2-Oxazolidinone, 3-ethenyl-5-methyl-

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Nov 2018 - 19 Mar 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see: Principles of method if other than guideline

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

yes

Remarks:

see: Principles of method if other than guideline

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: JMAFF No. 12-Nousan-8147, 2-1-9

Deviations:

yes

Remarks:

see: Principles of method if other than guideline

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Com. Reg. (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Reg. (EC) No 1907/2006;Part B: Subchronic oral toxicity test repeated dose 90-day oral toxicity study in rodents; Off, Jour- of the European Union, No. L 142

Deviations:

yes

Remarks:

see: Principles of method if other than guideline

Principles of method if other than guideline:

Deviations: Inadvertently, clinical observation was not recorded on study days 0, 7 and 14 prior to the administration. This deviation did not influence the validity as well as the outcome of the study.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-ethenyl-5-methyl-1,3-oxazolidin-2-one

EC Number:

809-852-5

Cas Number:

3395-98-0

Molecular formula:

C6H9NO2

IUPAC Name:

3-ethenyl-5-methyl-1,3-oxazolidin-2-one

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): 5-Methyl-3-vinyloxazolidin-2-one
- Physical state: liquid
- Analytical purity: ca. 99.3 %
- Lot/batch No.: DEIMLIB-00070

Specific details on test material used for the study:

Name of test substance: 5-Methyl-3-vinyloxazolidin-2-on
Test substance No.: 14/0031-4
Batch No.: Z019-2018
CAS No.: 3395-98-0
Content: 95.9 g/100 g (1H-NMR)
ldentity: Confirmed
Homogeneity: Given
Expiry date: 09 Feb 2020
Storage stability: The stability of the test substance under storage
conditions over the test period was guaranteed by the
Sponsor, and the Sponsor holds this responsibility.
Date of production: 09.02.2018
Storage conditions: ambient (RT)
Physical state/appearance: Liquid, yellow

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:Wl(Han)

Details on species / strain selection:

Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
The rat is a frequently used laboratory animal, and there is comprehensive experience with
this animal species. Moreover, the rat has been proposed as a suitable animal species by
the OECD and the EPA for this type of study.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Sex: males and females
Age when supplied: 34 ± 1 days
Age at study initiation: 42 ± 1 days
Weight at study initiation: male: 115.1 -117.0 g; female: 98.4 - 100.8 g
Diet: ad libitum
Water ad libitum

ENVIRONMENTAL CONDITIONS
Temperature 20-24°C
Relative humidity 45-65%,
15 air changes per hour
Illumination period: 12/12
Type of cage: H-Temp polysulfonate cages type 2000P
No. of animals per cage: 5
Enrichment: Wooden gnawing blocks
Bedding: Dust-free wooden bedding
Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY
TheThe food used was ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Granovit AG, Kaiseraugst, Switzerland food used was ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Granovit AG, Kaiseraugst, Switzerland.The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and by the Environmental
Analytics Water/Steam Monitoring Department of BASF SE as well as for the presence of microorganisms by a contract laboratory.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a solution. To prepare this solution, the
appropriate amount of test substance was weighed out depending on the desired
concentration. Then, corn oil was filled up to the desired volume, subsequently mixed with a
magnetic stirrer. The test-substance preparations were produced at least weekly and stored
at room temperature. The administration volume was 4 mL/kg body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses of the test substance were carried out as a separate study at the test facility.
The stability of 5-Methyl-3-vinyloxazolidin-2-on in corn oil at room temperature for a period of 7 days was proven before the start of the administration period.

Concentration control analyses of test-substance preparations were performed at the beginning and towards the end of the administration period in all concentrations. A homogeneity control analysis was not performed, because 5-Methyl-3-vinyloxazolidin-2-on was administered as a solution in corn oil.

Duration of treatment / exposure:

90 days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Starting on the day of arrival the animals were accustomed to the environmental conditions of the
study for an adaptation period during which they received ground diet and drinking water ad libitum.
Prior to the first detailed clinical observation, the animals were distributed according to weight among
the individual test groups, separated by sex. The weight variation of the animals used did not exceed
20 percent of the mean weight of each sex.
The test substance was administered daily by gavage for 3 months. Control animals received only the
vehicle. Additional animals in control group and high dose furthermore were kept untreated for
28 days after the administration period. All animals were sacrificed after a fasting period
(withdrawal of food) of at least 16 to 20 hours.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Mortality
A check for moribund and dead animals was made twice daily on working days and once daily on
Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed
and necropsied.
Clinical observations
All animals were checked daily for any abnormal clinically signs before the administration as well as
within 2 hours and within 5 hours after the administration. Abnormalities and changes were
documented for each animal.

