Registration Dossier

Administrative data

Description of key information

Two studies of reliability 1 were available.

Subacute toxicity study performed by the oral route in rat: NOAEL = 411 mg/kg bw/d based on effects observed at the highest tested dose (2057 mg/kg bw/d): mortality, effect on the digestive system (forestomach, stomach, small intestine, colon) and respiratory system.

Subchronic toxicity study (90 days) performed by the oral route in rat: NOAEL = 100 mg/kg bw/d based on effects observedat 300 and 1000 mg/kg bw/d: effect on digestive system and respiratory system.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
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
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
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
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
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.
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.


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
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
Critical effects observed:
not specified

No other information

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.

 

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-May-2016 to 31-July-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
Recognized by international guidelines as the recommended test system (e.g. EPA, FDA, OECD and EC).
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females nulliparous and non-pregnant: [yes]
- Age at study initiation: Approximately 6 weeks.
- Weight at study initiation: no data
- Fasting period before study: no
- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom). During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: At least 5 days before the start of treatment under laboratory conditions.

DETAILS OF FOOD AND WATER QUALITY:
Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24°C
- Humidity (%): 40-70%
- Air changes (per hr): at least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): a 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: 11 May 2016 To: 4 November 2016
Route of administration:
oral: gavage
Details on route of administration:
Oral gavage, using a plastic feeding tube.
Formulations were placed on a magnetic stirrer during dosing. A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures.
Vehicle:
water
Remarks:
Elix, Millipore S.A.S., Molsheim, France
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Method of formulation: Formulations (w/w) were prepared weekly, and were homogenized to visually acceptable levels. Adjustment was made for specific gravity of the test item (1.1086). A correction was made for purity/composition of the test item. A correction factor of 1.39 was used.
Storage conditions: In the refrigerator in the dark.

DIET PREPARATION
not apllicable

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch and on information from the Sponsor
- Concentration in vehicle: 0, 100, 300 and 1000 mg/kg
- Amount of vehicle (if gavage): 5 mL/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:
Analyses were conducted during the treatment phase, according to a validated method. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations, in Weeks 1, 6 and 13).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration for solutions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily, 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels are based on results of a 14-day oral range finding study with Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid by daily gavage in the rat
Dose levels in the range- finding study: 500 and 1000 mg/kg body weight. No clear peak effect of occurrence of clinical signs was observed in the range finding study.
Positive control:
Not required
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: Weekly. In addition some animals were weighed manually in order to monitor the health status of the animals.

FOOD CONSUMPTION:
- Food consumption : Yes
- Time schedule: Weekly.

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 : Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes
Following instillation of tropicamide solution (Tropicol 5 mg/ml solution, THEA Pharma, Wetteren, Belgium), both eyes will be examined by means of an ophthalmoscope (Beta 200S):
at pretest : All animals
at Week 13 : Group 1 and 4 animals
Since no treatment-related ophthalmologic findings were noted in Week 13, the eyes of the rats of Groups 2 and 3 were not examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the 90 day exposure
- Anaesthetic used for blood collection: Yes (isoflurane (Abbott B.V., Hoofddorp, The Netherlands))
- Animals fasted: Yes
- How many animals: all rats excluded those that died before the end
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the 90 day exposure
- Animals fasted: Yes
- How many animals: all rats excluded those that died before the end
- Parameters checked in table 1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During Week 12-13 of treatment, functional observation tests were performed on the first five animals/sex for Group 1-3 ( 0, 100, 300 mg/kg) and on the four surviving animals/sex for Group 4 (1000 mg/kg).
- Battery of functions tested:
• hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
• fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
• locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.

