Registration Dossier

Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
GLP compliance:
yes (incl. QA statement)
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
(2,2-dimethyl-1,3-dioxolan-4-yl)methyl prop-2-enoate
EC Number:
Cas Number:
Molecular formula:
(2,2-dimethyl-1,3-dioxolan-4-yl)methyl prop-2-enoate
Test material form:
Details on test material:
Analytical study no. 20L00019
Batch: 0812-HS-0030
Specific details on test material used for the study:
- Source and lot/batch number of test material: Manufacturer, Batch 0812-HS-0030
- Purity: 96.2%

- Storage condition of test material: refrigerator

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River Laboratories, Germany, Sulzfeld
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at beginning of treatment: males: 14-16 weeks, females: 13-14 weeks
- Weight at beginning of treatment: Males: app. 385g; Females: app: 217g
- Fasting period before study: no
- Housing: individually except during mating and after parturition until PND13
- Diet (e.g. ad libitum): ad lib.
- Water (e.g. ad libitum): ad lib.
- Acclimation period: 3 weeks
- Temperature (°C): 20-24°C
- Humidity (%): 45-65%
- Air changes (per hr):15
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:
oral: gavage
corn oil
Details on oral exposure:
For the preparation of the solutions the specific amount of test substance was weighed in a calibrated beaker, topped up with corn oil and intensely mixed with the magnetic stirrer. Afterwards the test substance preparations were stored at room temperature.

- Justification for use and choice of vehicle (if other than water): solubility of the test substance
- Concentration in vehicle: 1.5, 5.0, 15.0 g/100mL
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Analytical verifications of the stability of the test substance in corn oil for a period of 7 days at room temperature was proven (Project No. 01Y0245/18L062)

At the beginning (during pre-mating) and twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. These samples were used as a concentration control at the same time. At the time points mentioned above, additionally one sample from the mid concentration was taken for concentration control analysis.

Homogeneous distribution in the vehicle and accuracy of the concentrations was confirmed.
Duration of treatment / exposure:
30 days (males), 64 days (females)
Frequency of treatment:
once daily (except to females in labor)
Doses / concentrationsopen allclose all
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on a range finding study (included in IUCLID as supporting study), Clear toxicity (body weight gain, local effects and changes in organ weights and hematology and clinical chemistry) were observed at 750mg/kg and to a lesser extent at 250mg/kg.
- Fasting period before blood sampling for clinical biochemistry: yes


Observations and examinations performed and frequency:
- Time schedule: at least daily
- Cage side observations included: morbidity, pertinent behavioral changes, signs of overt toxicity before as well as within 2h and between 2-5 after administration

- Time schedule: weekly

- Time schedule for examinations: weekly, more frequent after parturition

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

- Time schedule for examinations: generally once a week for a period of 4 days
• Water consumption was not determined after the 2nd premating week (male parental animals)
• Water consumption of the females with evidence of sperm was determined on gestation days (GD) 0-1, 6-7, 13-14 and 19-20.
• Water consumption of the females, which gave birth to a litter was determined for PND 1-2, 3-4, 6-7, 9-10 and 12-13.

- Time schedule for examinations:
- Dose groups that were examined:

- Time schedule for collection of blood: males on day 31 shortly before sacrifice, females on PND14
- Anaesthetic used for blood collection: Yes (isofluran)
- Animals fasted: Yes
- How many animals: 5 per group
- Parameters examined: Leukocyte count, Erythrocyte count, Hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin (concentration), platelet count, differential blood count, reticulocytes, prothrombin time

- Time schedule for collection of blood: males on day 31 shortly before sacrifice, females on PND14
- Animals fasted: Yes
- How many animals: 5 per group
- Parameters examined: ALT, AST, ALP, GGT, sodium, potassium, chloride, inorganic phosphate, clacium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, bile acids

- Time of blood sample collection: parental males on day 31 shortly before sacrifice, pubs on PND13
- Animals fasted: Yes (only adults)
- How many animals: 1 pub per sex, parental males: 5 per group

- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.

- Time schedule for examinations: males (5 per group) on day 29, females (5 per group) on day 62
- Dose groups that were examined: all
- Battery of functions tested: sensory activity/ reflexes, touch response, visual placing response, pupillary reflex, pinna reflex, startle response, coordination, pain perception, grip strength, landing, motor activity
Sacrifice and pathology:

- organ weights (all animals): terminal body weight, epididymides, ovaries, prestate, seminal vesicles, testes, thyroid, uterus
- organ weights (5 per sex and group): adrenals, brain, heart, kidneys, liver, spleen, thymus

HISTOPATHOLOGY: Yes (see table below)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All males and females of the high-dose (600 mg/kg bw/d), all males and most of the female animals of the mid-dose (200 mg/kg bw/d) and five males of the low-dose (60 mg/kg bw/d) showed salivation immediately after dosing (up to 2 hours post dosing) during the treatment period. Because the finding ws transient and only occured directly after dosing, it is attributed to the irritant properties or bad taste of the test substance.

Female animal No. 139 of the high-dose (600 mg/kg bw/d) showed piloerection and a pale skin before the dam was found dead at the end of the gestation period.
mortality observed, treatment-related
Description (incidence):
2 high dose females were found dead at the end of the gestation period. Cause of death for 1 female could be determined: Adhesion between the stomach and the left lateral lobe and the diaphragm characterized by necrosis and inflammation. It resulted most likely from a transmural ulcer in the forestomach.
Forestomach findings were also reported for the second deceased female, but cause of death could not be determined.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
High dose males showed a decreased body weight change (-46% compared to control) on days 7-13.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period (Lactation days 10-13) food consumption in high dose females was reduced by 19% compared to the control. Combined with the trend towards reduced body weight in males, this finding was seen as a potential indication of systemic toxicity.

Food consumption in the high dose was significantly increased in one interval each for males (days 7-13) and females (GD14-20). This singular finding was considered incidental and unrelated to treatment.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Statistically significantly increased water consumption in the high-dose females (600 mg/kg bw/d) were observed on In-life days 7-11 (+40% compared to control), on GD 6-7 and 19-20 (up to +55% compared to control). This minor finding is most likely due to the bad taste of the test substance or local affection of the upper digestive tract and therefore considered not to be an adverse toxicologically relevant finding per se.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
600mg/kg, males:

- hemoglobin, hematocrit, MCHC decreased
- reticulocyte counts increased
These findings lead to the diagnosis of a microcytic normo-chromic, regenerative anemia.

- prothrombin time reduced
This is a consequence of changes in red and white blood cell counts and correlating changes in laminar blood flow in the vessels which in turn lead to increased coagulation factor synthesis.

- WBC, absolute and relative neutrophil counts and absolute monocyte counts increased

600mg/kg, females:
- WBC and absolute neutrophil counts increased
Clinical biochemistry findings:
no effects observed
Endocrine findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
High dose females:
- absolute (+21%) and relative (+26%) liver weight increase
- relative (+10.5%) kidney weight increase
- relative (+7%) heart weight increase
All changes were within historical control ranges and therefore regarded as incidental and not related to treatment.

High dose males:
- relative (+16.1%) liver weight increasee
- absolute (+26.9%) and relative (+28.2%) spleen weight increase
The increased mean absolute and relative weight of the spleen (0.688 g; 0.176%) correlated histologically with an increased amount of extramedullary hematopoiesis in this organ. These findings were interpreted as treatment-related.

The statistically significant increase in mean relative liver weight (2.644%) was minimally above the historical threshold (males: 2.200 – 2.382%). This finding was considered possibly treatment-related, but not adverse as no correlating histopathological changes were present.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Findings resulted from the local irritant properties of the test substance:
White foci, a thickening of the margo plicatus, and/or a thickening of the stomach wall was present in the forestomach of both sexes in test group 3 and to a lesser extent in test group 2. These findings correlated with the histological presence of erosions/ulcers and of squamous cell hyperplasia and were considered treatment-related.
In males and females of test group 3, the duodenum showed a thickened wall in 8/10 and 2/10 animals, respectively. This finding was also regarded as treatment-related and correlated with a thickening of the mucosa in histology in most cases. Additionally, the pancreatic lymph node was enlarged in size in all male and 9/10 female animals of test group 3. This enlargement correlated with an increased number of lymphocytes and plasma cells as well as with the presence of lymphangiectasis in histology and was likewise interpreted as treatment-related.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the forestomach of all high dose animals, ulcers of grade 3-4 (1 ulcer of grade 2) were observed with grade 4 being transmural ulcers. These were associated with varying degrees of inflammatory cell infiltrates and formation of granulation tissue. Minimal erosions/ulcers were also present in 2/10 animals per sex in the mid dose.

In addition, a slight to severe squamous cell hyperplasia was present in all high dose animals, characterized by a thickening of the stratum spinosum and the stratum corneum and a mostly exophytic growth pattern. In this test group, this finding was accompanied by proliferation of the basal cell layer with endophytic growth into the underlying tissue, but maintenance of the basal membrane (basal cell hyperplasia).
In the mid dose, 7/10 male and 4/10 female animals also showed a minimal to slight squamous cell hyperplasia.
This change is considered an adaptive response to continued irritation.

The thickening of the duodenal wall correlated with a thickening of the mucosa in 7/10 males and 1/10 females. The thickening included both an elongation of the villi as well as an enlarge ment of enterocytes along the villi.

The enlarged pancreatic lymph node in all male and 9/10 female animals of test group 3 was characterized histologically by a minimal to moderate increase in the number of lymphocytes and plasma cells. Further, most animals also showed minimal to severe lymphangiectasis. Five male and three female animals also showed increased amounts of erythrocytes in the sinuses (blood resorption). These changes are seen as a reaction to the lesions in the forestomach.

In the spleen of high dose males (10) and females (8, though evaluation of one spleen only partially possible), the number of hematopoietic cells, notably erythropoietic precursor cells in most cases, in the red pulp was increased. This increase was severe (grade 3-4) in 8 males and 2 females, while 5 females only showed a minimal increase. This is likely a consequence of the forestomach ulcers and loss of blood into the lumen. It also correlates to the regenerative anemia.

Effect levels

open allclose all
Dose descriptor:
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Dose descriptor:
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

Applicant's summary and conclusion