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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 April 2003 to 16 September 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to OECD 408. GLP study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
- Name of test material (as cited in study report): OS2200
- Physical state: liquid.
- Analytical purity: 95.3%.
- Lot/batch No.: X173A
- Expiration date of the lot/batch: 20 June 2005.
- Storage condition of test material: at 4ºC and in dry conditions due to the rapid hydrolysis of the test substance.

Test animals

Species:
rat
Strain:
other: Crl: CD® (SD)IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent, England
- Age at study initiation: 44 to 48 days.
- Weight at study initiation: 196 to 275 g for males and 150 to 216 g for females.
- Fasting period before study: no
- Housing: 5 of one sex per cage (stainless steel body with a stainless steel, mesh lid and floor and were suspended above absorbent paper) in a barriered rodent facility.
- Diet (e.g. ad libitum): Ad libitum, standard rodent diet.
- Water (e.g. ad libitum): Ad libitum, tap water.
- Acclimation period: 12 days before treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23 ºC, on rare occasions deviations outside this range occurred resulting in an overall range of 21-26ºC.
- Humidity (%): 40 to 70%, on rare occasions deviations outside this range occurred resulting in an overall range of 24-71%.
- Photoperiod: 12 hr light/ 12 h dark.

IN-LIFE DATES: From20 September 2004 (treatment commenced) to 22 December 2004 (necropsy completed, main study) or to 18 January 2005 (necropsy completed, recovery study).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amounts of test substance were measured out into the final formulation bottles for each dosage group. A sufficient amount of vehicle was supplied in a separate bottle. Within the animal facility just before daily dose administration, starting with the lowest concentration, the required amount of vehicle was withdrawn from the bottle using a syringe and the contents added to the respective final formulation bottle. The contents of the formulation bottle were then stirred with a spatula to remove any test substance adhering to the bottle walls, then mixed with a whirlimixer for approximately one minute to aid dissolution. Each subsequent dose concentration was formulated similarly, in ascending order. Formulation was stirred continuously using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle: All formulations were shown to be homogenous in the vehicle and stable at ambient temperature for 2 days and for 15 days following refrigerated storage (approximately 4ºC).
- Concentration in vehicle: 0, 2.5, 7.5, 25 mg test substance/mL
- Amount of vehicle (if gavage): 2 mL/kg bw.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Due to the rapid hydrolysis of OS2200, the hydrolysis product MIBKO was used to assess the concentration of OS2200. At week 1 and 12, MIBKO concentrations were analyzed in the OS2200 formulations giving at the most -9.2 and + 4.2% deviations from nominal concentration.
Duration of treatment / exposure:
13 weeks.
Frequency of treatment:
Once daily.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw/day (control)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
5 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
15 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
50 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 rats/sex/dose for the main study (13 weeks treatment).
5 rats/sex/dose for the recovery study (13 weeks treatment +4 weeks recovery).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosages were selected in conjunction with previous work with the compound performed in the same laboratories with the Huntingdon Life Sciences report numbers ALS 66/95011 and ALS 77/952298. Results from these studies indicated that a level of 150 mg/kg/day or above would not be tolerated over 13-weeks of treatment and it was predicted that treatment at 50 mg/kg/day for 13 weeks would result in tolerable toxicity.
- Rationale for selecting satellite groups: 3 groups of 5 males and 5 females were treated with 0 (control), 5, 15, 50 mg/kg bw/day for 13 weeks, followed by a 4 week period without treatment to assess recovery from any treatment related effect
- Post-exposure recovery period in satellite groups: 4 weeks

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of ill-health. During the acclimatisation period and recovery Week 1, observations of the animals and their cages were recorded at least once per day and during recovery Week 2 onwards, at least once each week.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily throughout the treatment period, detailed observations were performed immediately before dosing and between one and two hours after completion of dosing of all groups. A further observation was performed as late as possible in the working day during the first week of treatment. In addition, a more detailed weekly physical examination was performed on each animal to monitor general health.Animals were assessed for physical condition and behaviour during handling and after being placed in a standard arena. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behaviour.

