Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 April 2021 - 26 July 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 Jun 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
Aug 1998
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(Z)-4-[C11-13 (branched) alkylamino]-4-oxo-2-butenoic acid
EC Number:
701-116-0
Cas Number:
2156592-45-7
Molecular formula:
C17H31NO3
IUPAC Name:
(Z)-4-[C11-13 (branched) alkylamino]-4-oxo-2-butenoic acid
Specific details on test material used for the study:
Name of test substance: (Z)-4-[C11-13 (branched) alkylamino]-4-oxo-2-butenoic acid
CAS No.: 2156592-45-7
Test substance No.: 11/0632-5
Batch identification: EVO800002-0183
Content: 89.8 g/100 g +/- 0.3 g/100 g (Final Report, Study Code: 20L00194)
Identity: Confirmed
Homogeneity: Given
Storage stability: Expiry date: 25 Aug 2024
The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility. The test facility is organizationally independent from the BASF SE sponsor division.
Physical state/appearance: Viscous yellowish liquid, clear
Storage conditions: Room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-12 weeks
- Weight at study initiation: time-mated animals of comparable size and weight (171.5 - 226 g)
- Housing: individually in Polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm²)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: The animals were paired by the breeder and supplied on GD 0 (= detection of vaginal plug/sperm). After arrival of each cohort, the assignment of the animals to the different test groups was carried out by withdrawing them from the transport boxes and placing them into the cages at random. Each cohort was evenly distributed among the dose groups. After randomization the rats were identified consecutively and uniquely by ear tattoo.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: feed
Details on exposure:
The test substance preparations were prepared once and stored deep frozen immediately. The food was changed at least twice weekly in the morning of the working day after the food in the bag had reached room temperature.
For the test substance preparations, the required quantity of test substance was weighed and thoroughly mixed with a small amount of food. Then further amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations. Mixing was carried out for about 10 minutes in a laboratory mixer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical investigations of the test substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, 67056 Ludwigshafen, Germany.
The stability of the test substance in the diet over a period of 31 days deep-frozen plus 4 days at room temperature had been verified prior to the start of the study in a similar batch (Project No.: 01Y0632/11Y188).
Samples of the test substance preparations were sent once (at the beginning of treatment) to the analytical laboratory for verification of the concentrations. The samples were also used to verify the homogeneity of the low- and high-concentrations (700 and 7000 ppm).
All test samples, plus a duplicate set of reserve samples, were withdrawn by staff of the Central Food Mixing Laboratory. 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. Reserve samples were described by the suffix “R” in the report. Following finalization of the report, all analytical samples, including reserve samples, were discarded.
Details on mating procedure:
- Impregnation procedure: [purchased timed pregnant]
- Proof of pregnancy: The animals were paired by the breeder and supplied on GD 0 (= detection of vaginal plug/sperm).
Duration of treatment / exposure:
continuous via feed from GD 6 - 20.
Frequency of treatment:
continuous via feed from GD 6 - 20.
Duration of test:
continuous via feed from GD 6 - 20.
Doses / concentrationsopen allclose all
Dose / conc.:
57 mg/kg bw/day (actual dose received)
Remarks:
700 ppm
Dose / conc.:
185 mg/kg bw/day (actual dose received)
Remarks:
2300 ppm
Dose / conc.:
475 mg/kg bw/day (actual dose received)
Remarks:
7000 ppm
No. of animals per sex per dose:
25
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The high dose was selected based on signs of toxicity noted in a previously conducted test study (BASF project 10C0632/11S289) and in a maternal toxicity range-finding study (BASF project 11R0632/11R298) which preceded this definitive prenatal developmental toxicity study.
In the test study 4 male and 4 female Wistar rats per group were diet exposed to the test compound over 2 weeks, at concentrations of 0, 7500 and 15000 ppm (corresponding to 754 and 1224 mg/kg bw/d in the females). The 15000 ppm concentration caused a distinct reduction of water and food consumption (reduction by almost 50%) and body weight gain (by about 16%) in all females. In addition, a significant disturbance in a number of hematology and clinical chemistry parameters was noted: such as decreased hemoglobin, increased numbers of large unstained blood cells; increased alanine and aspartate aminotransferase, cholesterol and triglycerides; decreased creatinine and total protein. These changes corresponded to increased kidney and liver weights. At 7500 ppm a significant decrease of water and food consumption (by 30%) was noted in females during the first 10 days of treatment, accompanied by reduced body weight gain (about 7%).
In the maternal toxicity range‐finding study, 7 presumed pregnant Wistar rat females were administered the test substance via diet from gestational day (GD) 6 through GD 20, at concentrations of 0, 3500 and 10000 ppm (corresponding to doses of about 287 and 680 mg/kg bw/d).
The 10000 ppm concentration caused in the pregnant rats a significant reduction of water and food consumption (by about 30%) as well as body weight gain (by about 66%). When corrected for the weight of the pregnant uterus, the 10000 ppm females in fact lost weight, almost 20 g of their initial value. Hematology and clinical chemistry revealed some signs of hemoconcentration and metabolic disorder (increased red blood cell count; decreased proteins and triglycerides; increased aspartate aminotransferase, cholesterol and calcium) at 10000 ppm.
At 3500 ppm still a lower body weight gain was noted, when corrected for the weight of the pregnant uterus it was about 22% below control.
Organ weights and macroscopic pathology revealed no relevant findings.
The selected high concentration for the definitive study (7000 ppm) represented an approximate dose which caused a moderate reduction of food consumption and body weight gain in pregnant female rats. This procedure meets the principles of guidelines OECD 414 (adopted 2001) and OPPTS 870.3700 (US EPA), as well as ECHA practical guide 10 (“how to avoid unnecessary testing on animals”; chapter 4 “animal welfare”; ECHA-10-B-17-EN, 2010) which is in in compliance with EU Directive 2010/63/EU on animal protection.
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for (rat) dam thyroid hormones: none, feeding stuy
- Time of day for (rat) dam blood sampling: GD20