DETAILED CLINICAL OBSERVATIONS: Yes
Abnormal behavior when handled, fur, skin, posture, salivation, respiration, activity/arousal level,
tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure,
exophthalmos, assessment of the feces discharged during the examination
(appearance/consistency), assessment of the urine discharged during the examination, pupil size

BODY WEIGHT:
Body weight was determined before the start of the administration period in order to randomize the animals.
The body weight was determined on study day 0 (start of the administration period) and thereafter at
weekly intervals. The difference between the body weight on the respective day of
weighing and the body weight on study day 0 was calculated as body weight change.


FOOD CONSUMPTION AND COMPOUND INTAKE :
Food consumption was determined from day -2 to 0 and thereafter weekly (as representative value over 7 days) for each cage and calculated as mean food consumption in grams per animal and day.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
Drinking water consumption was monitored by daily visual inspection of the water bottles for any
changes in volume.


NEUROBEHAVIOURAL EXAMINATION: Yes
Functional observational battery:
A functional observational battery (FOB) was performed in all animals at the end of the administration period as well as in all animals left at the end of the recovery period starting in the morning. At least one hour before the start of the FOB the animals were transferred to single-animal cages. Drinking water was provided ad libitum, but no food was offered during the measurements. The FOB started with passive observations without disturbing the animals, followed by removal from the home
cage, open field observations in a standard arena and sensory motor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable.

Home cage observations:
The animals were observed in their closed home cages; during this period, any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals.
Attention was paid to: posture, tremors, convulsions, abnormal movements, gait, other findings


Open field observations:
The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes.
The following parameters were examined: behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors,
convulsions, abnormal movements/stereotypes, gait, activity/arousal level, feces excreted within 2 minutes (consistency/ color), urine excreted within 2 minutes (amount/color), rearing within 2 minutes, other findings
Sensory motor tests/ reflexes:
The animals were then removed from the open field and subjected to following sensory motor or reflex tests:
reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (startle response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), other findings, grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test

Motor activity assessment
Motor activity (MA) was also measured in the early afternoon onwards on the same day as the FOB was performed. For this purpose, the animals were placed in new clean cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts was counted over 12 intervals for 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to
place the animals in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal.


OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes of all animals were examined prior to the start of the administration period. At the end of the administration period, i.e. study day 91, the eyes of animals in test groups 0 (control) and 3 (175 mg/kg bw/d) were examined for any changes using after application of a mydriatic agent (Mydrum®, Bausch + Lomb GmbH, Berlin, Germany). An examination at the end of the recovery period was not necessary due to the animals showing no findings at the end of the administration period.

ESTROUS CYCLE EXAMINATION.
Vaginal smears for terminal vaginal cytology examinations were prepared in the morning of the day of sacrifice


HAEMATOLOGY: Yes
leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean
corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin
concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes (RETA),
prothrombin time (HQT)

CLINICAL CHEMISTRY: Yes
Enzyme:
alanine aminotransferase (ALT), aspartate aminotransferase (AST), Alkaline phosphate (ALP),
γ-Glutamyl-transferase (GGT)

Blood chemistry parameter:
sodium (Na), potassium (K), chloride (CL), inorganic phosphate (INP), calcium (Ca), urea (UREA), creatinine (CREA), glucose (GLUC), total bilirubin (TBIL), total protein (TPROT), albumin (ALB), globulins (GLOB), triglycerides (TRIG), cholesterol (CHOL), bile acids (TBA), HDL-cholesterol (HDL-CHOL), LDL-cholesterol (LDL-CHOL).

THYROID HORMONES
Total triiodothyronine (T3), Total thyroxine (T4), Thyroid stimulating hormone (TSH)

URINALYSIS: Yes
pH, protein (PRO), glucose (GLU), ketones (KET), urobilinogen (UBG), bilirubin (BIL), blood, specific gravity (SP.GR), sediment, color, turbidity (COL, TURB), volume (VOL)

Sacrifice and pathology:

Necropsy
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.
One male animal (control) died intercurrently and was necropsied and assessed by gross pathology as soon as possible after its death.

Organ weights:
anesthetized animals (final body weight), adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, pituitary gland, prostate, seminal vesicles incl. coagulating glands, spleen, testes, thymus, thyroid glands (with parthyroid glands), uterus with cervix.

