4,5-dihydroxy-1,3-dimethylimidazolidin-2-one

Endpoint summary

Description of key information

In the key oral 90-day repeated-dose toxicity study in rats according to OECD guideline 408, the test item did not cause adverse effects in male or female Han:WIST rats after 90-day consecutive oral (by gavage) administration of 100, 300 or 1000 mg/kg bw/day (corresponding to doses of 236, 710 and 2360 mg/kg bw/day of the product). There were no toxic changes in the examined parameters (clinical signs, body weight and body weight gain, food intake, ophthalmology, hematology, blood coagulation and clinical chemistry, serum levels of thyroid hormones, urinalysis, estrous cycle, necropsy findings, organ weights or histopathological findings). Based on the observations made in this toxicity study the NOAEL was 1000 mg/kg bw/day in male and female Han: WIST rats.

 

In the supporting range-finding study, 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one did not cause adverse effects in male or female Han: WIST rats after consecutive 14-day oral (by gavage) administration at the doses of 100, 300 or 1000 mg/kg bw/day. Based on the observations made in this toxicity study, the following dose levels for a 90-Day Repeated Dose Oral Toxicity Study of 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one in Rats (main study) are suggested: 100, 300 and 1000 mg/kg bw/day (corresponding to 236, 710 and 2360 mg/kg bw/day of the product).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-03-09 to 2021-12-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Version / remarks:

1998-08

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008-05-30

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

2018-06-25

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han:Wist

Details on species / strain selection:

The rat is commonly used species for toxicological studies in accordance with international
recommendations. The Wistar rat was the system of choice because it has been the preferred and most commonly used species for oral gavage toxicity tests and is a well-known laboratory model with sufficient historical data.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90.
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 255-277 g (males), 175-207 g (females)
- Fasting period before study: No
- Housing: Group caging, max. 2 or 3 animals of the same sex/ cage in type III polypropylene/polycarbonate cages with certified laboratory wood bedding
- Diet: Ad libitum (except for overnight food deprivation before blood sampling)
- Water: Ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used. Water quality control analysis and microbiological assessment are performed once in every six months by the National Public Health and Medical Officer Service. The quality control results are available at the laboratory's archives.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): above 10
- Photoperiod (hrs dark / hrs light): 12/12 (except for days of ophthalmology examinations)

IN-LIFE DATES: From day 0 to day 90/91 (males/females) and from day 0 to day 119 (males/females of the recovery groups)

Route of administration:

oral: gavage

Details on route of administration:

The route of application was selected in compliance with international guidelines (See "test guidelines"). The oral route is a possible route of human exposure to the test item.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
4,5-dihydroxy-1,3-dimethylimidazolidin-2-one was dissolved in the vehicle (distilled water) in concentrations of 47.2, 141.8 and 472.4 mg/mL, calculated by the content of 4,5- dihydroxy-1,3-dimethylimidazolidin-2-one and its organic impurities in the product (42.34 %). Formulations were prepared in the formulation laboratory of the test facility beforehand for not longer than 3 days and stored at room temperature (15 – 25°C) until use.

VEHICLE
- Concentration in vehicle: 47.2, 141.8 and 472.4 mg/mL
- Amount of vehicle: 5 mL/kg bw
- Lot/Batch Nos: 202010095, 202009092, 202011113, 202103012, 202102004

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing formulations (verification of concentrations and homogeneity) was performed in the analytical laboratory of the test facility three times during the study. Five aliquots of 10 mL of each formulation that was administered to the animals and five aliquots of 10 mL control substance (vehicle) was taken from different places (top, middle and bottom). During the formulation sampling the formulation was stirred by electromagnetic stirrer. 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one concentrations in the dose forms used for administration of animals varied within the range of 95.3 % to 99.6 % in comparison to the nominal values, thereby confirming proper dosing. The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front. 4,5-dihydroxy-1,3-dimethylimidazolidin-2- one proved to be stable in ultrapure water at the intended concentrations at room temperature.