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).
The body weight of the pregnant animals on day 0 varied between 171.5 – 226.0 g.
The animals continuously received the test substance at the appropriate dose levels in the diet from implantation to one day prior to the expected day of parturition (GD 6-20).
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.

Rationale for exposure route
The information required for the present test substance was a prenatal developmental toxicity study (Annex IX, Section 8.7.2; test method: OECD TG 414) by oral route, dietary exposure.
Dietary exposure was chosen as the test substance caused severe local effects in the respiratory tract and mortality in an OECD TG 408 study (BASF Project 50C0632/11S284) due to gavage-related reflux. Dietary route was supposed to prevent these effects and was agreed upon by ECHA.

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 for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity. If such signs occurred, the animals were examined several times daily (GD 0-20).

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.

Intake of test substance
The mean daily intake of test substance (group means) was calculated based upon individual values for body weight and mean food consumption per cage.
(FCx x C) / BWx = test substance intake for interval x
BWx = Average body weight during the food consumption interval x [g]
FCx = mean daily food consumption during interval x [g]
C = concentration in the diet during interval x [mg/kg]

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).

TERMINAL EXAMINATIONS OF THE DAMS
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, special attention being given to the reproductive organs.
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.

Pathology
Necropsy
On GD20 all surviving animals were sacrificed by decapitation under isoflurane anesthesia in a randomized sequence. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

Organ weights
The following weights were determined in all dams sacrificed on schedule:
1. Adrenal glands (fixed)
2. Kidneys
3. Liver
4. Spleen
5. Thyroid glands (with parathyroid glands) (fixed)
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 from all animals, sacrificed on schedule:
1. All gross lesions
2. Adrenal glands
3. Brain
4. Kidneys
5. Larynx
6. Liver
7. Lungs
8. Nasal cavity
9. Spleen
10. Thyroid glands (with parathyroid glands)
11. Trachea

Histopathology
Fixation of thyroid glands from all dams was followed by histotechnical processing, examination by light microscopy and assessment of findings in all dams/test group (hematoxylin and eosin (H&E) stain).

