Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 26, 2003 - March 17, 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3-(3-hydroxypropyl)oxazolidin-2-one
EC Number:
431-840-1
EC Name:
3-(3-hydroxypropyl)oxazolidin-2-one
Cas Number:
87010-29-5
Molecular formula:
C6 H11 N O3
IUPAC Name:
3-(3-hydroxypropyl)oxazolidin-2-one
Details on test material:
- Name of test substance: 3-(3-Hydroxypropyl)-oxazolidin-2-on
- Test substance number: 03/0046-1
- Batch number: 73653056 PO
- Date of production: August 09, 2002
- Purity: 94.0 9/1 00 g (analytical report 03L00170)
- Homogeneity: homogeneous (analytical report 03L00170)
- Stability: stable; proven by reanalysis (analytical report 04L00027)
- Physical state / colour: Iiquid/colourless-yellowish
- Storage conditions: room temperature
The analyses were carried out at the Analytical Department of BASF AG, Ludwigshafen/Rhein, Germany.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Strain: CrlGlxBrlHan:WI
- Age at study initiation: 35 ±1 days
- Fasting period before study: none
- Housing: singly in type OK III stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel, FRG (floor area about 800 cm2)
- Diet: ground Kliba maintenance diet mouse/rat ‹GLP", meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland ad libitum
- Water: drinking water (from water bottles) ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24° C
- Humidity (%): 30 - 70%
- Photoperiod (hrs dark / hrs light): 12 hours (12 hours light from 06.00 a.m. - 06.00 p.m., 12 hours dark from 06.00 p.m. - 06.00 a.m.)
Deviations from these ranges did not occur.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
TEST SUBSTANCE PREPARATION AND ANALYSES
Preparation
The test substance was weight out and thoroughly mixed with a small amount of food. Thereafter these premixes were adjusted to the desired concentrations with appropriate amounts of food and mixed for about 10 minutes in a Iaboratory mixer.

Food analyses
The food used in the study was assayed for chemical and microbiological contaminants by the supplier.

Drinking water analyses
The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and the Technical Services of BASF AG as well as for the presence of microorganisms by a contract Iaboratory.

Bedding analyses
The bedding is regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical investigations of the test substance preparations were carried out at the Analytical Chemistry Laboratory of the Experimental Toxicology and Ecology of BASF AG, Ludwigshafen, Germany. The stability of the test substance in the diet over a period of 32 days at room temperature was confirmed before the start of the study. This exceeded the interval from diet preparation to the end of the feeding period. Homogeneity analyses of the test substance preparations were performed in samples of the high and low concentrations at the start of the administration period. These samples also served for concentration control analyses. Additional concentration control analyses were performed with a sample drawn from the mid concentration at the start of the administration period.
Duration of treatment / exposure:
28 day with an additional recovery period
Frequency of treatment:
continuously
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
750; 3000; 12000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
1149.7; 285.2; 69.7 mg/kg bw/day (males)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
1171.3; 295.3; 74.9 mg/kg bw/day (females)
Basis:
nominal in diet
No. of animals per sex per dose:
0 (vehicle control) 10 animals per sex (5 animals per sex in the recovery group)
750 ppm dosing group: 5 animals per sex
3,000 ppm dosing group: 5 animals per sex
12,000 ppm dosing group: 10 animals per sex (5 animals per sex in the recovery group)
Control animals:
yes, concurrent vehicle
Details on study design:
AIM OF THE STUDY
The aim of the study was to determine the toxicological profile including the target organs and the NOAEL of the test substance after oral administration via the diet for 4 weeks. Additionally, animals of the highest dose and control group were maintained a recovery period of two weeks in order to prove reversibility of possible effects.

SELECTION OF DOSES
On request of the sponsor, the following dose levels were selected:
12,000 ppm: as high dose
3,000 ppm: as mid dose
750 ppm: as low dose

The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.

EXPERIMENTAL PROCEDURE
On the day of arrival the animals were subjected to an acclimatization period during which they received ground diet and drinking water ad libitum. Prior to the first open field observation, the animals were distributed according to weight among the individual test groups, separated by sex. The list of randomization instructions was compiled with a computer. The weight variation of the animals used did not exceed 20 percent of the mean weight of each sex. At the start of the administration period (day 0) the rats were 42 ±1 days old. The test substance was administered daily via the diet for 4 weeks. Control animals received ground diet only. At the end of the administration period the animals of the main groups were sacrificed. The animals of the recovery groups were maintained for another two weeks with ground diet. All surviving animals were sacrificed after a fasting period (withdrawal of food) tor at least 16-20 hours.
Positive control:
not investigated

Examinations

Observations and examinations performed and frequency:
Clinical observations
The animals were examined tor overt signs of toxicity or mortality twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) an Saturdays, Sundays and public holidays. Additionally, further clinical examinations were carried out daily, as well as once a day during the recovery period. Detailed clinical observations were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 x 37.5 cm with sides of 25 cm high). The following parameters were examined:

1. abnormal behavior during handling
2. fur
3. skin
4. posture
5. salivation
6. respiration
7. activity/arousal level
8. tremors
9. convulsions
10. abnormal movements
11. impairment of gait
12. lacrimation
13. palpebral closure
14. exophthalmos
15. feces (appearance/consistency)
16. urine
17, pupil size

Food consumption
Individual food consumption was determined weekly over a period of 7 days and calculated as mean food consumption (g/animal/day).

Water consumption
Water consumption was observed daily by visual inspection of the water bottles for any overt changes in volume.

Body weight data
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period and the recovery period the body weight was determined an day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight an the respective day of weighing and the body weight an day 0 was calculated as body weight change.

Food efficiency
Food efficiency (group means) was calculated based upon individual values for body weight and food consumption:

(BWx – BWy) / (FC y to x) x 100 = Food efficiency for day x

BWx = body weight on day x (g)
BWy = body weight on day y (last weighing date before day x) (g)

FC y to x: mean food consumption from day y to day x; calculated as mean daily food
consumption on day x, multiplied with the number of days from day y to day x.

Intake of test substance
The mean daily intake of test substance (group means) was calculated based upon individual values for body weight and food consumption.

(FCx x C) / (BWx) = Substance intake for day x

BWx = body weight on day x [g]
FCx = mean daily food consumption on day x [g]
C = concentration in the diet on day x [mg/kg]

Functional observational battery (FOB)
A functional observational battery was performed in all animals at the end of the administration period starting at about 10.00 a.m. 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 sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random

Home cage observations:
The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Beside other findings attention was paid to:
1. posture
2. tremor
3. convulsions
4. abnormal movements
5. impairment of gait
6. other findings

Open field observations:
The animals were transferred to a standard arena (50 x 50 cm with sides of 25 cm high) and observed for at least 2 minutes. Following parameters were examined:
1. behavior when removed from cage
2. fur
3. skin
4. salivation
5. nose discharge
6. Iacrimation
7. eyes/pupil size
8. posture
9. palpebral closure
10. respiration
11. tremors
12. convulsions
13. abnormal movements
14. impairment of gait
15. activity/arousal level
16. feces (number of fecal pellets/appearance/consistency) within two minutes
17. urine within two minutes
18. number of rearings within two minutes
19. other findings

Sensorimotor Tests/Reflexes:
The animals were removed from the open field and subjected to following sensorimotor
or reflex tests:
1. approach response
2. touch response
3. vision (“visual placing response")
4. pupillary reflex
5. pinna reflex
6. audition (“startle response")
7. coordination of movements (“righting response")
8. behavior during “handling"
9. vocalization
10. pain perception (“tau pinch")
11. grip strength of forelimbs
12. grip strength of hindlimbs
13. landing foot-splay test
14. other findings

Motor activity assessment
Motor activity was measured on the same day as FOB was performed. The measurement was performed in the dark using the Multi-Varimex-System (Columbus Instruments Int. Corp., Ohio, USA) with 4 infrared beams per cage. During the measurement the animals were kept in Polycarbonate cages with absorbent material. The animals were put into the cages in a randomized order. The measurements started at about 12.45 p.m. in the main groups and at about 2.00 p.m. in the recovery groups. The number of beam interrupts were counted over 12 intervals, each lasting 5 minutes. Measurement did not commence at the same instant tor all cages; the period of assessment tor each animal started when the first beam was interrupted by pushing the cage into the rack (staggered start). Measurements ended exactly 60 minutes thereafter. During the measurements the animals received no food and no water.

Statistics of clinical examinations
Groups 0, 1, 2 and 3
Food consumption, body weight, body weight change, food efficiency:
A comparison of each group with the control group was performed using DUNNETTs test (two-sided) for hypothesis of equal means.

Feces, rearing, grip strength forelimbs, grip strength hindlimbs, foot- splay test, motor activity:
Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). lf the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two sided) for the equal medians

Groups 0 and 3 (recovery period):
Food consumption, body weight, body weight change, food efficiency:
A comparison of each group with the control group was performed using the Welch t-test (two-sided) for the hypothesis of equal means.

Blood was taken from the retroorbital venous plexus in the morning from fasted animals. The animals were anaesthetized using isoflurane (Isoflo©, Essex GmbH, Munich, Germany) as anesthesia. The blood sampling procedure and the subsequent analysis of the blood and serum samples were carried out in a randomized sequence. The list of randomization instructions was compiled with a computer using a random number generator. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. The urine samples were evaluated in a randomized sequence. The assays of blood and serum parameters were performed under internal Iaboratory quality control conditions with commercial reference controls to assure reliable test results. The results of the clinical pathology examinations are expressed in units of the International System (SI). The following examinations were carried out in all animals per test group and sex at the end of the administration period. At the end of the recovery period only hematological parameters in females and urea in males were examined.

Hematology
The following parameters were determined in blood with EDTA-K 3 as anticoagulant using
a particle counter (ADVIA 120 model; Bayer, Fernwald, Germany):
- leukocytes
- erythrocytes
- hemoglobin
- hematocrit
- mean corpuscular volume
- mean corpuscular hemoglobin
- mean corpuscular hemoglobin concentration
- platelets
- differential blood count
- reticulocytes

Furthermore, differential blood smears were prepared and stained according to Wright without being evaluated.

Clinical chemistry
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters. The following parameters were determined:
- alanine aminotransferase
- aspartate aminotransferase
- alkaline phosphatase
- serum-y-glutamyltransferase
- sodium
- potassium
- chloride
- inorganic phosphate
- calcium
- urea
- creatinine
- glucose
- total bilirubin
- total protein
- albumin
- globulins
- triglycerides
- cholesterol
- magnesium

Urinalysis
With the exception of volume, color, turbidity, sediment examination and the specific gravity, ah the urine constituents were determined semi-quantitatively using test strips (Combur-9-test M, Roche, Mannheim, Germany) and a reflection photometer (Miditron M; Rache, Mannheim, Germany). The specific gravity was determined using a urine refractometer. The sediment was evaluated microscopically. The following examinations were carried out:

- volume
- colour
- turbidity
- pH
- protein
- glucose
- ketones
- urobilinogen
- bilirubin
- blood
- specific gravity
- sediment
Sacrifice and pathology:
Necropsy
All animals sacrificed at scheduled dates, were sacrificed by decapitation under CO2-anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. The animal, which died intercurrently, was necropsied as soon as possible after death and assessed by gross pathology.

Weight parameters
Weight assessment was carried out on all animals sacrificed at scheduled dates. The weights of the following organs were determined:
1. Anesthetized animals
2. Liver
3. Kidneys
4. Adrenal glands
5. Testes
6. Epididymides
7. Ovaries
8. Uterus
9. Spleen
10. Brain
11. Heart
12. Thymus

Organ / Tissue preservation list
The following organs / tissues were preserved in neutral buffered 4% formaldehyde
solution:
1. All gross lesions
2. Brain
3. Pituitary gland
4. Thyroid glands/ parathyroid glands
5. Thymus
6. Trachea
7. Lungs
8. Heart
9. Liver
10. Spleen
11. Kidneys
12. Adrenal glands
13. Testes/ ovaries
14. Oviducts, uterus, vagina
15. Epididymides, prostate, seminal vesicle
16. Stomach (tore- and glandular stomach)
17. Duodenum, jejunum, ileum
18. Cecum, colon, rectum
19. Urinary bladder
20. Lymph nodes (mesenteric and mandibular Iymph nodes)
21. Sciatic nerve
22. Bone marrow (femur)
23. Eyes
24. Spinal cord (cervical, thoracic and lumbar cord)
From all animals, parts of the liver dedicated tor routine investigation were fixed in Carnoy's solution. After fixation, the liver was embedded in paraplast.
Statistics:
- Clinical pathology parameters, except reticulocytes and differential blood count: Non-paramteric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pair-wise comparison of each dose-group with the control group was performed using the WILCOXON test (two-sided) for the equal medians.
- Clinical pathology parameters, except reticulocytes and differential blood count: Comparison of the dose-group with the control group using the WILCOXON test (two-sided) for the equal medians.
- Urinalysis, except volume, colour, turbidity and specific gravity: Pair-wise comparison of each dose group with the control group using FISHER's exact test for the hypothesis of equal portions.
- Weight parameters: Non-parametric one-way analysis using KRUSKAL- WALLIS test (two-sided). if the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using the WILCOXON test tor the hypothesis of equal medians.

Results and discussion

Results of examinations

Details on results:
ANALYSES
Stability analyses
The stability of the test substance in the diet over a period of 32 days at room temperature was confirmed before the start of the study. As the test substance preparations were stored no longer than this time period, the stability was guaranteed.

Homogeneity and Concentration control analyses
Considering the low standard deviation in the homogeneity analysis, it can be concluded that 3-(3-Hydroxypropyl)-oxazolidin-2-on was distributed homogenously in the diet. Concentration control analyses yielded 100.7 - 103.8% of the nominal values. These results demonstrated the correctness of the concentrations of 3-(3-Hydroxypropyl)-oxazolidin-2-on in the diet.

Diet analyses
On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants the diet was found to be suitable. Fed. Reg. Vol. 44, No. 91 of May 9,1979, p. 27354 (EPA), served as a guideline for maximum tolerable chemical contaminants. The number of microorganisms did not exceed 5*10e5/g food. Individual results can be found in the archives of the Experimental Toxicology and Ecology of BASF AG.

Drinking water analyses
On the basis of the analytical findings the drinking water was found to be suitable. German Drinking Water Regulation (Trinkwasserverordnung) served as a guideline for maximum tolerable contaminants. Individual results can be found in the archives of the Experimental Toxicology and Ecology of BASF AG.

Bedding analyses
On the basis of the analytical findings the bedding was found to be suitable. Levels given in Lab. Animal, Nov. - Dec. 1979, pp. 24 - 33, served as a guideline for maximum tolerable contaminants. Individual results are to be found in the archives of the Experimental Toxicology and Ecology of BASF AG.

CLINICAL EXAMINATIONS
Mortality
One female animal of group 3 died during blood sampling, spontaneously. This single occurrence was assessed as clearly not related to the test article.

Clinical examinations
No substance-related findings were observed.

Food consumption
No substance-related findings were observed.

Body weight data
No substance-related findings were observed.

Food efficiency
Food efficiency was statistically significantly increased an day 28 in males group 1 and on day 35 in males of group 3. These single occurrences were clearly not related to the treatment with the test article.

Intake of test substance
Test group - Nominal dose level (ppm) - Mean daily test substance intake in mg/kg body weight/day
750 ppm dosing group: Males: 69.7; Females: 74.9
3,000 ppm dosing group: Males: 285.2; Females: 295.3
12,000 ppm dosing group: Males: 1149.7; Females: 1171.3

Functional observational battery
Deviations from “zero values" were obtained in several animals. However, as most findings were equally distributed between treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental.

Home cage observations:
No substance-related findings were observed.

Open field observations.
No substance-related findings were observed.

Sensorimotor tests/reflexes:
No substance-related findings were observed.

Quantitative parameters:
No substance-related findings were observed.

Motor activity measurement
Regarding the overall motor activity (summation of all intervals) no substance-related findings were measured in both sexes.
Comparing the single intervals with the control groups, the motor activity was statistically decreased in males group 1 at interval 2, in females group 1 at interval 8 and group 3 at interval 7. These single occurrences were clearly not related to the treatment with the test substance.

CLINICAL PATHOLOGY

Hematology
At the end of the study slightly, but statistically significantly increased red blood cell counts and hematocrit values were found in the blood of the high dose females. Both effects normalized following cessation of test substance administration. No treatment-related changes were observed in the other hematological parameters for any of the treated groups.

Clinical chemistry
Compound-related differences in serum enzyme activities were not evident at any dose level in either males or females.
Blood chemistry examinations revealed statistically significantly decreased urea concentrations in the high dose group males, which were reversible during the treatment-free recovery period. In the serum of the high dose females a tendency towards decreased urea levels was observed. No treatment-related changes were present in the other blood chemistry parameters of both sexes.

Urinalyses
No test substance-related effects were observed in urine parameters of either sex.

Other deviations
There was one additional statistically significant intergroup difference in the results of clinical pathology testing. This deviation was marginal, incidental and inconsistent, when compared with the other sex, and lack dose-response relationship. Accordingly, this finding was considered to be of no toxicological significance.

PATHOLOGY
Weight parameters

Absolute weights
Final sacrifice group (F1)
When compared to control group 0, the mean absolute weight of the ovaries was significantly increased:
Female animals; Group 2: Ovaries +21.2%
All other mean absolute weight parameters did not show significant differences when compared to the control group. The increase of the mean absolute weight of ovaries in females of dose group 2 is considered incidental.
Recovery group (R1)
When compared to control group 0, the mean absolute weights of following organs were significantly increased:
Test group 3: Kidneys: +16.2 %; Spleen: +25.4 %
All other mean absolute weight parameters did not show significant differences when compared to the control group. The mean absolute kidney weight was significantly increased in males of dose group 3. The mean relative kidney weight of these males did not show significant differences. Therefore, the significant increase of the absolute kidney weights in high dose males is considered incidental.

Relative Organ Weights
Final sacrifice group (F1)
When compared to control group 0, the mean relative weight of the ovaries was significantly increased:
Female animals; Group 2: Ovaries +18.4%
All other mean relative weight parameters did not show significant differences when compared to the control groups. The increase of the mean relative weight of ovaries in females of dose group 2 is considered incidental.
Recovery group (R1)
When compared to control group 0, the mean relative spleen weight was significantly increased:
Male animals; Group 3: spleen +16.6%
All other mean relative weight parameters did not show significant differences when compared to the control groups.

Gross lesions
All gross lesions occurred singly.

Mortality:
All males survived until the scheduled end of the study. One female animal of group 3 (recovery group) died intercurrently.

Histopathology
All findings noted were either single observations or they were biologically equally distributed between control and treatment groups. All of them are considered to be incidental or spontaneous in origin and without any relation to treatment.

Mortality:
There were no microscopic findings, that could explain the cause of death of the female of group 3 (recovery group), that died intercurrently.

Effect levels

Dose descriptor:
NOAEL
Effect level:
3 000 other: ppm (285 mg/kg bw/day (males) and 295 mg/kg bw/day (females))
Sex:
male/female
Basis for effect level:
other: decreased urea in both sexes and increased red blood cells and hematocrit in females

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

3-(3-Hydroxypropyl)-oxazolidin-2-on was administered to male and female Wistar rats via the diet for 4 weeks at dose levels of 0 (group 0); 750 (group 1); 3,000 (group 2) and 12,000 (group 3) ppm; a recovery group (2 weeks) was additionally evaluated for the control (group 0) and high dose grup (group 3). During clinical examinations, including functional observational battery as well as measurement of motor activity, no signs of toxicity were found.

Regarding clinical pathology, mild increases in red blood cells and hematocrit were seen in high dose females and decreased urea concentrations were found in the serum of high dose animals of either sex at the end of the administration period. These findings were considered to be treatment-related and are indicative of a marginal adverse effect on erythrocytes in females and decreased production of urea in both sexes. All substance-related findings were reversible within a treatment-free recovery period of 2 weeks.

Concerning pathology, the mean absolute and relative spleen weights were significantly increased in males of dose group 3 in the recovery group. There was no histopathological correlate for the increased spleen weights. Moreover, regarding spleen weights, the male animals of the main group (F1) did not show any significant differences, when compared to control males. Therefore, the increased spleen weights in high dose males of the recovery group were considered as incidental.

In conclusion, only mild signs of toxicity were obtained at the top dose of 12,000 ppm that is equivalent to the limit dose of 1000 mg/kg bw/day. All findings were reversible after cessation of the test substance administration. Under the conditions of the present study, the no observed adverse effect level (NOAEL) was 3,000 ppm (285.2 mg/kg bw/day in males, 295.3 mg/kg bw/day in females) in both sexes.

 

Applicant's summary and conclusion