BODY WEIGHT: Yes
- Time schedule: The weight of each rat was recorded one week before treatment commenced, on the day that treatment commenced, weekly throughout the treatment and recovery periods before necropsy.

FOOD CONSUMPTION: Yes
- Time schedule: The weight of food supplied to each cage, that remaining and a estimate of any spilled was recorded for the week before treatment started, and each week throughout the treatment and recovery periods, from these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.

FOOD EFFICIENCY: Yes
- Overall group mean values were calculated as the overall group mean bodyweight gain (bodyweight table), divided by the total mean food consumption (calculated from individual cage values), the resultant fraction expressed as a percentage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment commenced, the eyes of all animals allocated to the study (including spare animals) were examined by means of a binocular indirect ophthalmoscope. During Week 12 of treatment the eyes of all animals of control and 50 mg/kg bw/day groups were examined. No ophthalmic examination was undertaken at the end of the recovery phase because no treatment related changes were observed at the Week 12 examination. Prior to each examination, the pupils of each animal were dilated using 0.5% tropicamide ophthalmic solution (Mydriacyl, Alcon Laboratories Ltd.). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

HAEMATOLOGY: Yes (peripheral blood)
- Time schedule for collection of blood: during week 5 and 13 of treatment (before dosing on each occasion).
- Anaesthetic used for blood collection: Yes, isoflurane.
- Animals fasted: Yes, overnight.
- How many animals: All animals.
- Parameters checked: Haematocrit (Hct), Haemoglobin (Hb), Erythrocyte count (RBC), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Total white cell count (WBC), Differential WBC count, Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (PLT), Abnormal morphology, Prothrhombin time (PT), Activated partial thromboplastin time (APTT).
Relating the recovery groups (based on 13 week haematology investigations): all red and white blood cell parameters were assessed for the control group, whereas only red cell parameterswere examined at treatment groups.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during week 5 and 13 of treatment (before dosing on each occasion).
- Animals fasted: Yes, overnight.
- How many animals: all animals.
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin (Bili), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Inorganic phosphorus (Phos), Total protein (Total Prot), Albumin (Alb), Albumin/globulin ratio (A/G Ratio).
Relating to the recovery groups (based on based on 13 week clinical chemistry investigations): Glucose for both sexes and bilirubin and potassium for females.

URINALYSIS: Yes
- Time schedule for collection of urine: during week 13 of treatment and week 4 of recovery.
- Metabolism cages used for collection of urine: Yes .
- Animals fasted: Yes, the afternoon prior to sampling.
- How many animals: all animals.
- Parameters checked: Appearance, Volume (Vol), pH, Specific gravity (SG), Protein (Prot), Glucose (Gluc), ketones (Keto), bile pigments (Bili), haem pigments (Blood) by Multistix, Microscopic examination of the urine sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 5 and 12 (+ recovery animals on week 4 of recovery).
- Dose groups that were examined: Sensory reactivity and grip strength: All recovery phase animals and the first five surviving main study animals from all groups. Motor activity: All recovery phase animals during week 4 of recovery.
- Battery of functions tested: sensory reactivity, grip strength and motor activity assessment.
Sacrifice and pathology:
NECROPSY: The animal killed during the recovery period and those surviving until the end of the scheduled treatment or recovery period were killed by carbon dioxide asphyxiation.

GROSS PATHOLOGY: Yes
All animals were subject to a detailed necropsy.
Full macroscopic examination of the tissues.
All external features and orifices were examined visually. The cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded.
The requisite organs were weighed and external and cut surfaces of the organs and tissues were examined as appropriate.
Organ weights: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Uterus with cervix.
FIxation: Adrenals, Aorta-thoracic, Brain, Caecum, Colon,Duodenum, Epididymides, Eyes#, Femurs with joint, Head#, Heart, Ilum (including Peyer's patch), Jejunum, Kidneys, Liver, Lungs (including bronchi), Lymph nodes (mandibular, mesenteric, caudal), Mammary area, Oesophagus, Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands (submandibular, sublingual), Sciatic nerves, Seminal vesicles, Skin, Spinal cord, Spleen, Sternum, Stomach, Testes, Thymus, Thyroid with parathyroid, Trachea, Urinary bladdes, Uterus und cervix, Any anormal tissue.
# Not process for examination.

HISTOPATHOLOGY: Yes
The relevant tissues were subject to histological processing for all animals.
Adrenals - cortex and medulla
Brain - cerebellum, cerebrum and midbrain
Femur with joint - longitudinal section including articular surface, epiphysial plate and bone marrow
Heart - included auricular and ventricular regions
Kidneys - included cortex, medulla and papilla regions
Liver - section from all main lobes
Lungs - section from two major lobes, to include bronchi
Spinal cord - transverse and longitudinal section at the cervical, lumbar and thoracic levels
Sternum - included bone marrow
Stomach - included keratinised, glandular and antrum in sections
Thyroid - included parathyroids in section where possible
Uterus - uterus section separate from cervix section

PATHOLOGY: Light microscopy:
All tissues preserved were examinated in all animals from control and 50 mg/kg bw/day. Tissues reported at macroscopic examination as being abnormal were examinated for all main and recovery animals.
Statistics:
All statistical analyses were carried out separately for males and females. The test substance was analysed in relation to the control group. Analyses were carried out using the individual animal as the basic experiment unit. Significant differences between Control and treated groups were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
(motor activity at 50 mg/kg bw/day)
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths during the treatment period. However, a single male rat receiving 15 mg/kg/day was killed in extremis due to poor clinical condition during the second week of the recovery period. Clinical signs observed before death comprised aggressive behaviour, a twisted left hind paw with skin encrustation on the digits, piloerection, partially closed eyelids, pale teeth, red discharge from the nose, elevated abnormal gait with a hunched posture, irregular respiration and slight muscle tremors. Macroscopic examination at the post-mortem of this animal also revealed dark adrenal glands, a small and dark thymus, gaseous distension of the gastrointestinal tract, congested lungs and bronchi. The histopathological investigation of this animal did not reveal a conclusive cause of death but ulcerative pododermatitis in the left hind paw was considered to be a contributory factor; thus the death of this animal is not considered to be associated with previous treatment. There were no post dose signs observed during the 13 week treatment period or 4 week recovery period that were considered to be attributable to treatment.

BODY WEIGHT AND WEIGHT GAIN
During the 13 week treatment period, bodyweight gains although variable showed no clear dosage related trend in either sex and it was considered not to have been affected by treatment. During the 4 week recovery period, the overall bodyweight gain was superior or similar to that of control among previously treated male and female groups.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The overall (Weeks 1 to 13) food consumption means among treated female groups were slightly, but statistically significantly, higher than control; although the mean values did not follow a dosage related trend and the differences from control were minor. The overall food consumption for the male treated groups was essentially similar to control, during the 13 week treatment period. During the 4 week recovery period food consumption was marginally higher than control for both sexes previously treated at 50 mg/kg/day and for males previously treated at 15 mg/kg/day. The food intake of the remaining recovery animals was similar to that of their respective controls.

FOOD EFFICIENCY
Overall, food conversion efficiency ratios for all treated groups were considered essentially similar to control. The food conversion efficiency recorded during the 4 week recovery period was variable, resulting in overall values that were superior to that of control for all male treated groups and were inferior to control for females previously treated at 15 mg/kg/day. The food conversion efficiency ratios for the remaining treated groups were considered to be essentially similar to that of their respective controls. The pattern of the overall ratios tended to follow a similar pattern to that of the recovery bodyweight gains. In the absence of any previous effect of treatment on food conversion efficiency these changes were considered not to be related to the previous treatment.

OPHTHALMOSCOPIC EXAMINATION
At the Week 12 ophthalmic examination there was no effect of treatment observed in the eyes of animals treated with OS2200. As such further ophthalmic investigations were not undertaken at the end of the 4 week recovery period.

HAEMATOLOGY
There were clear effects of treatment on red blood cell parameters for both sexes. These were confined to animals receiving 50 mg/kg/day and were observed mainly at the Week 13 investigation. Reversibility of these findings was evident after a 4-week period without treatment.
In Week 5 it was recorded a higher mean reticulocyte value recorded for females receiving 50 mg/kg/day. A similar difference was again noted for these females in Week 13, with the magnitude of differences from control being greater in Week 13 than in Week 5. A higher mean reticulocyte value was also recorded for males receiving 50 mg/kg/day in Week 13. In addition, in Week 13 both sexes treated at 50 mg/kg/day showed lower than control mean red blood cell values, with the males also showing lower than controls mean haemoglobin (Hb) and haematocrit (Hct) values. Associated with these effects on primary red blood cell parameters, in Week 13, were slightly higher than control mean cell haemoglobin (MCH) and mean cell volume (MCV) mean values recorded for females receiving 50 mg/kg/day. There were no treatment-related effects on blood film comments. Recovery from the red cell effects was clearly evident by the end of the 4-week period without treatment. At the end of the recovery period mean Hb and RBC values for females which had previously been treated at 50 mg/kg/day, were slightly higher than controls. This was considered to be due to an over compensation of red blood cell production following cessation of treatment, which was also reflected in the slightly lower than control mean reticulocyte value recorded for these females. At the recovery investigations females from other previously treated groups showed higher than control red cell parameters and lower reticulocyte values, of a similar magnitude to females previously treated at 50 mg/kg/day. In the absence of any effects in these groups during treatment, these differences were considered to be indicative of normal biological variation associated with the small group size rather than associated with previous treatment. There was considered to be no clear effect of treatment on clotting parameters for both sexes. In Week 13, females receiving 50 mg/kg/day also showed a higher than control mean platelet value, however, the degree of difference from controls was minor and individual values for these treated females were in the range of individual control values. Thus, this difference was not considered to be due to treatment. In Weeks 5 and 13 males receiving 50 mg/kg/day showed lower mean PT and APTT values, compared with controls. The magnitude of the difference from control for the PT values decreased with time, such that in Week 13 the mean was 96% of the control value and similar to the mean for males receiving 15 mg/kg/day. Consequently this difference from control was considered not to be of toxicological importance. Between Week 5 and 13, the degree of difference from control for mean APTT increased, however, the difference in Week 13 was considered to be mainly due to a low value for a single male (No. 69), unduly influencing the group mean; with the individual values for the remaining males at 50 mg/kg/day being similar to controls. There were no other differences from controls, including white blood cell parameters and other statistical significances, which were considered be treatment related.

CLINICAL CHEMISTRY
In Week 13 only, females receiving 15 or 50 mg/kg/day showed higher than control mean bilirubin values. The magnitude of the difference from controls was similar for both groups. Full recovery in bilirubin values were seen by the end of the recovery period. Females receiving 50 mg/kg/day showed lower than control mean potassium values in Weeks 5 and 13, with the difference from control increasing over time. There was no difference from control for potassium at the end of the recovery period. In addition, in Week 5 only, these females also showed a slightly lower than control mean sodium value. All but one individual sodium value for these females was within the concurrent control range, thus this difference from control was considered not to be treatment-related. Both sexes receiving 50 mg/kg/day showed differences from controls for mean glucose values in Week 13. Males showed a higher than control mean value, whilst conversely females showed a lower than control mean value of similar magnitude. At the end of the recovery period similar differences from controls were again exhibited for these groups, with females which had previously received 15 mg/kg/day also showing a lower than control mean value. Due to the sexes showing opposite trends, the wide degree of variation in individual values and the mean value for females previously treated with 15 mg/kg/day showing a lower mean at recovery than females which had received 50 mg/kg/day in Week 13, the differences from controls in glucose values were considered not to be associated with treatment. In Week 5 slightly lower than control mean creatinine values were noted for all male treated groups, which attained statistical significance, with all male treated groups having the same mean value. The majority of individual creatinine values for the treated male animals were, however, within the range of the individual control values, thus this change was not considered to be of toxicological importance. In Week 13 slightly lower than control AST values were recorded for all treated male groups, but this change was considered to be fortuitous as the mean values did not follow a dosage related trend and all the individual values (except for one male receiving 50 mg/kg/day) were within the range for the individual control values, thus this change was not considered to be related to treatment.
There were no other differences from controls, including those that attained statistical significances, which were considered to be treatment related.

URINALYSIS
There were no effects of treatment on urine parameters for males at the end of the treatment period. Females receiving 15 or 50 mg/kg/day did however show higher than control mean protein values both at the end of the treatment and recovery periods. These differences from controls did not follow a dosage relationship and the majority of individual values recorded for these females were within or close to the concurrent control ranges and thus an effect of treatment is considered to be doubtful. All other parameters are considered essentially similar to control.

NEUROBEHAVIOUR
At the week 5 and 12 assessments of sensor reactivity and grip strength, there were no effects of treatment observed in animals treated with OS2200, as no effects were observed, no further assessment was undertaken during the recovery period. Males treated at 50mg/kg/day showed higher low beam scores at several times during the week 12 assessment. Total scores for males receiving 50mg/kg/day were also significantly higher than control. However high beam scores were only marginally higher than control, no similar change was observed among treated females at this dose level and also no treatment related clinical signs were observed during the week 13 treatment period, thus these changes were considered to be of minor toxicological importance.

ORGAN WEIGHTS
Analysis of the organ weights for animals killed after completion of 13 weeks of treatment revealed a statistically significantly higher than control mean spleen weight (adjusted for bodyweight) for males receiving 50 mg/kg/day. Recovery was evident at the end of the 4 week recovery period with spleen weights for males previously treated at 50 mg/kg/day being comparable with control. Higher than control mean heart weights were recorded for females treated at 5 and 15 mg/kg/day, while heart weights for females treated at 50 mg/kg/day were comparable with control, thus, as no microscopic changes were observed in the heart, this change was not considered to be associated with treatment. Analysis of organ weights for animals killed at the end of the 4 week recovery period revealed a statistically significantly lower than control mean spleen weight (adjusted for bodyweight) for females previously treated at 50 mg/kg/day. In addition, slightly higher than control adrenal weights were recorded for previously treated male groups when compared with control. However, the mean values did not follow a dosage related trend; thus, in the absence of treatment related microscopic changes being observed in the adrenals, these differences from control are not considered to be associated with previous treatment. All other parameters are considered essentially similar to control

GROSS PATHOLOGY
The macroscopic examination for animals killed after completion of 13 weeks of treatment with OS2200, or 4 weeks of recovery, did not reveal any macroscopic changes which could be attributed treatment. The incidence and distribution of all the findings were considered to fall within the background range of macroscopic changes.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related findings:
Kidney: increased severity and/or incidence of cortical tubules with hyaline droplets and cortical tubular basophilia were seen in males treated at 15 or 50 mg/kg/day of OS2200, when compared with controls. Increased incidence only of cortical tubular basophilia was seen in males treated at 5 mg/kg/day OS2200 when compared with controls. Increased incidence and severity of medullary tubular basophilia was seen in males treated at 50 mg/kg/day OS2200, when compared with controls. Tubular basophilia was unaffected in female rats treated with OS2200. Spleen: Increased incidence and severity of extramedullary haemopoiesis was seen in all OS2200 treated groups in both sexes, when compared with controls. Dosage-relationships were not evident. Increased incidence and severity of haemosiderosis was seen in males and females receiving 50 mg/kg/day of OS2200, when compared with controls.
Recovery phase:
Kidney: Cortical tubules with hyaline droplets were not present in any of recovery animals. Recovery had occurred for cortical tubular basophilia or medullary tubular basophilia in all previously treated groups. Spleen: Extramedullary haemopoiesis and haemosiderosis had broadly resolved in both males and females.