Duration of treatment / exposure:

90/91 days (males/females), 119 days (males/females of the recovery groups)

Frequency of treatment:

Daily

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Doses and nominal concentrations determined on the basis of 4,5-dihydroxy-1,3 dimethylimidazolidin-2-one and its organic impurities (42.34 %) contained in the product. Dose of the product: 2360 mg/kg bw/day

Dose / conc.:

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

Remarks:

Doses and nominal concentrations determined on the basis of 4,5-dihydroxy-1,3 dimethylimidazolidin-2-one and its organic impurities (42.34 %) contained in the product. Dose of the product: 710 mg/kg bw/day

Dose / conc.:

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

Remarks:

Doses and nominal concentrations determined on the basis of 4,5-dihydroxy-1,3 dimethylimidazolidin-2-one and its organic impurities (42.34 %) contained in the product. Dose of the product: 236 mg/kg bw/day

Dose / conc.:

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

No. of animals per sex per dose:

10 animals per sex per group + 5 animals/ sex in the control and highest dose groups for recovery observations

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose setting with 0, 100, 300 and 1000 mg/kg bw/day was based on the results of a preliminary dose range finding study with 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one in rats (Study no. 805-400-5789, see IUCLID section 7.5). 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one did not cause adverse effects in male or female rat, Han: WIST of Wistar origin rats after the consecutive 14-day oral (by gavage) administration at 100, 300 or 1000 mg/kg bw/day. Thus, in this study, the limit dose of 1000 mg/kg bw/day was chosen as the highest dose (corresponding to 2360 mg/kg bw/day product concentration) defined in the applicable guidelines.
- Fasting period before blood sampling for clinical biochemistry: Food was deprived overnight
- Rationale for selecting satellite groups: Recovery groups were selected to test the reversibility of potential effects.
- Post-exposure recovery period in satellite groups: 29 days (males) or 28 days (females)
- Dose range finding studies: Study no. 805-400-5789, see IUCLID section 7.5

Positive control:

Not used

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once a day, after the administration at approxy the same time. Animals were inspected for signs of morbidity and mortality twice daily at the beginning and end of each working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On the day prior to the first treatment with the test item, and approximately once weekly thereafter.
- Parameters checked in table 1 were examined.

BODY WEIGHT: Yes
- Time schedule for examinations: Once during acclimatization, on day 0 (prior to study start) and once weekly during weeks 1-13 and during the recovery period.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes (food consumption was measured weekly)
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During the acclimation period, prior to test termination and on the last week of recovery period.
- Dose groups that were examined: All animals of the control and high dose group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: One day after the last treatment and, for the recovery groups, at the end of the recovery period
- Anaesthetic used for blood collection: Yes (Isofluran CP®)
- Animals fasted: Yes (approx. 16 hours)
- How many animals: All animals from the test and recovery groups
- Parameters checked in table 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: One day after the last treatment and, for the recovery groups, at the end of the recovery period
- Animals fasted: Yes (approx. 16 hours)
- How many animals: All animals from the test and recovery groups
- Parameters checked in table 3 were examined.

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: In the morning, approx. 1 day after the last treatment
- Animals fasted: Yes, overnight (approx. 16 hours)
- How many animals: All animals from the test and recovery groups

URINALYSIS: Yes
- Time schedule for collection of urine: At termination of the treatment and recovery periods (for approx. 16 hours before the blood collection)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table 4 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During the last exposure week and the end of recovery period
- Dose groups that were examined: All dose groups
- Battery of functions tested: Sensory activity / grip strength / motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 5)

HISTOPATHOLOGY: Yes (see table 5, preserved organs). Histological examination was performed on the preserved organs and tissues of the animals from both the control and high dose groups (Groups 1 and 4, respectively) including recovery groups. As there were no test item-related lesions in the organs or tissues of high dose animals, no general histopathological examination was performed for low and mid dose animals.