Organ/tissue fixation
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution:
all gross lesions, adrenal glands, aorta, bone marrow (femur), brain, cecum, cervix, coagulating
gland, colon, duodenum, epididymides (modified Davidson’s solution), esophagus, extraorbital lacrimal glands, eyes with optic nerve (modified Davidson’s solution), femur with knee joint, harderian glands, heart, ileum, jejunum (with Peyer’s patches), kidneys, larynx, liver, lungs, lymph nodes
(mesenteric and axillary lymph nodes), mammary gland (male and female), nose (nasal cavity), ovaries, oviducts, pancreas, parathyroid glands, pharynx, pituitary gland, prostate, rectum, salivary glands (mandibular and sublingual glands), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar cord), spleen, sternum with marrow, stomach (forestomach and glandular stomach), testes (modified Davidson’s solution), thymus, thyroid glands, trachea, urinary bladder, uterus, vagina.
The epididymides, eyes with optic nerve and testes of animal no 6 which died were fixed in 4%
neutral buffered formaldehyde solution.

Histopathology
Main groups (F1)
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings.
All gross lesion, adrenal glands, aorta, bone marrow (femur), brain, cecum, cervix, coagulating
glands, colon, duodenum, epididymides, esophagus, eyes with optic nerve, extraorbital lacrimal glands, femur with knee joint, harderian glands, heart, ileum, jejunum, kidneys, larynx, liver, lung, lymph nodes (axillary and mesenteric), mammary gland (female), nose (nasal cavity, level I, II, III, IV), ovaries, pancreas, parthyriod glands, Peyer`s patches, pharynx, pituitary gland, prostate, rectum, salivary glands (mandibular and sublingual), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, thoracic, lumbar), spleen, sternum with marrow, stomach (forestomach and glandular stomach), testes, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina.

Recovery groups (R1)
All gross lesions, harderian glands, liver, nasal cavity, level I, II, III. IV

Animals which died or were sacrificed intercurrently were processed histotechnically and assessed like control animals.

Special stain
Oil Red O staining for neutral fat was performed exemplarily on the livers of ine control male animal nos 1 and 1 animal (test group 3, 175 mg/kg bw/day).
Special attention was given to the synchrony of the morphology of the estrous cycle in ovaries, uterus, cervix, and vagina.
Special attention was given to the male reproductive organs, especially the stage of seminiferous tubules.
A correlation between gross lesions and histopathological findings was attempted.

Peer review
After completion of the histopathological assessment by the study pathologist an internal peer review was performed including liver and Harderian gland (males only) and nasal cavity (both sexes) in all main and recovery groups. Results presented in this report reflect the consensus opinion of the study pathologist and the peer review pathologist.

Statistics:

DUNNETT's test: Body weight, body weight change
KRUSKALWALLIS test; Posttest WILCOXON: Rearing, grip strengtof h fore- and hindlimbs, landing foot-splay test, motor activity,
blood parameters; urine pH, volume and specific gravity,
weights parameters
WILCOXON test: Urinalysis parameters (apart from pH, urine volume, specific gravity, color and
turbidity)
WILCOXON-test with Bonferroni-Holm adjustment (with several dose groups): Sperm analysis parameters.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test group 3 (175 mg/kg bw/d) and test group 2 (50 mg/kg bw/d)
slight to moderate salivation; it was considered to be related to a bad taste or irritant properties of the test substance preparation administrated by gavage.

Test group 2 ( 50 mg/kg/bw/d)
The left testis of 1 male animal was reduced in size which was recognized on study day 18. This isolated finding was considered as an incidental finding not related to treatment.

One male animal in test group 1 (15 mg/kg bw/d) had a skin lesion in the neck region
from study day 18 onwards spreading to the region behind the left ear on study day 29 and
lasting until study day 40. This may be caused by another animal due to group housing but it
was considered as not test-substance related.
Furthermore, one female animal of the control group had an injury and opacity of the
right eye which first was observed on study day 21 and later resulted in a small eye ball with
opacity on study day 51 onwards. This finding was considered as accidental and not treatment related.

