment groups corresponded to the expected values within the limits of the analytical method, i.e. were above 90% and below 110% of the nominal concentrations.
Details on mating procedure:
After an acclimatization period , the does were fertilized by means of artificial insemination. A synthetic hormone (0.2 mL), which stimulates release of LH and FSH from the anterior pituitary lobe (Receptal®) was injected intramuscularly to the female rabbits about 1 hour before insemination. The ejaculate samples used for the artificial insemination were obtained from male New Zealand White rabbits of the same breed as the females. Each female was inseminated with the sperm of a defined male donor. The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study.
The day of insemination was designated as GD 0 (beginning of the study) and the following day as GD 1.
Duration of treatment / exposure:
GD 6-28
Frequency of treatment:
Once daily
Duration of test:
GD0 (day of insemination) - GD29 (animal sacrifice)
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 (females)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In a previous test study (BASF project no.: 01R0267/10R201), Tetrahydrolinalool was tested orally by gavage at dose levels of 1000, 300 and 0 mg/kg bw/d in each three non-pregnant female New Zealand White rabbits. At 1000 mg/kg bw/d, all three animals showed reduced or no defecation and the mean food consumption was statistically significantly reduced during study days 0-6 (down to -98% below control). The body weight was decreased down to -15% compared to the control group from study day 4 onwards and the body weight change was also decreased (-219g versus -7g in controls on day 0-2). For animal welfare reasons, this dose group was terminated prematurely. The necropsy revealed an abnormal consistency of large intestine content. At 300 mg/kg bw/d, body weight change was decreased on study days 4-6 (-29g versus 24g in controls). No clinical signs were observed.

Based on the above-mentioned findings, dose levels of 600, 450 and 300 mg/kg bw/d were used in the following maternal toxicity study in pregnant rabbits (BASF project no.: 20R0267/10R211). At 600 mg/kg bw/d, food consumption was reduced during GD 6-28 (-21% compared to control), correspondingly, body weight change was decreased during GD 0-29 with a maximum on GD 6-9 (-166g versus 24g in controls). At 450 mg/kg, clinical findings (reduced/ no feces) were observed and the body weight change was reduced on GD 6-9 (-43g versus 48 g on controls). At 300 mg/kg bw/d, two females showed reduced defecation. Although the body weight was almost comparable to controls, the body weight change on GD 6-9 was statistically significantly reduced (-43g versus 48g in controls).
Thus, the doses 50, 150 and 500 mg/kg bw/d were chosen for the main test.

Examinations

Maternal examinations:
CHECK FOR MORTALITY: Yes
- twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-29).

CAGE SIDE OBSERVATIONS: Yes
- at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity

CLINICAL SYMPTOMS: Yes
- during the administration period (GD 6-28) daily for any abnormal clinical signs before the administration as well as within 5 hours after the administration.


BODY WEIGHT: Yes
- on GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29. The body weight change of the animals was calculated based on the obtained results.
- corrected (net) body weight gain was calculated after terminal sacrifice (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6).

FOOD CONSUMPTION: Yes
- daily during GD 0-29.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- does were necropsied and were assessed by gross pathology.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes

On GD 29, the surviving does were euthanized in randomized order by intravenous injection of pentobarbital and later, fetuses were removed from the uterus. All prematurely dead or sacrificed females were examined following the same procedures as for females sacrificed on schedule with the exception that no uterine weights were determined.

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)
Fetal examinations:
Examinations after dissection from the uterus
At necropsy each fetus was weighed and examined macroscopically for external findings. Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. Individual placental weights were recorded.
Thereafter, the fetuses were sacrificed by an intraperitoneal injection of pentobarbital.

Soft tissue examination
The abdomen and thorax were opened in order to examine the organs in situ before they were removed. The heart and the kidneys were sectioned in order to evaluate the internal structure.
The sex of the fetuses was determined by examination of the gonads in situ.
The heads of approximately one half of the fetuses per doe (and the heads of any fetus which revealed severe findings during the external examination, e.g. anophthalmia, microphthalmia or hydrocephalus) were severed from the trunk. These heads were fixed in BOUIN’s solution and were, after fixation, processed and evaluated according to WILSON’s method (WILSON and WARKANY). About 10 transverse sections were prepared per head.
All fetuses (including those without heads) were skinned and fixed in ethyl alcohol. After fixation for approx. 1-5 days, the intact fetuses were removed from the fixative and a transversal incision was made into the frontal/parietal head bones. The two halves of the calvarium were cautiously bent outward and the brain was thoroughly examined. Subsequently, these fetuses were placed back into the fixative for further fixation.