Optional endpoint(s):

ESTROUS CYCLE EXAMINATION
At necropsy (at the end of treatment and recovery periods), the oestrous cycle was monitored by examining vaginal smears from each female animal. These observations provided information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of estrogen sensitive tissues.

Statistics:

Statistical analysis was done with SPSS PC+ software for the following data: Body weight, Food consumption, Hematology, Blood coagulation, Clinical chemistry, Thyroid hormones, Urinalysis, Organ weight.

The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test. Frequency of clinical signs, ophthalmoscopy, pathological and histopathological findings by sex and dose was calculated. For evaluation of data obtained during the recovery period, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the Two-Sample t-test was performed. The use of the word “significant” or “significantly” indicates – where relevant – a statistically significant difference between the control and the experimental groups. Significance was judged at a probability value of p < 0.05 and < 0.01. Male and female rats were evaluated separately.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

300 mg/kg bw group: Piloerection and activity decreased was noted in one male animal. Piloerection was noted on day 73-81 and activity decreased was present on day 73-83.

As no clinical signs were observed in the 100 and 1000 mg/kg bw/day groups, this finding was considered incidential.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

300 mg/kg bw group: One female was found dead on day 48. Histological examination revealed acute purulent bronchitis (probably in connection with miss-swallowing) as the cause of death. The death was likely to be caused by an application error.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weight development was not affected at 100, 300 or 1000 mg/kg bw/day (also recovery groups) throughout the treatment period.

Sporadic statistically significantly higher or lower mean body weight gains were found minor or incidential as no dose-response could be observed, the weights remained within the historical control ranges and were not accompanied by supporting findings (pathology, histopathology, related clinical pathology parameters).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Sporadic statistically significant differences in the food intake between the control and test item treated groups (also recovery groups) were not dose-dependent or remained well within the historical control ranges.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Sporadic statistically significant differences in hematological parameters like RBC, MCV, MCH, RET, NEU in male animals or animals of the recovery group (MPV in females, MONO in males) were small, not dose-dependent or were well within the historical control range. No relation to changes in other examined parameters was observed. Moreover, there were no accompanying findings in relevant organs (necropsy, organ weight, histopathology). Therefore, they were considered to be without toxicological relevance.

No changes were found in female animals of the main study.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Sporadic statistically significant differences in clinical chemistry parameters like K+, ALB (males), K+, UREA, BUN (females), Na+ (males of the recovery group) and K+, A/G (females of the recovery group) were small, not dose-dependent or were well within the historical control range. No relation to changes in other examined parameters was observed. Moreover, there were no accompanying findings in relevant organs (necropsy, organ weight, histopathology). Therefore, they were considered to be without toxicological relevance.

Endocrine findings:

no effects observed

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

In male animals at 100, 300 or 1000 mg/kg bw/day the volume of the urine was statistically significantly higher compared to the control but no dose-dependency was observed.

In one male animal from the 1000 mg/kg bw/day group, bloody urine (RBC), high pH (11.0) and tripelphosphate (few) showed in the urine which was accompanied by higher absolute weight of the kidneys and pyelectasis on both sides. The effects were thus concluded to result from the pyelectasis which was evaluated as a spontaneous finding and not related to the test item.

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Sporadic statistically significant differences in organ weighs (liver, spleen, brain, testes, thyroids or pituitary) in male animals, thymus, pituitary, thyroids, brain and heart (females animals), testes (male animals of the control group) were small, not dose-dependent or were well within the historical control range. No relation to changes in other examined parameters was observed. Moreover, there were no accompanying findings in relevant organs (histopathology). Therefore, they were considered to be without toxicological relevance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the animal from the 300 mg/kg bw/day group that died on day 48, the lungs were mottled. This was considered to be the result of an application error.

In one male animal from the 300 mg/kg bw/day group, granulated kidney surface (both sides) was observed and in another male animal, a diameter 1.5 cm purulent abscess was observed in the thoracic cavity around the right side of the lung.

In one male animal from the 1000 mg/kg bw/day group, pyelectasis (both sides) and in another male animal, yellowish colored thymus could be observed.