In the recovery groups no treatment-related findings were observed.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male animal in the control group was found dead on study day 53 after showing slightly
labored respiration and piloerection after administration on study day 52. Based on these observations
the death of this animal is considered to be caused by a gavage error. Macroscopic findings at necropsy
(red discoloration of lungs and red effusion, approx. 4.0 mL in thoracic cavity) support this conclusion.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight was reduced significantly in males of test group 3 (175 mg/kg bw/d, main group)
on study day 91 (-5.8%) and in females of test group 3 on study days 42 (-4.6%), 70 (-4.1%)
and 84 (-4.5%). During the recovery period significant deviations of the body weights were
recorded for the remaining males of test group 3 on study days 98 and 119 (-6.7% and -6.3%)
as well as for the remaining females of this group on study day 112 (-5.7%).
Furthermore, body weight gain was significantly reduced from study day 77 onwards in males
of test group 3 (175 mg/kg bw/d, main group) up to a maximum of -7.7% on study day 91 and
on study day 42 only in females of test group 3 (-6.7%, main group). During the recovery period
no considerable deviations from control in body weight gain occurred. All these findings were
considered as treatment-related and adverse.
No test-substance related changes of mean body weights and mean body weight change
values were observed in both sexes of test groups 1 and 2 (15 and 50 mg/kg bw/d).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Before the start of the administration period a decrease of food consumption (-36.2%) was
recorded in females of test group 1 (15 mg/kg bw/d) and is considered as a side effect of first
time handling the animals after the acclimatization phase.
Minor deviations from the control were seen in females:
test group 3 (175 mg/kd bw/d : -13.7%, day 35 to 42 and -12.2% day 42 to 49
test group 2 ( 50 mg/kg bw/d): - 12.9% day 35 to 42 and -11.3% day 42 to 49
test group 1 (15 mg/kg bw/d); - 11.9% from day 49 - 56

Towards the end of the administration period a slight decrease of food consumption (-12.7%, day 77 to 84 and -11.1%, day 84 to 91) was seen in females of test group 3.
Based on temporary occurrence, these findings were considered to be not related totreatment.

During the recovery period no deviations from control group were seen in the remaining animals which could be related to former treatment.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

One female animal of the control showed a healed injury on study day 91 which is also mentioned before in the clinical observations.
All apparent findings were assessed as being incidental in nature since they occurred in individual animals only and did not show a dose-response relationship.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology
After a three-month administration period, in males of test group 3 (175 mg/kg bw/d) absolute neutrophil and monocyte counts were significantly increased and in females of the same test group platelet counts were significantly increased. These changes were regarded as treatment-related and adverse.

Significantly increased total white blood cell (WBC) counts and absolute large unstained cell (LUC) counts in males of test group 3 were within historical control ranges (males, WBC 4.38-6.73 Giga/L, LUCA 0.01-0.03 Giga/L). The same was true for significantly increased platelet counts in females of test groups 1 and 2 (platelets 601-808 Giga/L) and in males of test group 3 (platelets 592-955 Giga/L). Significantly shortened prothrombin times (HQT, Hepatoquick’s test) in males and females of test group 3 were also within historical control ranges (HQT males 35.0-42.3 sec; females 32.5-37.0 sec). Significantly decreased absolute lymphocyte counts in males of test group 1 were not dose-dependently changed. Therefore, the mentioned alterations were regarded as incidental and not treatment-related.

After the administration period, the following significantly changed values among the red blood cell parameters were all within historical control ranges and therefore the alterations were regarded as incidental and not treatment-related: decreased mean corpuscular hemoglobin content (MCH) and decreased mean corpuscular hemoglobin concentration (MCHC) in males of test group 3 decreased mean corpuscular volume (MCV) and MCH in females of test groups 1, 2 and 3; decreased hemoglobin values in females of test groups 2 and 3; decreased hematocrit values in females of test groups 1 and 2; decreased absolute reticulocyte counts in females of test group 1 (males, MCH 1.03-1.09 fmol; MCHC 20.56-22.07 mmol/L; females, MCV 50.5-53.8 fL; MCH 1.08-1.15 fmol; hemoglobin 8.1-8.9 mmol/L; hematocrit 0.378-0.415 L/L; absolute reticulocytes 126.4-184.7 Giga/L).

After the four-week recovery period, in males of test group 3 platelet counts were significantly increased. In females of the same test group MCH and MCV values as well as relative basophil counts were significantly decreased. All values were within historical control ranges (males, platelets 592-955 Giga/L; females MCH 1.08-1.15 fmol; MCV 50.5-53.8 fL; relative basophils 0.1-0.7 %). Therefore, these changes were regarded as incidental and not treatment-related.