The organs were trimmed according to the “Revised guides for organ sampling and trimming in rats and mice” (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004).
A correlation between gross lesions and histopathological findings was attempted.
An internal peer review was performed including the thyroid glands of dams of all test groups. Results presented in this report reflect the consensus opinion of the study pathologist and the peer review pathologist.
Ovaries and uterine content:
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 F0 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 (Advia 120 model; Bayer, Fernwald, Germany):
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
An automatic analyzer (Cobas c501; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters.
Alanine aminotransferase (ALT)
(L-alanine: 2-oxoglutarate aminotransferase; EC 2.6.1.2.)
Aspartate aminotransferase (AST)
(L-aspartate: 2-oxoglutarate aminotransferase; EC 2.6.1.1.)
Alkaline phosphatase (ALP)
(orthophosphoric acid monoester phosphohydrolase; EC 3.1.3.1.)
gamma-Glutamyltransferase (GGT)
(gamma-glutamyl) peptide: aminoacid-gamm-glutamyl-transferase; EC 2.3.2.2.)
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)

Thyroid hormones
The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). T3 and T4 Elisa was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, 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.
Statistics:
Detailed statistical analyses were performed
Indices:
The conception rate (in %) was calculated according to the following formula:
(number of pregnant animals) / (number of fertilized animals) x 100

The preimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
(number of corpora lutea – number of implantations) / (number of corpora lutea) x 100

The postimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
(number of implantations – number of live fetuses) / (number of implantations) x 100

anogenital index = (anogenital distance [mm]) / (cubic root of fetal weight [g])
Historical control data:
Detailed historical controls are available

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female of all test groups 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, 700, 2300 or 7000 ppm).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights of the high-dose dams (7000 ppm) were statistically significantly reduced from GD 8 onwards until scheduled sacrifice on GD 20 (up to a maximum of 10% below the concurrent control group on GD 20).
Consistent with lower food consumption, the body weight change of the high-dose dams was distinctly and statistically significantly reduced on several occasions during the treatment period: GD 6-13 and GD 17-20. If calculated for the entire treatment period (GD 6-20) or study
period (GD 0-20), the high-dose dams gained about 32% or 24% less weight than the controls (attaining statistical significance, respectively). This is regarded as a treatment-related adverse effect.
The mean body weights and the average body weight gain of the low- and mid-dose dams (700 and 2300 ppm) were generally comparable to the concurrent control group throughout the entire study period.

Corrected (net) body weight gain
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 (7000 ppm - about 81% below control). Furthermore, the carcass weight of the high-dose dams was reduced in comparison to the control group (about 11% below controls). These effects are assessed as test substance-related signs of maternal toxicity.
The corrected body weight gain of test group 1 (700 ppm) was also slightly impaired and about 16% below controls attaining statistical significance. However, mean carcass weights remained unaffected by the treatment. This effect appears without relation to the dose and can be evaluated as not treatment-related.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In test group 3 (7000 ppm), the mean food consumption was statistically significantly reduced during the entire treatment period (GD 6-20; up to a maximum of -32% in comparison to the concurrent control). If calculated for the entire treatment period (GD 6-20), the high-dose dams consumed about 21% less food than the controls. This finding is regarded as treatment-related and adverse.
The mean food consumption of the dams in test groups 1 and 2 (700 and 2300 ppm) was generally comparable to the concurrent control group throughout the entire study period. This includes the statistically significantly reduced food consumption value during GD 8-10 in test group 2.