In one female animal from the 100 mg/kg bw/day group, moderate hydrometra and in another female animal, pyelectasis (both sides) could be observed.

In three female animals from the 300 mg/kg bw/day group, slight to moderate hydrometra and in another female animal, pyelectasis (both sides) could be observed.

In one female animal from the recovery group (1000 mg/kg bw/day), pyelectasis (right
side) could be observed.

Pyelectasis and the hydrometra are alterations which are frequent occurrences in experimental rats. Furthermore, the occurence was not dose-dependent or related to other findings. In the deceased animal, the reddish mottled lungs are an alteration which can be connected with an acute circulatory insufficiency (as the cause of death). The alteration found in the thymus (yellowish colored), in the kidneys (granulated surface) and the purulent abscess in the thoracic cavity around the lung can be interpreted with the results of the histopathological examination.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the animals from the 300 mg/kg bw/day group that died on day 48, histological examination revealed acute purulent bronchitis (probably in connection with missswallowing) as the cause of death.

Pyelectasia in the kidney described as a gross pathological finding was found not to be accompanied by other histopathological lesions.

CPN (chronic progressive nephropathy) is an important spontaneous renal disease of the
commonly used strains of laboratory rat. CNP occured sporadically in this study and it could be considered as individual disorder.

The focal subcronic purulent pneumonia (accompanied with abscess formation) in the 300 mg/kg bw/day group is likely to be in connection with probable miss-swallowing.

The dilatation of the uterine horns in some animals is a slight neuro-hormonal phenomenon in connection with the sexual function – prooestrus phase – of the inner genital organs.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

A test item effect on the estrous cycle was not detected at any dose level.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

1000 mg/kg bw/day corresponds to the substance concentration determined on the basis of 4,5-dihydroxy-1,3 dimethylimidazolidin-2-one and its organic impurities (42.34 %) contained in the product. Dose of the product: 2360 mg/kg bw/day.

Key result

Critical effects observed:

no

Conclusions:

In an oral 90-day repeated-dose toxicity study in rats according to OECD guideline 408, the test item did not cause adverse effects in male or female Han:WIST rats after 90-day consecutive oral (by gavage) administration of 100, 300 or 1000 mg/kg bw/day (corresponding to doses of 236, 710 and 2360 mg/kg bw/day of the product). There were no toxic changes in the examined parameters (clinical signs, body weight and body weight gain, food intake, ophthalmology, hematology, blood coagulation and clinical chemistry, serum levels of thyroid hormones, urinalysis, estrous cycle, necropsy findings, organ weights or histopathological findings). Based on the observations made in this toxicity study the NOAEL was 1000 mg/kg bw/day in male and female Han: WIST rats.

Executive summary:

In an oral 90-day repeated dose toxicity study according to OECD guideline 408 and GLP, possible health hazards linked to repeated exposure of 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one over a 90-day period of time covering post-weaning maturation and growth well into adulthood followed by a 28-day recovery (post treatment) period were examined. The study intended to provide information on the major toxic effects, indicate target organs and the possibility of accumulation, and an estimate of a no-observed-adverse-effect level (NOAEL) of exposure. Four dose groups of Han:WIST rats consisting of 15 animals/sex in the control and highest dose groups – including recovery animals – and 10 animals/sex in the low and middle dose groups were administered orally (by gavage) once daily at 0 (vehicle only), 100, 300 and 1000 mg/kg bw/day (corresponding to 236, 710 and 2360 mg/kg bw/day of the product) corresponding to a 5 mL/kg bw dosing volume. A group of vehicle (Distilled water) treated animals (n= 15/sex) served as a control. The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front. 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one proved to be stable in ultrapure water at the intended concentrations at room temperature for up to 4 days. Mean concentrations of the Test item in the formulation samples were in the range of 95.3-99.6% of the nominal concentrations. General clinical cage side observations were made once a day, at approximately the same time after treatment and during the course of the recovery period. Detailed clinical observations were performed weekly during the treatment and recovery period and functional observations were made during the last week of treatment and recovery periods. Body weights and food consumption were determined weekly during the entire study. Ophthalmologic examinations were performed on all animals being considered for study before the first treatment and on all animals of the control and high dose groups during the last week of treatment and recovery periods. Clinical pathology (hematology, blood coagulation, clinical chemistry and thyroid hormones), urinalysis and gross pathology examinations were conducted on all animals one day after the last treatment and at the end of the recovery period. Selected organs were weighed. Full histopathological examinations were performed on the organs and tissues of the control and high dose groups (main and recovery groups). The estrous cycle of each female animal was examined on the day of necropsy.