Effect levels

Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: (red blood cell rate of turnover resulting in associated changes in the spleen)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Histopathology: Treatment-related findings

Kidney:

Dose (mg/kg bw/day

Male

0

5

15

50

Cortical tubules with hyaline droplets

Minimal

1

0

4

6

Slight

0

0

0

3

Total

1

0

4

9b

Cortical tubular basophilia

Minimal

2

6

7

7

Slight

0

0

1

1

Total

2

6

8a

8a

Medullary tubular basophilia

Minimal

2

4

2

4

Slight

0

0

0

1

Total

2

4

2

5

Total number of kidneys examined

10

10

10

10

Dose (mg/kg bw/day

Male (recovery)

0

5

15

50

Cortical tubules with hyaline droplets

Minimal

3

5

3

4

Slight

0

0

0

0

Total

3

5

3

4

Medullary tubular basophilia

Minimal

3

1

2

1

Slight

0

0

0

0

Total

3

1

2

1

Total number of kidneys examined

5

5

4

5

Spleen:

Dose (mg/kg bw/day

Male

Female

0

5

15

50

0

5

15

50

Extramedullary haemopoiesis

Minimal

2

6

4

7

3

3

7

5

Slight

0

2

2

2

1

6

2

5

Moderate

0

0

0

1

0

0

0

0

Total

2

8a

6

10b

4

9

9

10a

Haemosiderosis

Minimal

3

2

4

8

3

5

4

1

Slight

0

2

0

2

1

0

3

4

Moderate

0

0

0

0

1

0

0

5

Total

3

4

4

10b

5

5

7

10a

Total number of spleens examined

10

10

10

10

10

10

10

10

Dose (mg/kg bw/day

Male (recovery)

Female (recovery)

0

5

15

50

0

5

15

50

Extramedullary haemopoiesis

Minimal

4

2

4

3

3

2

1

1

Slight

0

0

0

0

1

1

0

0

Total

4

2

4

3

4

3

1

1

Haemosiderosis

Minimal

1

0

1

1

4

2

3

3

Slight

2

2

0

3

1

2

1

2

Total

3

2

1

4

5

4

4

5

Total number of spleens examined

5

5

4

5

5

5

5

5

a-p<0.05, b-p<0.01 with Fisher’s Exact test, on total incidences only.

After oral administration of OS2200 to the rats for 13 weeks, changes were seen in the kidney and spleen. In the male kidney, cortical tubules with hyaline droplets and cortical tubular basophilia were identified with a severity and/or higher incidence in rats treated at 15 or 50 mg/kg/day OS2200. In the spleen, there were increased incidence and severity of extramedullary haemopoiesis in all treated groups of both sexes, compared with controls. Also increased incidence and severity of haemosiderosis were seen in animals treated at 50 mg/kg/day of OS2200 in both sexes, when compared with controls. In the recovery, all lesions of kidney and spleen had resolved.

Applicant's summary and conclusion

Conclusions:
The NOAEL for OS-2200 after 13 weeks of oral exposure to rats was determined to be 15 mg/kg bw/day based on the effects on the red blood cell rate of turnover occurring mainly among animals receiving 50 mg/kg bw/day and resulting in associated changes in the spleen.
Executive summary:

A 13 week repeated dose toxicity test followed by a 4 week recovery period was performed to assess the systemic toxicity of OS 2200 under GLP conditions. The test was performed in rats by oral gavage, three groups, each comprising then male and ten female rats received OS2200 at dosages of 5, 15 or 50 mg/kg bodyweight/day. A similarly constituted group received the vehicle, corn oil, at the same volume-dosage. A further five male and five female rats were assigned to each of the groups, these animals were treated for 13 weeks, followed by a 4 week period without treatment to assess recovery from any treatment related effect. During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, ophthalmic examination, haematology, blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken. It was concluded that the principal action of OS2200 was to affect the red blood cell rate of turnover occurring mainly among animals receiving 50 mg/kg/day, resulting in associated changes in the spleen. Due to the high incidence of medullary haemopoesis observed in the spleen in both sexes among all treated groups, a NOEL was not established for OS 2200 in this study. A dosage level of 15 mg/kg/day, however, was considered to be the highest NOAEL for OS 2200 on this study, as the changes observed at 5 and 15 mg/kg/day were considered to be minor in nature and had shown full recovery after 4 weeks without treatment.