IMMUNOLOGY: No

OTHER:
Mortality / Viability: Yes
- Time schedule for examinations: At least twice daily.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see below)

HISTOPATHOLOGY: Yes (see below)
Other examinations:
no other examinations
Statistics:
The following statistical methods were used to analyze the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data.
• The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Rales were occasionally observed in 300 mg/kg and 1000 mg/kg male and females (most frequent in 1000 mg/kg animals), as well as labored respiration in 1000 mg/kg males. Deep respiration and quick breathing (males only), gasping, piloerection, hunched posture, squeaking and abdominal swelling/distention were also observed in 1000 mg/kg males and females, although these findings were less frequent. These breathing related ailments were observed in all Group 4 animals to varying degrees. No findings were noted during the arena observations in this study.
Salivation was seen after dosing across all treatment groups. Its severity increased in a dose-dependent manner with 1000 mg/kg animals showing the most robust levels of salivation. This effect was considered test item related, however not adverse, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. shortly after dosing). This sign was considered to be a physiological response related to the test item rather than a sign of systemic toxicity.
Any other clinical signs noted during the dosing period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
At 1000 mg/kg, a total of 13 out of 20 animals were sacrificed in extremis or found dead. One 1000 mg/kg female (no. 71) was found dead on Day 46. A further 6 males and 6 females at 1000 mg/kg (no. 33, 35-38, 40 and 75-80) were sacrificed in extremis between study Day 4 and 83. All these animals displayed respiratory problems (rales, quick breathing, gasping) in the days leading up to sacrifice.
No unscheduled mortality occurred at 100 and 300 mg/kg.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights and percentage body weight gain were lower in 1000 mg/kg males throughout the study when compared to vehicle-controls, with statistical significance being achieved on most occasions.
Bodyweight and bodyweight gain in females remained similar across groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption after correction for body weight remained similar to the controls over the study period. Slightly reduced absolute food consumption in 1000 mg/kg males was caused by the lower bodyweights and therefore not considered to be toxicologically relevant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmology findings that were considered to be related to treatment were noted.
The nature and incidence of ophthalmology findings noted during pretest and in Week 13 was similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg, females showed a statistically significant increase in platelet levels when compared to controls.
Any other statistically significant changes in haematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg, higher cholesterol level was recorded for males and females (not statistically significant in females).
Other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was considered unaffected by treatment.
A statistically significant increase in hindlimb grip strength in 300 mg/kg males occurred in a non-dose dependent fashion, and was therefore considered unrelated to treatment.
Motor activity was similar between treated and control males, however there was a small albeit statistically insignificant decrease in 1000 mg/kg females. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
All minor organ weight differences observed, including those that reached statistical significance, were considered incidental and not toxicologically relevant.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related macroscopic findings were noted in 1000 mg/kg animals that were subjected to unscheduled sacrifice (12 animals), that died spontaneously (1 animal) and that were sacrificed at the end of the dosing period (7 animals) in one or more of the following tissues: larynx, trachea, lungs, stomach, intestines, spleen, thymus and mesenteric lymph nodes. These findings consisted of the following:
Trachea
• Foamy content in 2 male and one female 1000 mg/kg animals (unscheduled sacrifice).
Lungs
• Swollen in 3 male and 4 female 1000 mg/kg animals (unscheduled sacrifice).
Stomach
• Distended with gas in 6 male and 6 female 1000 mg/kg animals (unscheduled sacrifice).
• Reddish focus/foci in 5 male and 6 female 1000 mg/kg animals that were subjected to unscheduled sacrifice, and in 3 males and 1 female of this group that were sacrificed at the end of the treatment dosing period.
• Thickened in 2 male and 3 female 1000 mg/kg animals (unscheduled sacrifice).
• Irregular surface in 2 male and 1 female 1000 mg/kg animals (unscheduled sacrifice).
• Gelatinous content in 1 male and 1 female 1000 mg/kg animal (unscheduled sacrifice).
Intestines (small and large)
• Distended with gas in 4 male and 6 female 1000 mg/kg animals (unscheduled sacrifice).
Spleen
• Reduced in size in 2 male and 2 female 1000 mg/kg animals (unscheduled sacrifice).
Thymus
• Reduced in size in 2 male and 1 female 1000 mg/kg animals (unscheduled sacrifice).
Mesenteric lymph node
• Reduced in size in 3 male and 1 female 1000 mg/kg animals (unscheduled sacrifice).
Furthermore, 2 male and 2 female 1000 mg/kg animals that were subjected to unscheduled sacrifice were considered emaciated.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings after treatment were noted in several animals of the 1000 mg/kg group that were subjected to unscheduled sacrifice, and in single animals given 300 mg/kg (scheduled sacrifice) in one or more of the following organs: stomach, duodenum, larynx, trachea, lung, thymus, spleen and mandibular lymph nodes. Test item-related microscopic findings in the 300 and 1000 mg/kg treated groups are combined in the following description, and comprised:
Stomach
• Focal/multifocal mucosal hemorrhages, mainly but not exclusively found in the mucosa of the glandular stomach were noted in 1 control male (of minimal severity) and in 8 males and 6 females of the 1000 mg/kg group. The severity of the hemorrhages in the 1000 mg/kg treated group ranged from minimal to moderate.
• Minimal to slight, focal/multifocal erosion/ulceration was observed in the forestomach, at the limiting ridge or in the glandular stomach in 3 males and 1 female of the 1000 mg/kg group.
Duodenum
• Minimal to slight mucosal hemorrhages (glandular part) were noted in one 300 mg/kg male and in 3 males and 1 female 1000 mg/kg animal.
• A minimal, focal erosion/ulceration was observed in a single female of the 1000 mg/kg group.
Trachea
• Minimal to slight inflammatory infiltrate present in the trachea of a single 300 mg/kg male and in 5 females treated at 1000 mg/kg.
• Epithelial necrosis observed in a single male treated at 300 mg/kg and in a single male and 4 females at 1000 mg/kg. The necrosis was graded minimal in the male treated at 300 mg/kg, while it was moderate to marked in the animals treated at 1000 mg/kg.
• Exudate was found to be present within the lumen of the trachea in a single male and 3 females given at 1000 mg/kg.
• Within the exudate and inflammatory debris in the tracheal lumen birefringent material was observed in 2 females at 1000 mg/kg.
Larynx
• Moderate necrotizing laryngitis (acute) was present in the larynx of 2 males and 3 females treated at 1000 mg/kg. Marked epithelial necrosis (without clear inflammation) was observed in a single male treated at 1000 mg/kg.
• Slight hyperplasia, squamous cell was observed in a single male at 1000 mg/kg, while minimal hyperplasia, epithelial was observed in a single female treated at 300 mg/kg.
• Minimal to slight metaplasia, squamous cell was observed in a single male and 3 females treated at 1000 mg/kg.
• Within the inflammatory debris in the laryngeal lumen, birefringent material was observed in 2 females at 1000 mg/kg.
Lung (Bronchus)
• Minimal or slight necrotizing bronchitis (acute) was present in the larynx of a single male and a single female treated at 1000 mg/kg. Minimal epithelial necrosis, bronchus (without clear inflammation) was observed in a single female and sloughing of bronchus epithelium in a single male treated at 1000 mg/kg.
Thymus
• Lymphocytolysis was observed in 3 males of the 300 mg/kg group, and in 7 males and 6 females of the group given 1000 mg/kg.
• In 4 males and 2 females of the 1000 mg/kg group, lymphocytolysis progressed to clear atrophy of the thymus (ranging from slight to marked).
Mandibular lymph nodes
 Lymphoid atrophy was recorded in 3 males of the 1000 mg/kg group (the severity ranged from slight to moderate).
Spleen
• Slight to moderate atrophy of the red pulp was observed in 3 males and 2 females treated at 1000 mg/kg.
The remainder of the recorded microscopic findings were considered to be incidental findings or within the range of background pathology encountered in rats of this age and strain.


Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Sperm analysis
No toxicologically relevant changes were noted in sperm analysis parameters, including sperm counts, motility or morphology.
Any statistically significant changes in sperm analysis parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Details on results:
Discussion / conclusion
Formulation analyses confirmed that formulations of test item in Water (Elix) were prepared accurately and homogenously.
At 1000 mg/kg, 6/10 males and 7/10 females did not survive the scheduled treatment duration. Among these animals, the clinical findings pointed to respiratory problems and reduced animal health. Body weight loss was noted in several individual animals prior to death.
Clinical signs as rales and labored respiration were noted predominantly in 1000 mg/kg males and less frequently among the animals at 300 mg/kg and females at 1000 mg/kg. Deep respiration and quick breathing (males only), gasping, piloerection, hunched posture, squeaking and abdominal swelling/distention were also observed in 1000 mg/kg males and females, although these findings were less frequent. These findings were accompanied with mean reduced body weight gain in males at 1000 mg/kg.
Histopathological examination of all animals revealed test item-related findings in the stomach, duodenum, larynx, trachea, lungs, spleen, thymus and mesenteric lymph nodes, predominantly at 1000 mg/kg.
In the stomach of several animals that were given 1000 mg/kg/day, signs of irritation of the mucosal surface were observed: those included minimal to slight erosions/ulcerations in 3 males and a single female, and minimal to moderate mucosal hemorrhages in 8 males and 6 females of the 1000 mg/kg/day treated group. Also within the duodenal mucosa erosions/ulcerations were found to be present in a single female and hemorrhages in 3 males treated at 1000 mg/kg/day. As a reaction to the irritant properties of the test-item, squamous cell hyperplasia developed in the forestomach of 2 females given 1000 mg/kg/day.
Within the upper respiratory tract (larynx, trachea, bronchus) also signs of irritation of the lining mucosa were noted and comprised necrosis, necrotizing laryngitis, necrotizing bronchitis, epithelial sloughing and exudate. Those changes are believed to cause the difficulty in breathing and rales noted clinically. In the few animals that showed lesions within the trachea microscopically, a foamy content was found to be present grossly.
The changes within the stomach in combination with the changes observed within the upper respiratory system in several animals strongly suggest that regurgitation of the content of the stomach (with the test-item) could be responsible for the changes in the respiratory system.
It is possible that the physical characteristics of the test item determined the structures affected after regurgitation, such as larynx, trachea and bronchus. Except for the bronchus tree, the other histological structures of lung were not affected. Although lungs were considered swollen in several animals given 1000 mg/kg/day, no signs of atelectasis or emphysema, or other pathologic processes were observed that could explain this gross change. It is possible that lungs were swollen because more air remained within the lung alveoli as a result of (partial) obstruction of the upper respiratory tract (note there was foamy material found within the trachea of few animals). Although the test-item consisted of a clear fluid, birefringent crystalline material was found to be present within the exudate and/or inflammatory debris within the laryngeal or tracheal lumen. Presence of such crystalline material suggests the test-item possibly crystalized in the acid environment of the stomach.
Secondary reactive changes, consisting of squamous cell hyperplasia, epithelial hyperplasia and/or squamous metaplasia were observed in the larynx of few animals.
The increased incidence and severity of lymphocytolysis within the thymus in several high dose animals leading to clear thymic atrophy, the lymphocytolysis observed in the mesenteric lymph node, and lymphoid atrophy in the mandibular lymph nodes in few of those animals, are considered to be the result of severe stress. A minimal degree of lymphocytolysis can be noted in control animals and is considered a normal background finding.
Atrophy of the red pulp of the spleen was noted in few males and females of the 1000 mg/kg/day treated group. This atrophy resulted in reduced size and weight of this organ in these animals.
Hematological and clinical biochemistry analysis showed increased platelet and cholesterol levels in 1000 mg/kg females and in 1000 mg/kg males and females, respectively. These finding are suggested to be test item related but not considered adverse in the absence of related findings.
Histopathologic examination of males at 300 mg/kg revealed toxicologically relevant findings, such as slight focal necrosis in the trachea and multifocal hemorrhages within the duodenal mucosa in two different single males. Since these findings were noted in only single animals and with minor severity and without corroborative findings in animal health, no adversity for these animals was considered.
There were no relevant histopathological findings in animals given 100 mg/kg/day.
No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. functional observations, ophthalmoscopy, food consumption, sperm parameters or organ weights).