Thyroid hormones
No treatment-related changes of T3, T4 and TSH levels were observed after the three-month administration period. In females of test group 3 significantly decreased T3 levels were observed. However, the values were within the historical control range (females, T3 0.60 – 1.53 nmol/L). T4 and TSH values were not changed among these individuals. Therefore, the T3 decrease in females of test group 3 was regarded as incidental and not treatment-related.
No treatment-related changes of T3, T4 and TSH levels were observed after the four-week recovery period.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

After the three-month administration period, in rats of both sexes of test group 3 (175 mg/kg bw/d) HDL-cholesterol was significantly increased. In females of the same test group cholesterol and triglyceride values were significantly increased. In males of test group 3 inorganic phosphate levels were significantly increased. These alterations were regarded as treatment-related and adverse.
The following significantly changed parameters were within historical control ranges and therefore, the alterations were regarded as incidental and not treatment-related: decreased total protein, albumin and globulin values in rats of both sexes of test groups 2 and 3 and in females of test group 1; increased cholesterol and potassium values in males of test group 3; increased glucose and total bilirubin values in females of test group 3; decreased chloride values in females of test group 3; increased triglyceride values in females of test groups 1 and 2 (males, total protein 59.69-66.25 g/L; albumin 33.21-39.19 g/L; globulins 23.49-30.71 g/L; cholesterol 1.63-2.15 mmol/L; potassium 4.51-5.11 mmol/L; females, total protein 63.22-70.49 g/L; albumin 35.85-42.68 g/L; globulins 23.56-30.98 g/L; total bilirubin 1.18-2.71 µmol/L; glucose 4.76-6.16 mmol/L; chloride 96.4-104.2 mmol/L; triglycerides 0.44-0.82 mmol/L).

The following significantly changed enzyme activities were not dose-dependently changed and therefore, these alterations were regarded as incidental and not treatment-related: females in test groups 1, 2 and 3 decreased alanine aminotransferase (ALT) activity; females in test group 1 and 2 decreased aspartate aminotransferase (AST) activity.

After the four-week recovery period in males of test group 3 LDL-cholesterol values were significantly increased. The values were above the historical control range (males, LDL-cholesterol 0.17-0.27 mmol/L). However, this was the only changed parameter among these rats and therefore this alteration was regarded as maybe treatment-related but non-adverse.

After the recovery period, the values of the following significantly changed parameters in test group 3 (175 mg/kg bw/d) were within historical control ranges and therefore these alterations were regarded as incidental and not treatment-related: males, decreased triglyceride values; females decreased ALT activities and calcium values and increased urea levels (males, triglycerides 0.71-1.36 mmol/L; females, ALT 0.49-0.74 µkat/L; calcium 2.48-2.67 mmol/L; urea 4.42-8.10 mmol/L).

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

After the administration period in male and female rats of test group 3 (175 mg/kg bw/d) significantly higher incidences of crystals were observed in the urine sediment. These were predominantly calcium oxalate crystals in females but additionally triple phosphate crystals in males. Additionally, in males of the same test group higher ketone body levels in the urine were measured. These alterations were regarded as treatment-related and adverse.

In females of test groups 2 and 3 urine volume was significantly decreased. Additionally, in females of test group 2 specific gravity of the urine was significantly increased. However, these changes were not dose-dependent and therefore they were regarded as incidental and not treatment-related.

After the recovery period, in males of test group 3 urine volume was significantly lower compared to controls. This isolated alteration was regarded as non-adverse if ever treatment related.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Home cage observations:No test substance-related adverse effects were observed.

Open field observations: No test substance-related adverse effects were observed.


Sensorimotor tests/ reflexes: No test substance-related adverse effects were observed.

Quantitative parameters:
In maIes of test group 3 (175 mg/kg bw/d)a significantly increased number of rearings (+94%) was observed during the administration period as well as in the remaining animals during recovery period (+61%) which are in the range of historical control data. Additionally, males of test group 3 showed slightly, but not significant, decreased distance in foot splay test (-11.1%) during the administration period as well as in the remaining animals during recovery period (-13.9%). In the females of test group 3 (175 mg/kg bw/d) a significantly reduced distance (-12.9%) was observed in the foot splay test. All these findings were within the historical control data or in case of the values for females of the recovery group at the lower boarder of the historical control range with minor deviation of 0.1 cm. Therefore, these findings were considered as not-related to treatment and not adverse.
Males of test groups 1 and 2 (15 and 50 mg/kg bw/d) and females of all test groups did not show any deviations from control group during the administration period.

