[No public or meaningful name is available]

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in compliance with the GLP and similarly to the OECD 407 guideline.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid

IUPAC Name:

Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid

Test material form:

other: brown liquid

Details on test material:

- Name of test material (as cited in study report): N-[2-(2-Oxo-1-imidazolidinyl)ethyl] methacrylamide and methacrylic acid combination, other name: WAM II
- Stability under test conditions: The current committee verified that there was stability during the administration period while the test substance stability test was conducted
- Storage condition of test material: Must be stored in a cool environment

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Co. LTD
- Age at study initiation: 5-week old
- Weight at study initiation: males: 147.9-166.4g; females: 122.4-138.8g
- Fasting period before study: not applicable
- Housing: individually housed in a stainless steel metal wire cage (Tokiwa Scientific Equipment Corporation, Ltd., 165W x 300D x 150 H mm) in a room where a barrier system was created (with controlled environmental conditions)
- Diet (e.g. ad libitum): MF solid feed (Oriental Processing Manufacturing Corporation)
- Water (e.g. ad libitum): filtered drinking water from the Hida City tap water were provided
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2°C
- Humidity (%): 55 ±10%
- Air changes (per hr): 10 - 15 times/hour
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: To: no data

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Since the actual purity of the test substance was 48.61 % w/w (based on the concentration of N-[2-(2-Oxo-1-imidazolidinyl)ethyl] methacrylamide), agitation was conducted by adding distilled water (Takamatsu Pharmaceutical Corporation) to the properly weighing test substance and the density was calculated adjusting it to 10.0 % w/v. Both the 2.0 and 0.4 % w/v were diluted from the 10.0 % w/v preparation using distilled water. These types of preparation were conducted once a week.

DIET PREPARATION
not applicable

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 3 dosing preparations: 10% , 2% and 0.4% w/v
- Amount of vehicle (if gavage): 10mL/kg bw
- Lot/batch no. (if required): no data
- Purity: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity test were conducted on the test substance solutions by the Chemical Inspection and Testing Institute and formulations were confirmed to be stable and homogeneous for seven days in the range of 0.1 - 10 % w/v.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
40; 200 and 1000 mg/kg bw/d
Basis:
other: based on N-[2-(2-Oxo-1-imidazolidinyl)ethyl] methacrylamide content

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The range-finding study was conducted using 3 separate doses: 50, 250 and 1000 mg/kg bw/d, during a 14-day period. As a result, lesions were observed in the 1000 mg/kg bw/d group during the histopathological examination. Therefore, the revised 3 doses for the main study were set to <200 and 40mg/kg bw/d with 1000mg/kg bw/d being the highest dose. Furthermore, concurrent control recovery groups were set up for the vehicle control group and 1000mg/kg bw/d group.
- Rationale for animal assignment (if not random): no data
- Rationale for selecting satellite groups: concurrent control recovery groups were set up for the vehicle control group and 1000 mg/kg bw/d group.
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): no data

Positive control:

not required

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day

BODY WEIGHT: Yes
- Time schedule for examinations: day -2 prior to the injections, on day 1, 3, 5, 8, 10, 12, 15, 17, 19, 22, 24, 26 and 28. During the recovery period on day 1, 3, 5, 8, 10, 12 and 14. In addition, one weight measure was taken prior to the autopsy so as to have the relative organ weight.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. The food consumption was measured twice a week during the exposure and the recovery periods.

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the 28 day exposure and after the end of the recovery period.
- Anaesthetic used for blood collection: Yes (identity not precised)
- Animals fasted: Yes
- How many animals: all rats excluded those that died before the end
- Parameters checked in table 7.5.1/1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the 28 day exposure and after the end of the recovery period.
- Animals fasted: Yes
- How many animals: all rats excluded those that died before the end
- Parameters checked in table 7.5.1/1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the 28 day exposure and after the end of the recovery period.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table 7.5.1/1 were examined.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 7.5.1/2)
HISTOPATHOLOGY: Yes (see table 7.5.1/2)

Other examinations:

no other examinations

Statistics:

With regards to the blood chemistry test, urine volume, hematology test, and the absolute organ weight of all subjects (excluding the Deceased Rats, amount of feed, and weight) the Bartlett method is utilized to officially check the equal dispersion homogeneity of variance. When it was significant at 5%, an analysis for monotonicity trend was conducted. When the analysis significance has been verified, both groups’ example numbers are equal by the Dunnett method between the control group and each treated group. If it has not been verified then it is determined using the Scheffe method.

When the homogeneity of variance cannot be verified, utilize the Kruskal-Wallis test, both groups’ parameters are equal by the non-parametric Dunnett method between the control group and each treated group. If it has not been verified then it is determined using the non-parametric Scheffe method.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

2/10 males and 1/10 female died in the highest dose group. Decrease in salivation, abnormal breathing sounds, decrease respiratory rate at the top dose.