In conclusion, adverse test item-related alterations were noted at histopathological examination of the stomach, duodenum and upper respiratory tract mainly at 300 and 1000 mg/kg. The lesions in the stomach and duodenum point to irritating properties of the compound and were mainly present in males and females treated at 1000 mg/kg. Possibly because of irritation of the gastric mucosa, the animals regurgitated, causing test- item to enter into the larynx, trachea and bronchi where it caused quite severe changes to the mucosa.
From the results presented in this report a definitive and conservative No Observed Adverse Effect Level (NOAEL) for Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid of 100 mg/kg was established based on digestive and respiratory findings.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: digestive and respiratory findings
Critical effects observed:
no
Conclusions:
Adverse test item-related alterations were noted at histopathological examination of the stomach, duodenum and upper respiratory tract mainly at 300 and 1000 mg/kg. The lesions in the stomach and duodenum point to irritating properties of the compound and were mainly present in males and females treated at 1000 mg/kg. Possibly because of irritation of the gastric mucosa, the animals regurgitated, causing test- item to enter into the larynx, trachea and bronchi where it caused quite severe changes to the mucosa.
From the results presented in this report a definitive and conservative No Observed Adverse Effect Level (NOAEL) for Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid of 100 mg/kg was established based on digestive and respiratory findings.
Executive summary:

Wistar rats were administered Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid for 13 weeks by daily oral gavage at dose levels of 0, 100, 300 and 1000 mg/kg according to OECD Guideline n° 408.

The test item, formulated in Water (Elix), was administered daily for at least 90 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females.

Chemical analyses of formulation preparations were conducted during the study to assess accuracy and homogeneity.

The following parameters were evaluated: mortality and clinical signs daily; functional observation tests in Week 12-13; body weight and food consumption weekly; ophthalmoscopy at pretest and in Week 13; clinical pathology and macroscopy at termination; sperm parameters (count, mobility, morphology) at termination; organ weights and histopathology on a selection of tissues.

Formulation analyses confirmed that formulations of test item in Water (Elix) were prepared accurately and homogenously.

At 1000 mg/kg, 6/10 males and 7/10 females did not survive the scheduled treatment duration. Among these animals, the clinical findings pointed to respiratory problems and reduced animal health. Body weight loss was noted in several individual animals prior to death.

Clinical signs as rales and labored respiration were noted predominantly in 1000 mg/kg males and less frequently among the animals at 300 mg/kg and females at 1000 mg/kg. Deep respiration and quick breathing (males only), gasping, piloerection, hunched posture, squeaking and abdominal swelling/distention were also observed in 1000 mg/kg males and females, although these findings were less frequent. These findings were accompanied with mean reduced body weight gain in males at 1000 mg/kg.

Histopathological examination of all animals revealed test item-related findings in the stomach, duodenum, larynx, trachea, lungs, spleen, thymus and mesenteric lymph nodes, predominantly at 1000 mg/kg.

In the stomach of several animals that were given 1000 mg/kg/day, signs of irritation of the mucosal surface were observed: those included minimal to slight erosions/ulcerations in 3 males and a single female, and minimal to moderate mucosal hemorrhages in 8 males and 6 females of the 1000 mg/kg/day treated group. Also within the duodenal mucosa erosions/ulcerations were found to be present in a single female and hemorrhages in 3 males treated at 1000 mg/kg/day. As a reaction to the irritant properties of the test-item, squamous cell hyperplasia developed in the forestomach of 2 females given 1000 mg/kg/day.

Within the upper respiratory tract (larynx, trachea, bronchus) also signs of irritation of the lining mucosa were noted and comprised necrosis, necrotizing laryngitis, necrotizing bronchitis, epithelial sloughing and exudate. Those changes are believed to cause the difficulty in breathing and rales noted clinically. In the few animals that showed lesions within the trachea microscopically, a foamy content was found to be present grossly.

The changes within the stomach in combination with the changes observed within the upper respiratory system in several animals strongly suggest that regurgitation of the content of the stomach (with the test-item) could be responsible for the changes in the respiratory system.

It is possible that the physical characteristics of the test item determined the structures affected after regurgitation, such as larynx, trachea and bronchus. Except for the bronchus tree, the other histological structures of lung were not affected. Although lungs were considered swollen in several animals given 1000 mg/kg/day, no signs of atelectasis or emphysema, or other pathologic processes were observed that could explain this gross change. It is possible that lungs were swollen because more air remained within the lung alveoli as a result of (partial) obstruction of the upper respiratory tract (note there was foamy material found within the trachea of few animals). Although the test-item consisted of a clear fluid, birefringent crystalline material was found to be present within the exudate and/or inflammatory debris within the laryngeal or tracheal lumen. Presence of such crystalline material suggests the test-item possibly crystalized in the acid environment of the stomach.