Motor activity measurement:
One single interval was increased in males of test group 1 (15 mg/kg bw/d, interval 5) and in males of test group 2 (50 mg/kg bw/d, interval 2). Furthermore, the overall interval was increased in test group 2. During the recovery period one single interval was increased in the remaining males of test group 3 (175 mg/kg bw/d, interval 7). Based on the isolated occurrence and the missing dose-dependency of the findings, these observations were considered as not treatment-related.
Regarding the overall motor activity as well as single intervals, no deviations were noted for all female animals in test groups 1, 2 and 3 during administration and recovery period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

MAIN GROUPS /F1)
Absolute organ weights:
When compared with control group 0 (=100%), the following mean absolute weights (female animals) were significantly increased or decreased in one test group (percent deviation from control;
test group 1 (15 mg/kg bw/d). heart -1.1%; liver +2.1%
test group 2 (50 mg//kg bw/d) heart -8.8 %**; liver - 1%
test group 3 (175 mg/kg bw/d) heart -4.3 %; liver + 9.1 % **
**: p <= 0.01
All other mean absolute weight parameters in females and all absolute weight parameters in males did not show significant differences when compared to the control group 0.

Relative organ weights
When compared with control group 0 (=100%), the mean relative liver weights were significantly increased test group 3 males and females
test group 1 (15 mg/kg bw/d) liver +2.3% male animals, + 3.2% female animals
test group 2 (50 mg//kg bw/d) liver +4.7 % male animals, + 3.2% female animals
test group 3 (175 mg/kg bw/d) liver + 12.7 %** male animals, + 12.7 %** female animals
**: p <= 0.01
All other mean relative weight parameters did not show significant differences when compared to the control group 0.

The increased mean relative liver weights of males and the increased mean absolute and relative liver weights in females of test group 3 are regarded to be treatment – related, although a histopathological correlate could only be detected in male animals of test group 3.
The decreased mean absolute heart weight of females of test group 2 is considered to be incidental as there is no dose response, and there are no statistically significant changes in either the mean relative heart weight of test group 2 females or the mean absolute or relative heart weights of test group 3 females. Additionally, there were no treatment-related histopathological findings in test group 3 females.

RECOVERY GROUPS
Absolute organ weights
When compared with the control group 0 (=100%), the following mean absolute weights were significantly decreased (female animals) test group 3 : Final body weight -5.8%*, Adrenal glands -14.6 %*; Heart -7.7 %*; Ovaries -8.7 %**
* : p <= 0.05, **: p <= 0.01

Relative organ weights:
When compared with the control group 0 (=100%), the following mean absolute weights were significantly increased (female animals) test group 3: liver +5 %*
* : p <= 0.05,
The increased mean relative liver weight in test group 3 females was assumed to be treatment-related.

The decreased absolute weights of adrenal glands, heart and ovaries were regarded to be related to the decreased terminal body weight as there were no statistically significant changes in relative weights in these organs.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

MAIN GROUPS
All findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Adipose tissue: 1 male/test group 3
Adrenal cortes: 1 female/test group1
Eyes with opt.nerve; glandular stomach, pelvic dilation, unilateral; thyroid gland, size reduced; ureter dilation: 1 female/control group
Liver: 1 female/test group 3
Lungs, discoloration; thoracic cavity,effusion: 1 male/control group
Testes; 1 male/group 2

Male animal no. 6 (control) which died intercurrently showed macroscopically approximately 4 ml red effusion in the thoracic cavity and red discolored lungs.

RECOVERY GROUPS
All findings in the recovery groups occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Testes, size reduced: 1 male/test group 2
Thoracic cavity, effusion 1 male/control group
Thyroid gl., size reduced; Ureter, dilation: 1 female/control group

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

NASAL CAVITY; LEVEL I - IV

Test group 3: 175 mg/kg bw/d:
Nasal cavity, level I
Atrophy, respiratory epithelium, multifocal in all male and female animals;
Concretion in 9/10 male and 4/10 female animals;
Inflammation neutrophilic in 5/10 male animals;
Metaplasia, squamous cell in 4/10 female animals

Nasal cavity, level II
Atrophy, respiratory epithelium, multifocal in 8/10 female animals;
Concretion in all male and female animals;
Degeneration/regeneration of the olfactory epithelium in all male and female animals;
Inflammation neutrophilic in 2/10 male and 5/10 female animals;
Metaplasia, respiratory in 8/10 male and all female animals;

Nasal cavity, level III
Atrophy, respiratory epithelium, multifocal in 3/10 female animals;
Concretion in all male and 9/10 female animals;
Degeneration/regeneration of the olfactory epithelium in all male and female animals;
Metaplasia, respiratory in 6/10 female animals