 

The results of this study were summarized as follows:

 

Mortality: One female animal of middle dose (300 mg/kg bw/day) was found dead during the study.

 

Clinical observations: No clinical signs related to the test item were found at general daily or detailed weekly clinical observations at 100 and 1000 mg/kg bw/day. However, piloerection and activity decreased was noted in one male animals at 300 mg/kg bw/day. The functional observations did not reveal any test item influence on the animal behavior or neurological functions.

 

Body weight and body weight gain: There were no test item related changes in the mean body weight and body weight gain in the male and female animals at 100, 300 and 1000 mg/kg bw/day.

 

Food consumption: No adverse effects on the mean food consumption development were observed at 100, 300 or 1000 mg/kg bw/day in male or female animals (main and recovery groups).

 

Ophthalmoscopy: No alterations were detected in the eyes of the animals of the high dose group at the end of treatment or recovery periods.

 

Hematology and blood coagulation: There were no test item related adverse pathological changes in the investigated hematological or blood coagulation parameters in any group (main and recovery groups).

 

Clinical chemistry: Pathological test item effects were not detected upon the evaluation of the clinical chemistry parameters in male or female animals at 100, 300 and 1000 mg/kg bw/day (main and recovery groups).

 

Determination of serum levels of thyroid hormones: In male and female animals at 100, 300 and 1000 mg/kg bw/day the level of FT3 (free T3) and FT4 (free T4) thyroid hormones were normal at the end of treatment and recovery period.

 

Urine: No toxicologically changes in urinalysis parameters were seen in 4,5-dihydroxy-1,3- dimethylimidazolidin-2-one treated animals during the study.

 

Necropsy: Macroscopic alterations related to the test item were not detected at the necropsy at the termination of the treatment and at the end of the recovery period.

 

Organ weight: No treatment related alterations were found in the organ weight results (absolute weights, relative to the body weight and brain weight) in male and female animals compared to the control parameters (main and recovery groups).

 

Histopathology: There were no test item related lesions in the organs or tissues of high dose animals (no general histopathological examination was performed for low and mid dose animals).

 

Estrous cycle examination: A test item effect on the estrous cycle was not detected at any dose level.

 

Under the conditions of the present study, 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one did not cause adverse effects in male or female Han:WIST rats after 90-day consecutive oral (by gavage) administration of 100, 300 or 1000 mg/kg bw/day doses (corresponding to 236, 710 and 2360 mg/kg bw/day of the product, calculated by the content of 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one and its organic impurities in the product (42.34 %)). There were no toxic changes in the examined parameters (clinical signs, body weight and body weight gain, food intake, ophthalmology, hematology, blood coagulation and clinical chemistry, serum levels of thyroid hormones, urinalysis, estrous cycle, necropsy findings, organ weights or histopathological findings). Based on the observations made in this toxicity study the No Observed Adverse Effect Level (NOAEL) was determined as follows: NOAEL: 1000 mg/kg bw/day for male and female Han: WIST rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Reliable without restrictions

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Key, 90-day repeated dose toxicity, rat, RL1