Secondary reactive changes, consisting of squamous cell hyperplasia, epithelial hyperplasia and/or squamous metaplasia were observed in the larynx of few animals.

The increased incidence and severity of lymphocytolysis within the thymus in several high dose animals leading to clear thymic atrophy, the lymphocytolysis observed in the mesenteric lymph node, and lymphoid atrophy in the mandibular lymph nodes in few of those animals, are considered to be the result of severe stress. A minimal degree of lymphocytolysis can be noted in control animals and is considered a normal background finding.

Atrophy of the red pulp of the spleen was noted in few males and females of the 1000 mg/kg/day treated group. This atrophy resulted in reduced size and weight of this organ in these animals.

Hematological and clinical biochemistry analysis showed increased platelet and cholesterol levels in 1000 mg/kg females and in 1000 mg/kg males and females, respectively. These finding are suggested to be test item related but not considered adverse in the absence of related findings.

Histopathologic examination of males at 300 mg/kg revealed toxicologically relevant findings, such as slight focal necrosis in the trachea and multifocal hemorrhages within the duodenal mucosa in two different single males. Since these findings were noted in only single animals and with minor severity and without corroborative findings in animal health, no adversity for these animals was considered.

There were no relevant histopathological findings in animals given 100 mg/kg/day.

No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. functional observations, ophthalmoscopy, food consumption, sperm parameters or organ weights).

 

In conclusion, adverse test item-related alterations were noted at histopathological examination of the stomach, duodenum and upper respiratory tract mainly at 300 and 1000 mg/kg. The lesions in the stomach and duodenum point to irritating properties of the compound and were mainly present in males and females treated at 1000 mg/kg. Possibly because of irritation of the gastric mucosa, the animals regurgitated, causing test- item to enter into the larynx, trachea and bronchi where it caused quite severe changes to the mucosa.

From the results presented in this report a definitive and conservative No Observed Adverse Effect Level (NOAEL) for Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid of 100 mg/kg was established based on digestive and respiratory findings.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The study was performed in compliance with the GLP and similarly to the OECD 408 guideline (Klimish score = 1)

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

Reaction mass of methacrylic acid and N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide was assessed for repeated dose toxicity in two studies of reliability 1 (one subacute oral repeated dose toxicity study and one subchronic oral repeated dose toxicity study).

In a GLP-compliant subacute oral repeated dose toxicity study performed 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. Althought believed to be related to the test substance by the authors, these changes in testis in only one animal is likely incidental since it was not observed in any animals in the reproduction/developmental toxicity screening test (F. Spezia, 2013). An increase in eosinophilic bodies in the kidneys of the 1000 mg/kg bw/d males was also noted in the OECD 407 study and 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.

In a GLP-compliant subchronic oral repeated dose toxicity study performed to the OECD 408 guideline, Reaction mass of methacrylic acid and N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide diluted in water was administered by gavage to SPF-bred Wistar rats for 13 weeks at doses of 0, 100, 300 and 1000 mg/kg.

At 1000 mg/kg, 6/10 males and 7/10 females did not survive the scheduled treatment duration. Among these animals, the clinical findings pointed to respiratory problems and reduced animal health. Body weight loss was noted in several individual animals prior to death.

Clinical signs as rales and labored respiration were noted predominantly in 1000 mg/kg males and less frequently among the animals at 300 mg/kg and females at 1000 mg/kg. Deep respiration and quick breathing (males only), gasping, piloerection, hunched posture, squeaking and abdominal swelling/distention were also observed in 1000 mg/kg males and females, although these findings were less frequent. These findings were accompanied with mean reduced body weight gain in males at 1000 mg/kg.

Histopathological examination of all animals revealed test item-related findings in the stomach, duodenum, larynx, trachea, lungs, spleen, thymus and mesenteric lymph nodes, predominantly at 1000 mg/kg.