Nasal cavity, level IV
Concretion in 6/10 male and 8/10 female animals;
Degeneration/regeneration of the olfactory epithelium in all male and female animals;
Inflammation neutrophilic in 3/10 male and 1/10 female animals;
Metaplasia, respiratory in 3/10 male and 1/10 female animals;

RECOVERY GROUPS R1
Nasal cavity, level I
Concretion in 4/10 male and 5/10 female animals;
Inflammation neutrophilic in 1/10 male and 2/10 female animals
Metaplasia, squamous cell in 2/10 female animals

Nasal cavity, level II
Atrophy, respiratory epithelium, multifocal in 1/10 female animals;
Concretion in all male and female animals
Degeneration/regeneration of the olfactory epithelium in 7/10 male and 7/10 female animals;
Inflammation neutrophilic in 6/10 male and 5/10 female animals;
Metaplasia, respiratory in 9/10 male and 8/10 female animals;
Metaplasia, squamous cell in 1/10 female animals;

Nasal cavity, level III
Concretion in all male and 6/10 female animals;
Degeneration/regeneration of the olfactory epithelium in all male and 9/10 female animals;
Metaplasia, respiratory in 7/10 male and 2/10 female animals;

Nasal cavity, level IV
Concretion in all male and 8/10 female animals;
Degeneration/regeneration of the olfactory epithelium in all male and female animals;
Inflammation neutrophilic in 3/10 male and 1/10 female animals;
Metaplasia, respiratory in 8/10 male and 2/10 female animals;

Test group 2: 50 mg/kg bw/d
Nasal cavity, level I
Atrophy, respiratory epithelium, multifocal in all male and female animals;
Concretion in 2/10 male and 5/10 female animals;

Nasal cavity, level II
Concretion in 6/10 male and 8/10 female animals;
Degeneration/regeneration in 6/10 male and 3/10 female animals;

Nasal cavity, level III
Concretion in 4/10 male animals;
Degeneration/regeneration in 9/10 male and 6/10 female animals;

Nasal cavity, level IV
Concretion in 5/10 male and 5/10 female animals;
Degeneration/regeneration in all male and 9/10 female animals;

Test grouop 1; 15 mg/kg bw/d
No treatment-related, adverse effects were observed


Treatment-related findings were observed in the liver in male animals of test group 3, which showed a full recovery and in the nasal cavity, of all levels in male and female animals of test groups 2 and 3, which were mostly not reversible after the recovery period.
In the liver, 9 out of 10 male animals of main test group 3 showed a minimal centrilobular hepatocellular hypertrophy grade1 accompanied by minimal microvesicular fatty change grade 1 also in a centrilobular distribution in 4 out of 10 males.
The presence of neutral fat was confirmed exemplarily by Oil Red O staining in male animal 45 (test group 3).
Treatment - related findings were no longer noted in the liver of male animals of recovery test group 3.

Histopathological findings: neoplastic:

not examined

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The administration of 5-Methyl-3-vinyloxazolidin-2-on by gavage to male and female Wistar rats for 3 months caused signs of toxicity. These findings had been observed in males and females of test groups 3 (175 mg/kg bw/d) and 2 (50 mg/kg bw/d).

After a recovery period of 28 days, no clinical (pathology) findings related to treatment were observed. In the recovery test group 3, some treatment-related, adverse histopathological findings in the nasal cavity were still present (e.g. degeneration/regeneration of the olfactory epithelium) whereas other histopathological findings in the nasal cavity, such as a multifocal atrophy of the respiratory epithelium, showed lower incidences compared to the main group.
Therefore, under the conditions of the present study, the no observed adverse effect level (NOAEL) was 15 mg/kg bw/d for male and female rats.

Executive summary:

5-Methyl-3-vinyloxazolidin-2-on was administered by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0, vehicle control), 15 (test group 1), 50 (test group 2) and 175 mg/kg body weight/day (mg/kg bw/d; test group 3) over a period of 3 months.

Additional animals of the high dose and control group (10 males and 10 females each) were kept untreated for 28 days after the administration period in order to demonstrate reversibility of possible effects.

With regard to clinical examinations, signs of general systemic toxicity were observed in test group 3 animals. In males of test group 3 (175 mg/kg bw/d), body weight (study day 91,
-5.8%) and body weight change (study day 77 onwards, up to -7.7%) were significantly decreased. In females of test group 3, body weight and therefore body weight change were temporarily decreased during the administration period (e.g.up to -4.6% on study day 42).During the recovery period significant deviations of the body weights were recorded for the remaining males of test group 3 on study days 98 and 119 (-6.7% and -6.3%) as well as for the remaining females of this group on study day 112 (-5.7%).The significant findings were regarded as treatment-related and adverse.