The mean daily test substance intake in mg/kg bw/d over the entire administration period (GD 6-20) was calculated:
700 ppm - 57 mg/kgw bw/d
2300 ppm - 185 mg/kg bw/d
7000 ppm - 475 mg/kg bw/d
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
see food consumption
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At gestation day 20, in dams of test group 3 (7000 ppm) hemoglobin values and absolute reticulocyte counts as well as mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) were significantly decreased. Hemoglobin mean and absolute reticulocyte count values were below historical control range (dams, hemoglobin 6.5-6.9 mmol/L, absolute reticulocytes 115.7-181.0 Giga/L). These alterations were regarded as treatment-related and adverse.
In dams of test group 2 (2300 ppm) absolute reticulocyte counts were already significantly lower compared to controls, but the values were within the historical control range (dams, absolute reticulocytes 115.7-181.0 Giga/L). In dams of test groups 1 and 3 (700 and 7000 ppm) relative eosinophil counts were significantly decreased, but the alteration was not dose dependent. Therefore, the changes described in this paragraph were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At GD 20 in dams of test group 3 (7000 ppm), alkaline phosphatase (ALP) activities were significantly increased whereas total protein, albumin and globulin as well as total bilirubin, cholesterol, triglyceride and calcium values were significantly decreased. These alterations
were regarded as treatment-related and adverse.
Additionally, in dams of test group 3 alanine aminotransferase (ALT) activities and urea values were significantly increased whereas in dams of test group 2 (2300 ppm) besides significant ALT and ALP activity triglyceride and total bilirubin values were significantly lower compared
to controls. However, the values of nearly all changed parameters in this paragraph were within historical control ranges. Only ALP activities in dams of test group 2 were marginally above this range (dams, ALT 0.72-1.38 μkat/L, urea 5.07-6.52 mmol/L, ALP 0.84-1.37 μkat/L, triglycerides 3.63-7.11 mmol/L, total bilirubin 0.79-1.30 μmol/L). Therefore, these alterations were regarded as incidental and not treatment-related, apart from the ALP increase, which was maybe treatment-related, but non-adverse because it was the only relevantly changed parameter in dams of test group 2 (ECETOC Technical Report No. 85, 2002).
Endocrine findings:
effects observed, treatment-related
Description (incidence and severity):
Thyroid hormones
At GD 20 in dams of test group 3 (7000 ppm) T4 and TSH values were significantly increased whereas T3 values were significantly decreased. T3 was already significantly lower in dams of test group 2 (2300 ppm) compared to controls. T3 values in dams of test groups 2 and 3 were within the historical control range, whereas T4 and TSH values in dams of test group 3 were above their ranges (dams GD 20, T3 0.56-1.31 nmol/L, T4 23.17-33.22 nmol/L, TSH 4.92-8.54 μg/L). Regarding the slight thyroid hyperplasia, an activation of the Hypothalamus-Pituitary- Thyroid axis was considered and therefore, T4 and TSH value increases in dams of test group 3 were evaluated 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):
Uterus weight

The mean gravid uterus weights of the animals of test groups 1-3 (700, 2300 and 7000 ppm) 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.

For absolute and relative organ weights, please refer to the attached PDF (Tables_and_Figures.pdf).

The significantly decreased terminal body weight in test group 3 animals was regarded as treatment-related.

The increased absolute and relative liver weights in test groups 2 and 3 were regarded as treatment-related. All significantly changed weight parameters in both test groups lay above the mean value of historical control values, relative liver weights in test group 3 lay above the range of historical control values.
The significantly decreased spleen weights in test group 3 were regarded as a secondary, treatment-related effect, based on the significantly decreased terminal body weight. They lay below the range of historical control values.
The decreased spleen weights in test groups 1 and 2 were interpreted to be without relation to the treatment, because there was neither a clear dose-response relationship, nor correlating hematological findings, and the weight parameters lay within the range of historical control data.
The significantly decreased absolute kidney weights in test group 3, the significantly increased relative weights of adrenal glands in test groups 2 and 3 and the significantly increased relative kidney weights in test group 3 were regarded as secondary due to the decreased terminal body weight.
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:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in thyroid glands of females of test group 3, please refer to the attached PDF (Tables_and_Figures.pdf).

Some test group 3 animals (4 of 25) showed a minimal diffuse follicular hypertrophy and hyperplasia of thyroid glands (Grade 1), which was regarded as treatment-related.
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:
no effects observed
Description (incidence and severity):
Reproduction data
The conception rate was 96% in test group 2 (2300 ppm) and 100% in test groups 0, 1 and 3 (0, 700 and 7000 ppm). 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.
A 100% post-implantation loss was noted in one low-dose dam (No. 50 - 700 ppm), where two early resorptions were recorded after staining the apparently empty uterus at C-section (i.e. pregnant by stain). This isolated finding is regarded as incidental and not treatment-related.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
185 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
haematology
organ weights and organ / body weight ratios

Maternal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
thyroid gland
Description (incidence and severity):
Minimal diffuse follicular hypertrophy and hyperplasia of thyroid glands in 4/25 animals

Results (fetuses)

Fetal body weight changes:
not examined
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 (700, 2300 and 7000 ppm) was comparable to the control fetuses.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
No effetcs on litter size were observed.

