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

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

No effects on reproduction parameters in an OECD422 screening study.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

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)

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 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

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:

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

Parental animals: Observations and examinations:

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
- Animals fasted: Yes
- How many animals: 5 males 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

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:

STANDARDISATION OF LITTERS
- 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.

PARAMETERS EXAMINED
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.

SERUM HORMONES: Yes (T4, TSH)
- Time of blood sample collection: PND13
- Animals fasted: No
- How many animals: 1 pub per sex

GROSS EXAMINATION OF DEAD PUPS: yes

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals [Day 31]
- Maternal animals: All surviving animals [Day 64]

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

HISTOPATHOLOGY / 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
- 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

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

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: 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.

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

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

200 mg/kg bw/day

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

60 mg/kg bw/day

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

no

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.

Sex distribution did not differ between the dose groups.

T4 and TSH levels were not altered by treatment.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

200 mg/kg bw/day

Sex:

male/female

Basis for effect level:

body weight and weight gain

Critical effects observed:

no

Reproductive effects observed:

no

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Toxicity to reproduction: other studies

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.

There were no effects on mating, gestation, number of pups, and viability. High does male pups (trend in females but not significant) gained slightly less weight mainly between days 7-13, which led to a decreased (-8%) final body weight when compared to controls. Values were still well in the range of the historical controls, but the difference is likely secondary to severe maternal toxicity at this dose level, which also led to significantly reduced food consumption in the parental females during this lactation interval.

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

Justification for classification or non-classification

There was no direct adverse effect of the test substance on reproduction. Consequently, no classification according to Regulation (EC) No 1272/2008 (CLP) for toxicity to reproduction.