In an oral 90-day repeated dose toxicity study according to OECD guideline 408 and GLP, possible health hazards linked to repeated exposure of 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one over a 90-day period of time covering post-weaning maturation and growth well into adulthood followed by a 28-day recovery (post treatment) period were examined. The study intended to provide information on the major toxic effects, indicate target organs and the possibility of accumulation, and an estimate of a no-observed-adverse-effect level (NOAEL) of exposure. Four dose groups of Han:WIST rats consisting of 15 animals/sex in the control and highest dose groups – including recovery animals – and 10 animals/sex in the low and middle dose groups were administered orally (by gavage) once daily at 0 (vehicle only), 100, 300 and 1000 mg/kg bw/day (corresponding to 236, 710 and 2360 mg/kg bw/day of the product) corresponding to a 5 mL/kg bw dosing volume. A group of vehicle (Distilled water) treated animals (n= 15/sex) served as a control. The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front. 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one proved to be stable in ultrapure water at the intended concentrations at room temperature for up to 4 days. Mean concentrations of the Test item in the formulation samples were in the range of 95.3-99.6% of the nominal concentrations. General clinical cage side observations were made once a day, at approximately the same time after treatment and during the course of the recovery period. Detailed clinical observations were performed weekly during the treatment and recovery period and functional observations were made during the last week of treatment and recovery periods. Body weights and food consumption were determined weekly during the entire study. Ophthalmologic examinations were performed on all animals being considered for study before the first treatment and on all animals of the control and high dose groups during the last week of treatment and recovery periods. Clinical pathology (hematology, blood coagulation, clinical chemistry and thyroid hormones), urinalysis and gross pathology examinations were conducted on all animals one day after the last treatment and at the end of the recovery period. Selected organs were weighed. Full histopathological examinations were performed on the organs and tissues of the control and high dose groups (main and recovery groups). The estrous cycle of each female animal was examined on the day of necropsy.

 

The results of this study were summarized as follows:

 

Mortality: One female animal of middle dose (300 mg/kg bw/day) was found dead during the study.

 

Clinical observations: No clinical signs related to the test item were found at general daily or detailed weekly clinical observations at 100 and 1000 mg/kg bw/day. However, piloerection and activity decreased was noted in one male animals at 300 mg/kg bw/day. The functional observations did not reveal any test item influence on the animal behavior or neurological functions.

 

Body weight and body weight gain: There were no test item related changes in the mean body weight and body weight gain in the male and female animals at 100, 300 and 1000 mg/kg bw/day.

 

Food consumption: No adverse effects on the mean food consumption development were observed at 100, 300 or 1000 mg/kg bw/day in male or female animals (main and recovery groups).

 

Ophthalmoscopy: No alterations were detected in the eyes of the animals of the high dose group at the end of treatment or recovery periods.

 

Hematology and blood coagulation: There were no test item related adverse pathological changes in the investigated hematological or blood coagulation parameters in any group (main and recovery groups).

 

Clinical chemistry: Pathological test item effects were not detected upon the evaluation of the clinical chemistry parameters in male or female animals at 100, 300 and 1000 mg/kg bw/day (main and recovery groups).

 

Determination of serum levels of thyroid hormones: In male and female animals at 100, 300 and 1000 mg/kg bw/day the level of FT3 (free T3) and FT4 (free T4) thyroid hormones were normal at the end of treatment and recovery period.

 

Urine: No toxicologically changes in urinalysis parameters were seen in 4,5-dihydroxy-1,3- dimethylimidazolidin-2-one treated animals during the study.

 

Necropsy: Macroscopic alterations related to the test item were not detected at the necropsy at the termination of the treatment and at the end of the recovery period.

 

Organ weight: No treatment related alterations were found in the organ weight results (absolute weights, relative to the body weight and brain weight) in male and female animals compared to the control parameters (main and recovery groups).

 

Histopathology: There were no test item related lesions in the organs or tissues of high dose animals (no general histopathological examination was performed for low and mid dose animals).

 

Estrous cycle examination: A test item effect on the estrous cycle was not detected at any dose level.