Skeletal examination
After fixation in ethyl alcohol, the skeletons (with and without skulls) were stained according to a modified method of KIMMEL and TRAMMELL. The stained skeletons were placed on an illuminated plate, investigated and archived 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 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 survival or health.
- Variation: A change that also occurs in the fetuses of control animals and/or is unlikely to adversely affect survival or health. This includes delays in growth or morphogenesis that have otherwise followed a normal pattern of development.
- Unclassified observation: fetal findings, which could not be classified as malformations or variations.


Statistics:
- DUNNETT-test (two-sided): Food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight.
- FISHER'S EXACT test (one-sided): Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings.
- WILCOXON-test (one-sided): Proportions of fetuses with malformations, variations and/or unclassified observations in each litter
Indices:
Conception rate (in %) = number of pregnant animals / number of fertilized animals x 100

Preimplantation loss (in %) = (number of corpora lutea – number of implantations) / number of corpora lutea x 100

Postimplantation loss (in %) = (number of implantations – number of live fetuses) / number of implantations x 100


Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental/ non-treatment related findings:
- Blood in bedding in 2, 1 (only after treatment), 2, 2 females of the control, low, mid and high dose group, respectively.
- Reduced defecation in 9, 3, 4, 10 females of the control, low, mid and high dose group, respectively.
- No defecation in 2, 0, 3, 4 females of the control, low, mid and high dose group, respectively.

Incidence and distribution do not indicate a relationship to the test substance. There were no further clinical findings in the other does in this study.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Test substance-related adverse effects
- High dose: 1 dam found dead after abortion + 2 dams were sacrificed after abortion

Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights
Test substance-related adverse effects
- High dose: Reduced on GD 11-21 (BW)

Body weight gains
Test substance-related adverse effects
- High dose: Reduced on GD 6-11; -31% vs ctrl. during treatment (GD 6-28). Dams lost weight on GD 6-9 (-77.1 g vs. 47.9 g in control) and GD 9-11 (-3.4 g vs. 29.9 g).

The mean body weights and the average body weight gain of the low- and mid-dose groups were generally comparable to the concurrent control group throughout the entire study period.

Corrected (net) body weight gain
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.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test substance-related adverse effects

- High dose: Reduced during GD 6-13 and GD 14-18 (down to -47% vs. ctrl.), 22% less food consumed during treatment (GD 6-28) than control.

The food consumption of the low- and mid-dose rabbits was comparable to the concurrent control throughout the entire study period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental/ non-treatment related findings:
- Mean gravid uterus weight: 0%, -10%, -16%* vs. ctrl in low, mid and high dose group, respectively.
*p<0.05
The mean gravid uterus weight of high dose rabbits was statistically significantly reduced. This decrease was coherent with a slightly lower number of corpora lutea and implantation sites and, consequently, a lower number of viable fetuses in this test group. As the origin of these findings lies before the start of the treatment, an association to the test substance can be excluded.
The mean gravid uterus weight of the rabbits of the low and mid dose groups were not statistically significantly influenced by treatment. The differences between these groups and the control group were assessed to be without biological relevance.


The mean placental weights (4.9g, 5.2g, 5.2g, 5.2 g in ctrl., low, mid and high dose groups, respectively) were not influenced by the test substance and were comparable to the control value.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Findings associated with unscheduled maternal death or sacrifice:
High dose:
- Watery feces in 1 doe (died after abortion on GD 28).
- Stomach mucosa detached from stomach wall and dry, hard feces in rectum in 1 doe (sacrificed after abortion on GD 27).
- Stomach filled with very dry, hard food and watery feces in 1 doe (sacrificed after abortion on GD 27).