No findings were observed in test group 1 and 2 animals.

Regarding clinical pathology, higher HDL-cholesterol values in males and female rats of test group 3 (175 mg/kg bw/d,(main groups) as well as higher cholesterol and triglyceride values in females and increased levels of ketone bodies in the urine of males of the same test group indicate a changed liver cell metabolism. This might have effects on the hemostasis in females resulting in increased platelet counts. In males of test group 3 higher absolute neutrophil and monocyte counts were due to an acute phase reaction. The cause of higher incidences of calcium oxalate and triple phosphate crystals in rats of both sexes of test group 3 could not be elucidated. The mentioned changes after the three-month administration period recovered completely after four weeks.

Regarding pathology, target organs were the liver and nasal cavity, all levels, in both sexes.

In the liver of main group animals,increased mean relative (+12.4%) liver weights in males and increased mean absolute (+9.1%) and relative (+12.7%) liver weights in females of test group 3 (175 mg/kg bw/d) were observed and regarded to be treatment-related. A histopathological correlate could only be detected in male animals of test group 3: minimal centrilobular hepatocellular hypertrophy was seen in 9 out of 10 animals accompanied by centrilobular fatty change in 4 of these animals (confirmed with Oil Red O staining in animal No 45).

Findings in the liver of test group 3 male and female animals were regarded as adverse in main group animals, in combination with the findings observed in clinical pathology.

After the recovery period, the mean relative liver weight in test group 3 (175 mg/kg bw/d) females was still slightly increased (+5.0%) and was assumed to be questionably treatment-related and assessed as non - adverse, as there were no other findings in clinical chemistry or histopathology. It might be related to the decreased terminal body weight in recovery group 3 females (-5.8%).

 

In the nasal cavity, the following findings were noted:

Atrophy,respiratory epithelium, multifocalwas observed predominantly in level I, where all animals of both sexes of test groups 2 and 3 were affected. In levels II and III, this was seen in females only with lower incidence in the more posterior levels, which is due to the anatomical distribution of this epithelium. Only one recovery female animal of test group 3 was minimally affected.

 Degeneration/regeneration was seen in many animalsof both sexesof main test groups 2 and in all animals of test group 3 with varying severity and multifocally in the olfactory epithelium in all areas of the nasal cavity of levels II, III and IV. It was most pronounced dorsally and along the septum. In recovery animals of both sexes of test group 3 this was still present without a reduction in incidence or severity.

Concretions were seen in test groups 2 and 3 of male and female main group animals and also in recovery animals of test group 3in all levels. This finding was, however, most prominent in levels II and III, where most or all animals were affected with similar severity in both main and recovery test group 3.

Eosinophilic globules were seen in all levels of the nasal cavity. In main group animals there was no treatment-related change in incidence, while in recovery groups, there was a slight increase in male animals in levels III and IV and in females in level II. Eosinophilic globules can be a nonspecific response to irritation of the nasal epithelium and were, therefore, assumed to be treatment - related even in absence of an increased incidence of this finding in main group animals.

Mostly minimal Inflammation, neutrophilic, multifocalwas seen in a few animals of test group 3 of both main and recovery groups.

Metaplasia, squamous cell, multifocalwas noted in very few animals in test group 3 of both main and recovery groups.

Metaplasia, respiratory, multifocal was often found in areas where there was compression by concretions. This was only noted in test group 3 animals of both main and recovery groups, with more male than female animals affected.

In general, the observed changes did not specifically target particular cell types or tissues within the nasal cavity, but they showed a rather broad and diffuse damage of the nasal mucosa. In addition, minimal to slight squamous metaplasia was noted within the nasal mucosa, indicative of prolonged irritation. Considering substance properties as strongly irritating to mucous membranes, it is likely that the effects on respiratory tract observed after oral exposure are linked to local irritation and may be related to administration pathway (gavage) combined with vehicle effect (corn oil): upon removal of the gavage tube from the esophagus, test formulation may be transported upward and deposited in the oropharyngeal region. During expiration, the expiratory airflow may carry parts of the test formulation from the oropharynx into the nasopharyngeal duct, and finally into the nasal cavity (retrograde aspiration into the nasal passages; cf. Damsch et al.: Gavage-Related Reflux in Rats: Identification, Pathogenesis, and Toxicological Implications (Review), Toxicologic Pathology, 39: 348-360, 2011).

 

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.