 

Under the conditions of the present study, 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one did not cause adverse effects in male or female Han:WIST rats after 90-day consecutive oral (by gavage) administration of 100, 300 or 1000 mg/kg bw/day doses (corresponding to 236, 710 and 2360 mg/kg bw/day of the product, calculated by the content of 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one and its organic impurities in the product (42.34 %). There were no toxic changes in the examined parameters (clinical signs, body weight and body weight gain, food intake, ophthalmology, hematology, blood coagulation and clinical chemistry, serum levels of thyroid hormones, urinalysis, estrous cycle, necropsy findings, organ weights or histopathological findings). Based on the observations made in this toxicity study the No Observed Adverse Effect Level (NOAEL) was determined as follows: NOAEL: 1000 mg/kg bw/day for male and female Han: WIST rats.

 

Supporting, 14-day dose-range finding study, RL2

The purpose of this study is to obtain first information on the toxic potential of 4,5-dihydroxy- 1,3-dimethylimidazolidin-2-one in rats at three dose levels to allow a dose-setting for a 90-Day Repeated Dose Oral Toxicity Study of 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one in Rats (main study). The dose setting was based on the LD50 value of 4,5-dihydroxy-1,3-dimethylimidazolidin-2- one (> 2000 mg/kg bw/day). 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one was administered as formulations in distilled water. Doses of 0 (vehicle only), 100, 300 and 1000 (236, 710 and 2360 mg/kg bw/day of the product, calculated by the content of 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one and its organic impurities in the product (42.34 %)) were orally administered (by gavage) to four groups of Han:WIST rats consisting of five animals per group and sex at a dosing volume of 5 mL/kg in concentrations of 47.2, 141.8 and 472.4 mg/mL. A group of vehicle (distilled water) treated animals (n= 5/sex) served as a control. The suitability of the chosen vehicle for the test item was assessed on basis of preliminary test item formulation experiment. Analytical control of dosing formulations (control of concentration and homogeneity) was not performed during the course of this study. Each formulation was administered to the animals were prepared by careful weight measurement just prior to administration daily and was used up within four hours after preparation. Detailed clinical observations were performed daily after the treatment. On Day 0, animals were observed continuously for 30 minutes then 1, 2, 3 and 5 hours after the administration to estimate the peak period of effects. Body weights were recorded twice weekly. The food consumption was determined weekly to coincide with body weight measurements during the study. Clinical pathology (hematology, blood coagulation and clinical chemistry) and gross pathology examinations were conducted one day after the last treatment (on Day 14). Selected organs were weighed. The results of this study were summarized as follows:

 

Mortality

There was no mortality in control, 100, 300 or 1000 mg/kg bw/day groups.

Clinical observations

4,5-dihydroxy-1,3-dimethylimidazolidin-2-one did not induce clinical signs at 100, 300 or 1000 mg/kg bw/day in male or female animals during the two weeks treatment period. The behavior and physical condition of all animals were normal.

Body weight and body weight gain

The body weight development was undisturbed in male or female animals at 100, 300 or 1000 mg/kg bw/day during the observation period.

Food consumption

The mean daily food consumption was comparable in the control and test item treated groups 100, 300 or 1000 mg/kg bw/day, male and female.

Hematology and blood coagulation

Hematological evaluation did not reveal test item related changes in the examined parameters at 100, 300 or 1000 mg/kg bw/day (male and female).

Clinical chemistry

There were no test item related alterations in the investigated clinical chemistry parameters at 100, 300 or 1000 mg/kg bw/day (male and female).

Organ pathology

Specific macroscopic alterations of the organs or tissues or changes in the investigated organ weights related to the test item were not detected.