The mean fetal body weights in test group 3 (7000 ppm) were significantly reduced (about 8% below the concurrent control value, if both sexes were combined). The change is assessed to be a treatment-related secondary effect and a consequence of the significant reduced food consumption and effects on body weight (gain) of the dams.
The mean fetal weights of test groups 1 and 2 (700 and 2300 ppm) did not show any biologically relevant differences in comparison to the control group.
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.
The statistically significantly reduced anogenital distance in male fetuses of test group 2 (2.8 mm vs 3.0 mm in control) is assessed as spontaneous in nature due to the lack of dose response relationship and the fact that all values lay well within the historical control range (mean 2.9 mm [2.3 - 3.4]).
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
External malformations were recorded in one control, two mid-dose fetuses and one high-dose fetus. One fetus, each, of the control and the mid-dose group had multiple external malformations associated with (multiple) skeletal malformations.
Furthermore, one high-dose fetus had an associated visceral malformation. Since there was no relation to dose, these findings are assessed as not treatment-related. The overall incidences of external malformations were comparable to those found in the historical control data.

For details, please refer to the attached PDF (Tables_and_Figures.pdf).
Skeletal malformations:
no effects observed
Description (incidence and severity):
Skeletal malformations were detected in one fetus, each, of test groups 0 and 2, in two fetuses of test group 1 and in three fetuses of test group 3. Male mid-dose fetus No. 52-08 (2300 ppm) had multiple skeletal malformations affecting the skull, the vertebral column and the sternum compared with additional multiple external malformations. All findings are assessed as not treatment-related since they occurred in single fetuses without a relation to dose. Most of them (except ‘split scapula’ and ‘fused rib’) can be found in the historical control data at comparable incidences. The total incidences of skeletal malformations did not differ significantly from
control and were comparable to the historical control data.
Visceral malformations:
no effects observed
Description (incidence and severity):
Fetal soft tissue malformations
Soft tissue malformations were detected in single fetuses of test groups 0, 1 and 3. One high-dose fetus had an associated external malformation. These findings were single events in single fetuses and can be found in the historical control data at comparable incidences. Thus, they are assessed as not treatmentrelated.
The total incidence of soft tissue malformations in treated animals did not differ significantly from the concurrent control group and was also covered by the historical control data.

For details, please refer to the attached PDF (Tables_and_Figures.pdf).
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Weight of the placentae
The mean placental weights in test groups 1-3 (700, 2300 and 7000 ppm) were comparable to the corresponding control group.

Fetal external variations
No external variations were recorded.

Fetal external unclassified observations
No external unclassified observations were recorded.

Fetal soft tissue variations
Three soft tissue variations were detected, i.e. short innominate in one control fetus, dilated renal pelvis in several fetuses of all test groups and dilated ureter in fetuses of test groups 0 and 3. The incidences of these variations were neither statistically significantly nor dosedependently 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.
For details, please refer to the attached PDF (Tables_and_Figures.pdf).

Fetal soft tissue unclassified observations
No soft tissue unclassified observations were recorded.

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 affected fetuses/litter incidence of skeletal variations was statistically significantly increased in test groups 2 and 3, with only the high-dose incidence outside the historical control range (HCD: mean% 96.4 [90.8 - 98.5]).
All skeletal variations with statistically significant differences between the control and any treated group were compiled in the table (For details, please refer to the attached PDF (Tables_and_Figures.pdf)). 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 findings in test group 3 were related to thoracic and sacral vertebrae, ribs and sternum.
The statistically significantly increased mean value of ‘misshapen sacral vertebra’ was inside the historical control range. Therefore, this particular finding is assessed as incidental and not
treatment-related.
For all other findings (for details, please refer to the attached PDF (Tables_and_Figures.pdf)), the statistically significantly increased mean values in test group 3 were outside the respective historical control range. These findings are considered to be treatment-related. The finding ‘supernumerary rib (14th) with cartilage not present’ was significantly increased also in test group 2. 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 (which is high anyway), it is evaluated as of no toxicological relevance.
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 ((for details, please refer to the attached PDF (Tables_and_Figures.pdf)). The observed unclassified cartilage findings were related to the skull, the vertebral column, the forelimbs, the ribs and the sternum and did not show, in the majority of cases, any relation to dosing.
However, the incidence of ‘fused cervical arch cartilage’ was statistically significantly increased in test group 3 (2.0 / 0.0 / 0.0 / 19.4**). This finding is not present in the historical control data and is assessed as treatment-related.
Details on embryotoxic / teratogenic effects:
Assessment of all fetal external, soft tissue and skeletal observations