Spontaneous findings

Mid dose:
- Multiple erosions and ulcerations, as well as very dry, hard food in stomach and watery feces in 1 doe
- Watery feces in 1 doe

Controls:
- Malpositioned kidney combined with a short ureter 1 doe
- Watery feces and dry, hard feces in rectum in 1 doe
- Watery feces and a single ulceration in stomach in 1 doe
- Watery feces in 1 doe
Neuropathological findings:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
- Number of females aborted: 0, 0, 0, 3 in the control, low, mid and high dose groups, respectively.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
- Preimplantation loss: 5.7%, 10.8%, 10.5%, 11.8% in the control, low, mid and high dose groups, respectively.
- Postimplantation loss: 5.9%, 6.3%, 7.4%, 5.0% in the control, low, mid and high dose groups, respectively.

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
Description (incidence and severity):
Dams with all resorptions: 0, 0, 0, 0 in the control, low, mid and high dose groups, respectively.
Early or late resorptions:
no effects observed
Description (incidence and severity):
- Early resorptions 0.5, 0.6, 0.5, 0.2 in the control, low, mid and high dose groups, respectively.
- Late resorptions: 0.2, 0.0, 0.2, 0.1 in the control, low, mid and high dose groups, respectively.

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
Description (incidence and severity):
No dead fetuses were oberved in any treated and control group.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Dams with premature births: 0, 0, 0, 0 in the control, low, mid and high dose groups, respectively.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
- Number of females pregnant: 24, 22, 24, 25 in the control, low, mid and high dose groups, respectively.
- Conception rate: 96%, 88%, 96%, 100% in the control, low, mid and high dose groups, respectively.
- Pregnant at terminal sacrifice: 96%, 88%, 96%, 88% in the control, low, mid and high dose groups, respectively.

A sufficient number of pregnant females was available for the purpose of the study (according to the test guidelines).

There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate.
Other effects:
not examined
Details on maternal toxic effects:
- Corpora lutea: 11.4, 10.2, 9.8, 9.0 in the control, low, mid and high dose groups, respectively.
- Implantation sites: 9.9, 9.1, 8.8, 8.0 in the control, low, mid and high dose groups, respectively.


The mean number of corpora lutea and as a result of this, mean number of implantation sites were lower and below their historical control ranges (HCD corpora lutea: mean 10.3 [9.5-11.5]; HCD implantation sites: mean 9.6 [8.9-10.7]). As ovulation, fertilization and formation of corpora lutea take place about 6 days before start of the treatment, a relationship of these apparent findings to the test substance can be excluded. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.



Effect levels (maternal animals)

Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
- Fetal weights (on a litter basis): 36.7g, 39.8g, 37.7g, 36.8g in the control, low, mid and high dose groups, respectively.

The mean fetal weights of low, mid and high dose groups 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):
- Nr. of live fetuses/dam: 9.3, 8.5, 8.1, 7.6 in the control, low, mid and high dose groups, respectively.

The number of live fetuses (both sexes combined) was lower in the high dose group (HCD viable fetuses [litter size]: mean 9.1 [8.4-10.1]). In line, the mean numbers of corpora lutea and the related mean numbers of implantation sites were lower (below their historical control ranges) in this dose group. As ovulation, fertilization and formation of corpora lutea take place about 6 days before start of the treatment, a relationship of these apparent findings to the test substance can be excluded.
Furthermore, no dead fetuses were oberved in any treated and control group.



Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
The sex distribution of the fetuses in low, mid and high groups 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):
see above
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Total fetal external malformations
- Fetal incidence: 0.5%, 0.5%, 0.5%, 0% in the control, low, mid and high dose groups, respectively.
- Litter incidence: 4.2%, 4.5%, 4.2%, 0% in the control, low, mid and high dose groups, respectively.
- Affected fetus/litter: 0.6%, 0.6%, 0.6%, 0% in the control, low, mid and high dose groups, respectively.
No statistically significant differences of overall incidences were noted between the groups.

Individual fetal external malformations:
- mid dose: 1 fetus with malrotated paw
- low dose: 1 fetus with short nose, domed head
- control: 1 fetus with cleft palate, short nose, malpositioned and closed nares

These findings were isolated events in single fetuses, thus, they are considered to be incidental.