 

Under the conditions of the present study, 4,5-dihydroxy-1,3-dimethylimidazolidin-2- one did not cause adverse effects in male or female Han: WIST rats after consecutive 14-day oral (by gavage) administration at the doses of 100, 300 or 1000 mg/kg bw/day. Based on the observations made in this toxicity study, the following dose levels for a 90-Day Repeated Dose Oral Toxicity Study of 4,5-dihydroxy-1,3-dimethylimidazolidin-2-one in Rats (main study) are suggested:

Group 1: Vehicle control

Group 2: 100 mg/kg bw/day (corresponding to 236 mg/kg bw/day of the product)

Group 3: 300 mg/kg bw/day (corresponding to 710 mg/kg bw/day of the product)

Group 4: 1000 mg/kg bw/day (corresponding to 2360 mg/kg bw/day of the product)

 

Supporting, read-across, 90-day repeated dose toxicity, rat, RL2

IRDC (1981) reported a subchronic toxiciy oral study in rats with dimethyloldihydroxyethylene urea (CAS 1854-25-8) which is basically identical with 4.5 -Dihydroxy-1.3-dihydroxymethylimidazolid-2-one and a possible metabolic sequel product of the 4.5 -Dihydroxy-1.3-dimethylimidazolidine-2-one.

Male and female Sprague-Dawley rats were administered (gavage) doses of 1000, 3000 or 6000 mg/kg for 13 weeks. Three males of the 6000 mg/kg/day dosage level were found dead on study day 3. The cause of one of the deaths was aspiration pneumonia. The cause of death of the other animals was not determined. The males in the 6000 and 3000 mg/kg/day dosage level groups exhibited a lower mean body weight gain and had lower body weights at termination (314 +/- 18.1 g and 331 +/-27.1 g, respectively) than controls (358 +/- 31.1 g). The mean body weights of the males of the 1000 mg/kg/day dosage level and of the treated female rats were comparable to controls throughout the study. Pharmacotoxic signs noted for male and female animals in the 3000 and 6000 mg/kg/day dosage level groups included primarily yellow discoloration of the fur in the anogenital region and stool. In addition, male animals in the 6000 mg/kg/day dosage level group exhibited yellow discoloration of fur in the abdominal region as well as soft stool. One male animal in the 6000 mg/kg/day dosage level group was noted for hypoactivity, decreased grasping reflex, extremities hypothermic to touch, and ataxia on study day 3. Other signs noted among rats of various dosage level groups (or controls) were considered incidental and unrelated to the test article. No toxicologically significant organ weight changes occurred in this study. Macroscopically, one male from the 6000 mg/kg/day dosage level group was found at the post-mortem examination to have multiple yellowish linear macroscopic lesions in the right testis. Microscopically, the lesions were found to be moderate bilateral mineralization of testes. Microscopically, treatment-related mild mineralization in the heart was seen in this male and another male in the 6000 mg/kg/day dosage level group. Mineralization in the testes and heart were considered to be test article-related lesions. No other macroscopic or microscopic findings were considered to be related to the test article. The NOAEL for male animals was found to be 3000 mg/kg and the NOAEL for female animals was found to be 6000 mg/kg bw/day.

 

Supporting, read-across, 90-day repeated dose toxicity, mouse, RL2

Another IRDC report (1981) reported about the subchronic oral toxicity with dimethyloldihydroethylene urea (CAS 1854-25-8) in mice. Male and female B6C3F1 mice were administered (gavage) concentrations of 1000, 3000 or 6000 mg/kg for 13 weeks. The following results were obtained: Mortality: 1 male animal in 3000 mg/kg/day group died during the third week of treatment from a dosing accident, which initiated an abscess in the thoracic region. Body weight gain: All dosed females showed an increased weight compared to the controls; the males of the 1000 and 6000 mg/kg/day group had the same or increased weight compared to the controls. The 6000 mg/kg/day group and controls showed no microscopically visible changes (the animals of the 1000 and 3000 mg/kg/day doses were not examined). Males and females in the control (2/10 and 4/10, respectively) and 1000 mg/kg groups (8/10 males and 8/10 females) exhibited alopecia. Chronic interstitial pneumonia in the control and animals of the 6000 mg/kg/day group was seen in correlation with the positive finding of the Sendai-virus. The NOAEL for male and female mice was 6000 mg/kg bw/day.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data, the test item is not classified and labelled for specific target organ toxicity - repeated dose (STOT-RE) according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.