2-Propenoic acid, (2,2-dimethyl-1,3-dioxolan-4-yl)methyl ester

Endpoint summary

Description of key information

OECD 422, oral, gavage (corn oil): severe local effects leading to systemic changes and death at 600mg/kg

NOAEL systemic: 200mg/kg

NOAEL local: 60mg/kg

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Dose Range Finder

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Principles of method if other than guideline:

The test substance was administered by gavage to 2 dose groups daily for 14 days. A concurrent vehicle control was performed. A limited set of parameters was evaluated with the goal to select relevant dose levels for subsequent studies.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Manufacturer, Batch 0812-HS-0030
- Purity: 96.2%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 days

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

750 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

Animals were checked daily for clinical signs of toxicity. Water and food consumptions were determined on study days 0, 3, 7, 10 and 14. Body weight was examined on study days 0, 3, 7, 10, and 14.

On the day of sacrifice, blood samples for hematological and clinico-chemical examinations were taken from all fasted animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia.

Sacrifice and pathology:

All animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

To provide more information for dose selection in subsequent studies, the weights of the anesthetized animals, adrenal glands, kidney, liver and spleen were determined in all animals sacrificed on schedule.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

750mg/kg: Decreased body weight change values from SD 0 to 3 (up to 86% below control) in male animals

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

750mg/kg:
• Decreased hemoglobin level and increased absolute reticulocytes in female animals
• Increased absolute polymorphonuclear neutrophils, decreased relative lymphocytes in male
animals and decreased relative eosinophils in male and female animals
• Decreased creatinine levels in male animals
• Increased cholesterol levels in male and female animals

250mg/kg:
• Increased white blood cells and absolute polymorphonuclear neutrophils

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

750mg/kg:
• Decreased creatinine levels in male animals
• Increased cholesterol levels in male and female animals

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

750mg/kg:
• Increased mean relative kidney weights (19% above control)
• Increased mean relative liver weights (up to 34% above control)
• Increased mean absolute liver weights (26% above control)

250mg/kg:
• Increased mean relative kidney weights (6% above control)
• Increased mean relative liver weights (6% above control)
• Increased mean absolute liver weights (11% above control)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

750mg/kg:
• Duodenum, thickened wall in 3 of 4 male animals and in all female animals
• Forestomach (focus and thickened margo plicatus or thickened wall) in all animals

250mg/kg:
• Duodenum, thickened wall in 1 of 4 male animals
• Forestomach (focus and thickened margo plicatus) in some male and female animals
• Glandular stomach (focus) in one male animal

Conclusions:

The administration of IPGA (2-Propenoic acid, (2,2-dimethyl-1,3-dioxolan-4-yl)methyl ester) by gavage to male and female Wistar rats for two weeks did cause dose dependently significant signs of toxicity in both administered dose levels of 250 and 750 mg/kg body weight/day.

Based on the results of this range-finding study, the dose levels of the subsequent OECD 422 study were set to 600-200-60-0 mg/kg bw/d.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

OECD422

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Manufacturer, Batch 0812-HS-0030
- Purity: 96.2%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: at least weekly
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.

VEHICLE
- 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:

yes

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)

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:

10

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:

CAGE SIDE OBSERVATIONS: Yes
- 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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

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

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

WATER CONSUMPTION: 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.

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- 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

CLINICAL CHEMISTRY: Yes
- 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

SERUM HORMONES: Yes (T4, TSH)
- 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

URINALYSIS: No
- 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.

NEUROBEHAVIOURAL EXAMINATION: Yes
- 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:

GROSS PATHOLOGY: Yes

ORGAN WEIGHTS
- 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)

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:

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.

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

mortality

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

60 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

guideline study

Additional information

Isopropylidenglycerinacrylat was administered in an OECD 422 guideline study at doses of 60, 200, and 600mg/kg in corn oil. Male rats were treated for 31 days, females to app. day 13 of lactation. Two high dose females died. Cause of death could be determined for one female and was due to a forestomach ulcer. All high dose animals showed severe local effects (grade 3-4, partly transmural forestomach ulcers, adaptive responses of the basal layer (stomach), thickening of the duodenal wall) accompanied by bleeding into the stomach. As a consequence, hematology showed changes indicative of continued inflammation and regenerative anemia. The latter also matched findings in the spleen. Anemia was more pronounced in males than females, but affected all high dose animals.

Minimal erosions and squamous cell hyperplasia was still observed in some mid dose animals.

The following NOAELs were obtained: parental, systemic: 200mg/kg, parental, local: 60mg/kg

Justification for classification or non-classification

There was no direct organ toxicity and all changes are very likely secondary to severe local effects. Besides, the LOAEL of 600mg/kg in a subacute study is above the proposed classification limit of 300mg/kg according to Regulation (EC) No 1272/2008 (CLP). Consequently, the registered substance does not need to be classified for specific target organ toxicity.