In the stomach of several animals that were given 1000 mg/kg/day, signs of irritation of the mucosal surface were observed: those included minimal to slight erosions/ulcerations in 3 males and a single female, and minimal to moderate mucosal hemorrhages in 8 males and 6 females of the 1000 mg/kg/day treated group. Also within the duodenal mucosa erosions/ulcerations were found to be present in a single female and hemorrhages in 3 males treated at 1000 mg/kg/day. As a reaction to the irritant properties of the test-item, squamous cell hyperplasia developed in the forestomach of 2 females given 1000 mg/kg/day.

Within the upper respiratory tract (larynx, trachea, bronchus) also signs of irritation of the lining mucosa were noted and comprised necrosis, necrotizing laryngitis, necrotizing bronchitis, epithelial sloughing and exudate. Those changes are believed to cause the difficulty in breathing and rales noted clinically. In the few animals that showed lesions within the trachea microscopically, a foamy content was found to be present grossly.

The changes within the stomach in combination with the changes observed within the upper respiratory system in several animals strongly suggest that regurgitation of the content of the stomach (with the test-item) could be responsible for the changes in the respiratory system.

It is possible that the physical characteristics of the test item determined the structures affected after regurgitation, such as larynx, trachea and bronchus. Except for the bronchus tree, the other histological structures of lung were not affected. Although lungs were considered swollen in several animals given 1000 mg/kg/day, no signs of atelectasis or emphysema, or other pathologic processes were observed that could explain this gross change. It is possible that lungs were swollen because more air remained within the lung alveoli as a result of (partial) obstruction of the upper respiratory tract (note there was foamy material found within the trachea of few animals). Although the test-item consisted of a clear fluid, birefringent crystalline material was found to be present within the exudate and/or inflammatory debris within the laryngeal or tracheal lumen. Presence of such crystalline material suggests the test-item possibly crystalized in the acid environment of the stomach.

Secondary reactive changes, consisting of squamous cell hyperplasia, epithelial hyperplasia and/or squamous metaplasia were observed in the larynx of few animals.

The increased incidence and severity of lymphocytolysis within the thymus in several high dose animals leading to clear thymic atrophy, the lymphocytolysis observed in the mesenteric lymph node, and lymphoid atrophy in the mandibular lymph nodes in few of those animals, are considered to be the result of severe stress. A minimal degree of lymphocytolysis can be noted in control animals and is considered a normal background finding.

Atrophy of the red pulp of the spleen was noted in few males and females of the 1000 mg/kg/day treated group. This atrophy resulted in reduced size and weight of this organ in these animals.

Hematological and clinical biochemistry analysis showed increased platelet and cholesterol levels in 1000 mg/kg females and in 1000 mg/kg males and females, respectively. These finding are suggested to be test item related but not considered adverse in the absence of related findings.

Histopathologic examination of males at 300 mg/kg revealed toxicologically relevant findings, such as slight focal necrosis in the trachea and multifocal hemorrhages within the duodenal mucosa in two different single males. Since these findings were noted in only single animals and with minor severity and without corroborative findings in animal health, no adversity for these animals was considered.

There were no relevant histopathological findings in animals given 100 mg/kg/day.

No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. functional observations, ophthalmoscopy, food consumption, sperm parameters or organ weights).

In conclusion, adverse test item-related alterations were noted at histopathological examination of the stomach, duodenum and upper respiratory tract mainly at 300 and 1000 mg/kg. The lesions in the stomach and duodenum point to irritating properties of the compound and were mainly present in males and females treated at 1000 mg/kg. Possibly because of irritation of the gastric mucosa, the animals regurgitated, causing test- item to enter into the larynx, trachea and bronchi where it caused quite severe changes to the mucosa.

From the results No Observed Adverse Effect Level (NOAEL) for Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid of 100 mg/kg was established based on digestive and respiratory findings.

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.


Justification for classification or non-classification

Harmonized classification:

No harmonized classification is available according to the Regulation (EC) No 1272/2008 including ATP3.

Self classification:

Reaction mass of methacrylic acid and N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide is not classified for repeated dose toxicity according to the Regulation (EC) 1272/2008 (CLP) and to the Directive 67/548/EEC as the LOAEL (300 mg/kg bw/d) obtained in the subchronic toxicity study is higher than the concentration limit for classification.