Total fetal external variations
- Fetal incidence: 0%, 0.5%, 0%, 0% in the control, low, mid and high dose groups, respectively.
- Litter incidence: 0%, 4.5%, 0%, 0% in the control, low, mid and high dose groups, respectively.
- Affected fetus/litter: 0%, 0.5%, 0%, 0% in the control, low, mid and high dose groups, respectively.

One external variation (paw hyperflexion) was recorded in one low-dose fetus. This is a common finding which can be found in the historical control data at comparable incidences and is considered to be incidental.

No external unclassified observations were recorded.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Total fetal skeletal malformations
- Fetal incidence: 0.5%, 0%, 0.5%, 0.6% in the control, low, mid and high dose groups, respectively.
- Litter incidence: 4.2%, 0%, 4.2%, 4.5% in the control, low, mid and high dose groups, respectively.
- Affected fetus/litter: 0.5%, 0%, 0.4%, 0.5% in the control, low, mid and high dose groups, respectively.

The overall incidences were well within the historical control range of the test facility.

Individual fetal external malformations:
- high dose: 1 fetus with absent interparietal
- mid dose: 1 fetus with severely malformed vertebral column and/or ribs
- control: 1 fetus with sternebrae severely fused (bony plate), branched rib

No statistically significant differences between the test substance-treated groups and the control were noted.

Total fetal skeletal variations
- Fetal incidence: 91%, 92%, 92%, 96% in the control, low, mid and high dose groups, respectively.
- Litter incidence: 100%, 100%, 100%, 100% in the control, low, mid and high dose groups, respectively.
- Affected fetus/litter: 92.4%, 92.6%, 89.8%, 95.1% in the control, low, mid and high dose groups, respectively.

The overall incidences of skeletal variations appeared without a relation to dosing and were comparable to the historical control data.

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.
The incidence of ‘supernumerary thoracic vertebra’ (21.7/19.4/20.1/34.4* [p≤0.05] mean% affected fetuses per litter in ctrl, low, mid and high dose respectively) was statistically significantly increased in the high dose group and outside the historical control range (HCD: mean% 20.3 [11.6-33.5]).

Total fetal skeletal unclassified observations
- Fetal incidence: 7.2%, 16%, 11%, 5.4% in the control, low, mid and high dose groups, respectively.
- Litter incidence: 46%, 50%, 50%, 27% in the control, low, mid and high dose groups, respectively.
- Affected fetus/litter: 7.0%, 16.5%, 10.3%, 4.8% in the control, low, mid and high dose groups, respectively.

The overall incidences of unclassified cartilage observations appeared without a relation to dosing and were comparable to the historical control data.

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 sternum and the ribs and did not show any relation to dosing.
The incidence of ‘cartilaginous part of ribs not connected with sternum’ was statistically significantly increased in the low-dose group (mean% 7.8*/3.7/3.1 affected fetuses per litter in low, mid and high dose, respectively, vs. 2.0 % in the control [p≤0.05]). However, since this was not related to dose, it is assessed as incidental.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Total fetal soft tissue malformations
- Fetal incidence: 0.5%, 0.5%, 0%, 0% in the control, low, mid and high dose groups, respectively.
- Litter incidence: 4.2%, 4.5%, 0%, 0% in the control, low, mid and high dose groups, respectively.
- Affected fetus/litter: 0.6%, 0.6%, 0%, 0% in the control, low, mid and high dose groups, respectively.
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 external malformations:
- low dose: 1 fetus with hydrocephaly
- control: 1 fetus with misshapen naso-pharyngeal tract, hydrocephaly

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.

Total fetal soft tissue variations
- Fetal incidence: 3.2%, 3.2%, 3.1%, 3.6% in the control, low, mid and high dose groups, respectively.
- Litter incidence: 17%, 18%, 21%, 23% in the control, low, mid and high dose groups, respectively.
- Affected fetus/litter: 3.0%, 3.1%, 4.2%, 3.5% in the control, low, mid and high dose groups, respectively.
The overall incidences were within the historical control range.

The examinations of the soft tissues revealed malpositioned carotid branches and an absent lung lobe (Lobus inferior medialis) in all test groups including the control. Other variations, such as dilated cerebral ventricle, cystic dilatation of the brain, supernumerary artery, dilated aorta, dilated aortic arch and narrowed aorta, occurred in individual fetuses of the different test groups. No statistically significant or toxicologically relevant differences between the groups were noted.