Mortality:

mortality observed, treatment-related

Description (incidence):

2/10 males and 1/10 female died in the highest dose group. Decrease in salivation, abnormal breathing sounds, decrease respiratory rate at the top dose.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

On day 5 and 8 there was a slight decrease in weight observed in males within the 1000 mg/kg bw group which was found to be related to the death of two subjects which exhibited a decrease in weight. There were no changes in females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

On day 4 there was a decrease in consumption observed in the 1000 mg/kg group which was found to be related to the death of two subjects who had exhibited a decrease in consumption. There were no changes in females.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Significant increase in the eosinophile percentage in white blood cell count of the 1000 mg/kg female group. During the recovery period, significant increase in the mean red cell hemoglobin concentration in the top dose female group. No changes in males.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Significant decrease in Chloride in the top dose female group during the admistration period. During the recovery period, significant decrease in serum glucose in the top dose female group. No changes in males.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Significant increase in the urine volume in the females during the recovery period.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Significant increase in relative kidney weight in the top dose female group. No changes in males. No changes in either males or females at the end of the recovery period.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

See details below

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See details below

Histopathological findings: neoplastic:

no effects observed

Details on results:

GROSS PATHOLOGY:
In the 1000 mg/kg male group the following was observed: whitening of the testes (1/4 animals) and elevation of mucosa on the forestomach (limiting lidge) (3/4); within the 200 mg/kg group the following was observed: adherence of liver and kidney (1/6); hair loss was observed in the cervical region in the 40 mg/kg group (1/6). In the 1000 mg/kg female group, animals experienced an elevation of mucosa on the forestomach (limiting lidge) (6/6). At the end of the recovery period, there were no changes in the males whereas blackish mucosa regions were observed in the glandular stomach (1/6) in the female control group. In animals who died during the administration period, dark reddish lesions on the lungs (1/2), gas buildup in the stomach (1/2), gas buildup in the small intestine (2/2) and gas buildup in the colon (1/2) were observed in the 1000 mg/kg male groupwhereas map-like dark redness on the lungs (1/1) and stomach buildup in the colon (1/1) were observed in the 1000 mg/kg female group.

HISTOPATHOLOGY: NON-NEOPLASTIC
Increase in eosinophilic bodies in the kidney (+: 1/4), hyperkeratosis of the forestomach (+: 3/4), decrease in sperm formation (+++:1/4) and sperm granuloma (+; ¼) were observed in the 1000 mg/kg male group; a slight necrosis of the adhered region in macroscopic findings (1/1) with calcium deposition in the kidney, and a slight necrosis of the adhered region in macroscopic findings (1/6) in the liver were observed in the 200 mg/kg male group; basophilic change of tubular epithelium with cell reaction was observed in the kidney (+; 1/6) in the control male group. Hyperkeratosis was observed in the forestomach in the 1000 mg/kg female group (+; 6/6). At the end of the recovery period, there were no changes in males whereas necrosis of the mucous membrane of the glandular stomach was observed in the control female group (+; 1/6). In animals who died during the administration period, atrophy of the Spleen (++;1/2), congestion in the lung (+;1/2, ++;1/2), lung edema (++;1/2) and hyperkeratosis of the forestomach (+; 1/1) were observed in the 1000 mg/kg male group while congestion in the lung (+;1/2, ++;1/2), lung edema (++; 1/2), and hyperkeratosis of the forestomach (+; 1/1) were observed in the 1000 mg/kg female group.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

other: dose adjusted for content of the main constituent N-[2-(2-oxo-1-imidazolidinyl)ethyl]methacrylamide (48.61%)

Sex:

male/female

Basis for effect level:

other: Mortality, effect on the digestive system (forestomach, stomach, small intestine, colon), respiratory system and testes

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

other: Dose for the substance as registered (including impurities and residual water necessary for the stability)

Sex:

male/female

Basis for effect level:

other: Mortality, effect on the digestive system (forestomach, stomach, small intestine, colon), respiratory system and testes

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

No other information

Applicant's summary and conclusion

Conclusions:

Under the test conditions, Reaction mass of methacrylic acid and N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide induced mortality in the highest tested dose groups (1000 mg/kg bw/d), alterations in the respiratory and digestive system and also in testes in males. The NOAEL was determined to be of 200 mg/kg bw/d when expressed as the amount of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and as 411 mg/kg bw/d when considering the substance as registered (including impurities and residual water necessary for stability).

Executive summary:

In a subacute oral repeated dose toxicity study performed in compliance with the GLP and similarly to the OECD 407 guideline, Reaction mass of methacrylic acid and N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide diluted in water was administered by gavage to Sprague Dawley rats (10 animals/sex/dose) at doses of 40; 200 and 1000 mg/kg bw/d (based on N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide content i.e. 82, 411 and 2057 mg/kg bw/d in terms of registered substance).The exposure period was of 28 days, and a recovery period of 14 days was performed for the vehicle control and the highest dose groups.

Mortality was observed in the highest tested dose groups where 2/10 males died on day 8 and 1/10 died on day 14. During an autopsy to determine the cause of death, the lungs were either darkened in a reddish color or exhibited a slight darkening, and the stomach and/or the intestines were filled with gas. The histopathological examination revealed hyperkeratinosis of the forestomach, decrease in the size of the spleen, and a congestion and edema in the lung, thus, related to alterations in the respiratory system and digestive system leading to death. In the surviving animals, the test item induced an increased thickness of the mucous membrane of the forestomach, correlated histopathologically with hyperkeratosis of the forestomach, in both males and females given 1000 mg/kg bw/day. One male of the 1000 mg/kg bw/d group exhibited whitening of the left testis associated by microscopic observation of decrease in sperm formation, and sperm granuloma. Although noted in only one animal, the testes finding is believed to be related to the test substance. In addition, an increase in eosinophilic bodies in the kidneys of the 1000 mg/kg bw/d males was believed to be related to the test substance. During the recovery period, there was no effect related to the test item. 

Based on the results of this study, 411 mg/kg bw/d of test item was established as the no-observed-adverse effect-level (NOAEL) in males and females when considering the substance as registered.

Therefore, the registered substance is not classified for repeated dose toxicity according to the classification criteria of the Regulation (EC) 1272/2008 (CLP) and of the Directive 67/548/EEC. This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 407.