External, soft tissue and skeletal malformations were noted in two control, three low-dose, two mid-dose and five high-dose fetuses (0, 700, 2300 and 7000 ppm).
Three fetuses carried more than one malformation: female control fetus No. 16-03 had multiple external and one skeletal malformations, such as anasarca, malrotated hindlimbs and anal atresia, combined with a cleft sternum. For mid-dose male fetus No. 52-08 (2300 ppm) multiple external and multiple skeletal malformations were recorded, such as exencephaly, microphthalmia, cleft palate, severely malformed skull bones, severely malformed vertebral column and ribs, severely malformed sternum and a fused cervical arch. Furthermore, the findings in female high-dose fetus No. 78-09 (7000 ppm) consisted of a meningoencephalocele, cleft palate and anophthalmia. Further malformations, i.e. misshapen liver, hydronephrosis, misshapen basisphenoid, misshapen thoracic vertebra, split scapula, malpositioned and bipartite sternebra and fused rib, were observed in individual fetuses, unrelated to dose and nearly all of them can be found in the historical control data.
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.
For details, please refer to the attached PDF (Tables_and_Figures.pdf).

External variations did not occur in any of the fetuses in this study. A spontaneous origin is assumed for three soft tissue variations which were observed in fetuses of all test groups including the control.

A broad range of skeletal variations occurred in all test groups including controls.
The majority of skeletal variations were equally distributed about the different test groups including controls, if normal biological variation is taken into account, and can be found in the historical control data at comparable frequencies.
However, the overall affected fetuses/litter incidence of skeletal variations was statistically significantly increased in test groups 2 and 3, with only the high-dose incidence being outside the historical control range (HCD: mean% 96.4 [90.8 - 98.5]).
Specifically, the affected fetuses/litter incidences of ‘supernumerary thoracic vertebra’ (affect. fetuses/litter incidences: 4.2 / 4.5 / 7.3 / 24.5** [p <= 0.01]), ‘supernumerary rib (14th) (cartilage present)’ (8.4 / 7.2 / 10.9 / 22.5**), ‘supernumerary rib (14th) (cartilage not present)’ (41.3 / 49.5 / 66.5** / 85.0**) and ‘cervical rib (cartilage not present)’ (4.7 / 0.8 / 2.1 / 8.8*) were statistically significantly increased in test group 3 (7000 ppm). The mean incidences of these variations were outside the historical control range, thus they were assessed as treatment-related. These thoracic vertebra and rib changes appear at high background incidences in the population of the used rat breed which may qualify them to be of relatively low toxicological concern.
However, the high incidences of them under treatment at 7000 ppm, affecting a defined skeletal area, may indicate beginning developmental toxicity.
In contrast, the statistically significantly increased observed findings ‘unossified sternebra (unchanged cartilage)’ (5.2 / 3.3 / 3.1 / 12.0*) and ‘incomplete ossification of sternebra (unchanged cartilage)’ (61.1 / 64.7 / 72.1 / 89.2**) in affected fetuses/litter, respectively, in test group 3 (7000 ppm) were assessed as treatment-related, but not as adverse by themselves.
These findings represent slight delays of ossification which did not affect morphology, as the underlying cartilage model was completely intact in all these cases. This assessment is supported by the fact, that the mean fetal body weights in test group 3 (7000 ppm) were significantly reduced (about 8% below the concurrent control value, if both sexes were combined) which indicates a delay in overall development going along with the delay in ossification.
It should, however, be noted that these minor disturbances/delays of ossification occurred in the presence of clear maternal toxicity, such as a reduction in food consumption, a decrease in (corrected) body weight gain, a mild normochromic, microcytic anemia and a changed liver cell metabolism.
If all different types of variations are summarized, the incidence of affected fetuses/litter in the high-dose group was statistically significantly increased. For details, please refer to the attached PDF (Tables_and_Figures.pdf).