Total fetal soft tissue unclassified observations
- Fetal incidence: 0%, 1.1%, 4.1%, 0% in the control, low, mid and high dose groups, respectively.
- Litter incidence: 0%, 4.5%, 21%*, 0% in the control, low, mid and high dose groups, respectively.
- Affected fetus/litter: 0%, 1%, 5%**, 0% in the control, low, mid and high dose groups, respectively.
*p ≤ 0.05 (Fisher’s exact test [one-sided])
**p ≤ 0.01 (Wilcoxon-test [one-sided])

Two unclassified soft tissue observations were recorded. Discolored thymus was seen in three mid-dose fetuses; furthermore, a blood coagulum around urinary bladder was recorded in two low-dose and five mid-dose fetuses. Both findings can be found in the historical control data at comparable incidences, therefore, they are assessed neither as treatment-related nor as adverse.
In summary, these events added up to a slightly increased affected fetuses per litter-incidence of soft tissue unclassified observations in the mid dose group, which attained statistical significance. However, as there was no dose response-relationship and the overall incidences were well within the historical control range (HCD total: affected fetuses/litter mean% 3.0 [0.0-7.8]), this finding is not considered as treatment-related.
Details on embryotoxic / teratogenic effects:
Total fetal malformations
- Fetal incidence: 0.9%, 0.5%, 1.0%, 0.6% in the control, low, mid and high dose groups, respectively.
- Litter incidence: 8.3%, 4.5%, 8.3%, 4.5% in the control, low, mid and high dose groups, respectively.
- Affected fetus/litter: 1.1%, 0.6%, 1.0%, 0.5% in the control, low, mid and high dose groups, respectively.

There were noted external, soft tissue and skeletal malformations in all test groups including the control. The distribution of total malformations about the groups was not related to dose.
Four fetuses had more than one malformation:
- Mid-dose fetus with a severely malformed vertebral column and/or ribs (i.e. thoracic center misshapen and fused, small vertebral arch, rib partly absent or displaced).
- Low-dose fetus with a short nose and a domed head which was confirmed during visceral examination (hydrocephaly).
- Control fetus with multiple external malformations, such as cleft palate, short nose, malpositioned and closed nares, combined with a misshapen naso-pharyngeal tract and a hydrocephaly.
- Control fetus with severely fused sternebrae (bony plate) and a branched rib.
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.
Further malformations, such as malrotated paw (1 mid dose fetus) and absent interparietal (1 high dose fetus) were scattered observations in single fetuses which occurred without a relation to the dose, thus, a relation to the treatment is not assumed.

Total fetal variations
- Fetal incidence: 91%, 92%, 92%, 97% in the control, low, mid and high dose groups, respectively.
- Litter incidence: 100%, 100%, 100%, 100% in the control, low, mid and high dose groups, respectively.
- Affected fetus/litter: 92.4%, 92.6%, 89.8%, 95.7%* in the control, low, mid and high dose groups, respectively.
*p ≤ 0.05 (Wilcoxon-test [one-sided])
One external variation, several soft tissue variations and a range of skeletal variations were noted in all test groups including controls.
The incidence of ‘supernumerary thoracic vertebra’ was statistically significantly increased in the high dose group and outside the historical control range. This finding might be associated with the treatment, but since this is the only increased skeletal variant in this group and only slightly exceeds the historical background incidence, it is evaluated as of no toxicological relevance.
If all different types of variations are summarized, the total rate of affected fetuses per litter was statistically significantly increased in the high-dose group. Nevertheless, the overall incidences of all classified fetal variations were well within the historical control range (HCD: affected fetuses/litter mean% 91.96 [86.02-97.16) and is therefore assessed as not treatment-related.

Total fetal unclassified observations
External unclassified observations did not occur in any fetus in this study. A spontaneous origin is assumed for the unclassified soft tissue (and unclassified skeletal cartilage observations, which were observed in several fetuses of control and treatment groups. 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 (500 mg/kg bw/d).

Effect levels (fetuses)

Dose descriptor:
NOAEL
Remarks:
fetal developmental toxicity
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: i.e. highest dose tested

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion