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Diss Factsheets

Administrative data

screening for reproductive / developmental toxicity
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)

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 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
Details on mating procedure:
- M/F ratio per cage: 1:1
- mated after 2 weeks of treatment
- Length of cohabitation: max. of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
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


Parental animals: Observations and examinations:
- 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
- Animals fasted: Yes
- How many animals: 5 males 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
Oestrous cyclicity (parental animals):
For all females in a pool of up to 50 animals, estrous cycle normality was evaluated before the randomization.

For a minimum of 2 weeks prior to mating estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats. Determination was continued throughout the pairing period until the female exhibited evidence of copulation.

At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice. Special attention was given to the synchrony of the morphology of the estrous cycle in ovaries, uterus, cervix, and vagina.
Sperm parameters (parental animals):
Parameters examined in F1males:
testis weight, epididymis weight, stages of spermatogenesis, histopathology of interstitial testicular cell structure
Litter observations:
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups.

- Time of blood sample collection: PND13
- Animals fasted: No
- How many animals: 1 pub per sex


Postmortem examinations (parental animals):
- Male animals: All surviving animals [Day 31]
- Maternal animals: All surviving animals [Day 64]

- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

- 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
- for histopathological examinations, refer to the table below
Postmortem examinations (offspring):
On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations (see 3.9.3.). Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution, transferred to the Pathology Laboratory, and archived without further processing.
Reproductive indices:
Male/female mating index
Male/female fertility index
Gestation index
Offspring viability indices:
Live birth index
Postimplantation loss
Viability index
Survival index
Sex ratio

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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
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.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Spermatogenesis stages were unaffected by treatment.
Reproductive performance:
no effects observed
Description (incidence and severity):
The two dead high dose females resulted in a lower gestation index for this group which is not related to the test substance.

Details on results (P0)

TSH and T4 levels were not altered (only measured in males)

Effect levels (P0)

open allclose all
Dose descriptor:
Effect level:
200 mg/kg bw/day
Basis for effect level:
body weight and weight gain
Dose descriptor:
Effect level:
60 mg/kg bw/day
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity (P0)

Critical effects observed:

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Rates of liveborn and stillborn pubs as well as the number of dead pubs were evenly distributed about the test groups. The viability index (PND 0 - 4) varied between 99.0%/ 98.6% / 100% and 99.1% in test groups 0 - 3, respectively.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights were statistically significantly decreased on PND 13 in male pups of test group 3 (-8.5% compared to control) and also if male and female pups were combined (-8.0% compared to control).

The mean body weight change was statistically significantly decreased between PND 1 and 13 in male pups of test group 3 (-9.7% compared to control) and also if male and female pups were combined (-8.7% compared to control).
The mean body weight change was statistically significantly decreased between PND 7 and 13 in male and female pups of test group 3 (-12% males and -10.6% females versus control group) and also if male and female pups were combined (-11.3% versus control group).

These findings are considered secondary of maternal toxicity (reduced food consumption, chronic inflammation / ulcers in digestive tract, beginning regenerative anemia) and not a direct developmental effect. All values remained within the historical control range.
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Six male and five female pups in test group 3 showed post mortem autolysis and in one female pup of test group 2 a small spleen was detected.

Details on results (F1)

Sex distribution did not differ between the dose groups.

T4 and TSH levels were not altered by treatment.

Effect levels (F1)

Dose descriptor:
Effect level:
200 mg/kg bw/day
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:

Overall reproductive toxicity

Reproductive effects observed:

Applicant's summary and conclusion