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 majority of unclassified skeletal cartilage observations which were observed in several fetuses of all test groups (0, 700, 2300 and 7000 ppm). The distribution and type of these findings do not suggest any relation to treatment.
However, the incidence of ‘fused cervical arch cartilage’ was statistically significantly increased in test group 3 (2.0 / 0.0 / 0.0 / 19.4**). This finding is not present in the historical control data and was therefore assessed as a treatment-related.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
185 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: supernumerary rib
other: supernumerary thoracic vertebra, cervical rib, fused cervical arch cartilage
Description (incidence and severity):
The affected fetuses/litter incidences of ‘supernumerary thoracic vertebra’ (affect.
fetuses/litter incidences: 24.5** [p <= 0.01]), ‘supernumerary rib (14th) (cartilage present)’: (22.5**), ‘supernumerary rib (14th) (cartilage not present)’ (85.0**) and ‘cervical rib (cartilage not present)’ (8.8* [p <= 0.05]) were statistically significantly increased in the high dose group.

Overall developmental toxicity

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

Any other information on results incl. tables

Discussion

In a prenatal developmental toxicity study, the test substance (Z)-4-[C11-13 (branched) alkylamino]-4-oxo-2-butenoic acid was administered to pregnant Wistar rats as a constant homogeneous addition to the food at dose levels of 0, 700, 2300 and 7000 ppm from implantation to one day prior to the expected day of parturition (GD 6-20) 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 food mixture. During administration period the concentrations in feed correspond to about 57, 185 and 475 mg/kg bw/d.

Regarding clinical examinations, signs of maternal toxicity were observed at the high-dose level (7000 ppm). High-dose dams showed a reduction in mean food consumption (overall 21% below control) and a decrease in body weight/body weight gain (up to 10 and 32% below control, respectively). Corrected body weight gain of the high-dose dams was statistically significantly decreased (81% below control), which resulted in decreased carcass weights (11% below controls). The above-mentioned findings were assessed as treatment-related and adverse. At 700 and 2300 ppm, no treatment-related, adverse effects were observed.

Regarding clinical pathology, in dams of test group 3 (7000 ppm) a marginal normochromic, microcytic anemia was present because of decreased hemoglobin and mean corpuscular volume (MCV) values, but unchanged mean corpuscular hemoglobin concentration (MCHC) values). At this stage, the anemia did not seem to be regenerative, because absolute reticulocyte counts were significantly decreased.

Additionally, a changed liver cell metabolism was observed in dams of this test group because of decreased total protein, albumin and globulin values as well as lower cholesterol, triglyceride and total bilirubin values. Total calcium levels were secondarily decreased because of lower levels of the transport globulins and albumin. Due to this changed liver cell metabolism the increased alkaline phosphatase (ALP) levels in dams of test group 3 were most probably also caused by a liver cell enzyme induction. T4 values in dams of test group 3 (7000 ppm) were increased and not decreased.

Regarding pathology, target organs were the liver and thyroid glands.

The significantly decreased terminal body weight in test group 3 animals was regarded as treatment-related. The increased liver weights in test groups 2 and 3 were regarded as treatment-related. The weight increase was only minimal. However, in combination with adverse findings of liver parameters in clinical chemistry in test group 3, the weight increase was judged as adverse. A histopathologic examination was not performed. The significantly decreased spleen weights in test group 3, the significantly decreased absolute kidney weights in test group 3, the significantly increased relative weights of adrenal glands in

test groups 2 and 3 and the significantly increased relative kidney weights in test group 3 were regarded as a secondary effect, based on the decreased terminal body weight. All weight changes were regarded as not adverse. A histopathologic examination of these organs was not performed. The minimal diffuse follicular hypertrophy and hyperplasia of thyroid glands, occurring in 4/25 test group 3 animals, was regarded as treatment-related. Since it correlated to aberrant hormone values (e.g. increased T4 and TSH values), this finding was interpreted to be adverse, although it was minimal. 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 (700, 2300 or 7000 ppm) 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.

The overall affected fetuses/litter incidence of skeletal variations was statistically significantly increased in test groups 2 and 3, with only the high-dose incidence being outside the historical control range (HCD: mean% 96.4 [90.8 - 98.5]). Specifically, the affected fetuses/litter incidences of ‘supernumerary thoracic vertebra’, ‘supernumerary rib (14th) (cartilage present)’, ‘supernumerary rib (14th) (cartilage not present)’, ‘cervical rib (cartilage not present)’ and ‘fused cervical arch cartilage’ were statistically significantly increased in test group 3 (7000 ppm). The mean incidences of these findings were outside the

historical control range, thus they were assessed as treatment-related. Almost all of these vertebra and rib variations appear at high background incidences in the population of the used rat breed, are unlikely to be detrimental for survival or health and are at least to some extent being repaired postnatally, which qualifies them to be of relatively low toxicological concern. For example, short supernumerary ribs represent ossification centers lateral to the vertebrae and are not of developmental importance. They usually resolve as the animal matures. Long supernumerary ribs may represent developmental field alterations and appear to be permanent, at least in some studies. However, it is not clear that they have implications for the health of the offspring. However, the high incidences of variants under treatment at 7000 ppm, affecting a defined skeletal area, may indicate beginning developmental toxicity. This sort of variation pattern was not observed at 2300 ppm, thus the slightly increased overall affected fetuses/litter incidence of skeletal variations in this dose group, inside the historical control range, was considered to be of no toxicological relevance.

In contrast, the statistically significantly increased observed findings ‘unossified sternebra (unchanged cartilage)’ (5.2 / 3.3 / 3.1 / 12.0*) and ‘incomplete ossification of sternebra (unchanged cartilage)’ (61.1 / 64.7 / 72.1 / 89.2**) in affected fetuses/litter, respectively, in test group 3 (7000 ppm) were assessed as treatment-related, but not as adverse by themselves.

These findings represent slight delays of ossification which did not affect morphology, as the underlying cartilage model was completely intact in all these cases. This assessment is supported by the fact, that the mean fetal body weights in test group 3 (7000 ppm) were significantly reduced (about 8% below the concurrent control value, if both sexes were combined) which indicates a delay in overall development going along with the delay in ossification.

It should, however, be noted that these minor disturbances/delays of ossification occurred in the presence of clear maternal toxicity. 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). Systemic toxicity such as reduction in food consumption, a decrease in (corrected) body weight gain, a mild normochromic, microcytic anemia and a changed liver cell metabolism noted at 7000 ppm are clear evidence for distinct maternal stress which in turn had an impact on fetal ossification status.

There was, however, no evidence that the test substance was teratogenic at any of the tested doses.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this prenatal developmental toxicity study, the oral administration of (Z)-4-[C11-13 (branched) alkylamino]-4-oxo-2-butenoic acid via the diet to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-20) provided evidence of maternal toxicity, such as a reduction in food consumption, a decrease in (corrected) body weight gain, a mild normochromic, microcytic anemia and a changed liver cell metabolism at the highest tested dose level of 7000 ppm (475 mg/kg bw/d). In addition, the high-dose group showed an incipient mild effect on the thyroid glands, which is indicated by aberrant thyroid hormone levels and minimal diffuse hypertrophy/hyperplasia of the thyroid tissue. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is the mid-dose level of 2300 ppm (185 mg/kg bw/d).
The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 2300 ppm (185 mg/kg bw/d) based on increased incidences of skeletal variations in the area of vertebrae and ribs at the high-dose level (7000 ppm, 475 mg/kg bw/d), which indicate beginning developmental toxicity. It should be noted that these minor disturbances/delays of ossification occurred in the presence of clear maternal toxicity.
There was no evidence that the test substance was teratogenic at any of the tested doses.