3,10-diamino-6,13-dichloro-2-((6-(((4-(1,1-dimethylethyl)phenyl)sulfonyl)amino)-2-naphthalenyl)sulfonyl)-4,11-triphenodioxazinedisulfonic acid, lithium potassium sodium salt

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 18, 2001 to May 24, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

Directive 96/54/ EC, B. 7. "Repeated dose (28 days) toxicity (oral)", September 30, 1996.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

"Repeated Dose 28-Day Oral Toxicity Study in Rodents", OECD Guidelines for the Testing of Chemicals, Section 4, Health Effects, Number 407, July 27, 1995.

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines for Screening, Toxicity Testing of Chemicals : Testing Methods for new Substances, enacted July 13, 1974, amended December 5, 1986

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

440-770-9

EC Name:

-

Cas Number:

371921-63-0

Molecular formula:

C38 H29 Cl2 N5 O12 S4 .x K .x Li .x Na

IUPAC Name:

3,10-diamino-2-{[6-(4-tert-butylbenzenesulfonamido)naphthalen-2-yl]sulfonyl}-6,13-dichloro-5,12-dioxa-7,14-diazapentacene-4,11-disulfonic acid lithium hydride potassium hydride sodium hydride

Test material form:

solid

Test animals

Species:

rat

Strain:

other: HanBrLWIST (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd; Biotechnology & Animal Breeding Division CH-4414; Fulllinsdorf/Switzerland
- Age at study initiation: 6 weeks
- Weight at study initiation: At acclimatization: Males: 118-148 grams (mean 133 grams); Females: 99- 122 grams (mean 110.5 grams)
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding ('Lignocel' Schill AG, CH-4132 Muttenz/Switzerland).
- Diet (e.g. ad libitum): Pelleted standard Provimi Kliba 3433 (batch no. 74/01 and 76/01) rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/ Switzerland) was available ad libitum. The feed batch was analyzed for contaminants
- Water (e.g. ad libitum): Community tap-water from Itingen was available ad libitum in water bottles. Results of bacteriological assay, chemical and contaminant analyses of representative samples are attached to the report

- Acclimation period: October 2001: 25-31

DETAILS OF FOOD AND WATER QUALITY: None of the contaminants analyzed in the water and diet is considered to have been present at a concentration which would have affected the validity of the results

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
Allocation A (toxicity testing: termination after 28 treatment days) : 25 October 2001 to 29 November 2001
Allocation B (recovery testing: termination after an additional 14-day recovery period): 25 October 2001 to 13 December 2001

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

Rate of preparation of test item formulations: weekly.
BLUE GS 5664.80 was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using a magnetic stirrer and stored at room temperature (17-23°C) in glass beakers.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- PEG 300
- Lot/batch no. 424718/I 1370 I

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability (after 2 hours and 7 days) of the dose formulations were determined in samples taken after experimental start. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment. The analyses were performed by RCC Ltd (Environmental Chemistry & Pharmanalytics Division) according to HPLC method.

The mean concentrations of the homogeneity samples were found to be 110.1%, 112.1%, and 112.6% of the nominal concentrations of dose group 2 (10 mg/ml), dose group 3 (40 mg/ml), and dose group 4 (200 mg/ml), respectively. The individual concentrations varied in the range from -2% to +1 % of the mean concentrations. Therefore, the test item was found to be homogeneously distributed in the vehicle.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 for Allocation A (toxicity testing: termination after 28 treatment days)
5 additional only for 0 and 1000 mg/kg bw/d groups for Allocation B (recovery testing: termination after an additional 14-day recovery period)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based upon the results of a non-GLP 5-day dose- range-finding study (RCC Study Number 816028) in which BLUE GS 5664.80 was administered by gavage to 2 rats per group and sex.
- Rationale for selecting satellite groups: not specified
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were observed for clinical signs once before commencement of administration; twice daily on days 1-3; as well as once daily on days 4-28; and once daily during days 29-42 (recovery).
- Cage side observations checked: Appearance (piloerection, salivation and hunched posture); Motor (ataxia, tremor/twitching and prostration); Behavioral (hyperactivity and somnolence); Respiration (dyspnea, tachypnea and bradypnea); Miscennaneous (feces dark)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly during pretest, treatment and recovery and before necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: The food consumption was recorded once during the pretest period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.

FOOD EFFICIENCY: No

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 and 6 weeks
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes: The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum
- How many animals: all animals
- Parameters checked: Erythrocyte count; Hemoglobin; Hematocrit; Mean corpuscular volume; Mean corpuscular hemoglobin; Mean corpuscular hemoglobin concentration; Platelet count; Reticulocyte count; Reticulocyte fluorescence ratios; Nucleated erythrocytes (normoblasts); Heinz bodies; Methemoglobin; Total leukocyte count; Differential leukocyte count; Red blood cell morphology; Thromboplastin time (=prothrombin time); Activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 and 6 weeks
- Anaesthetic used for blood collection: Yes (isoflurane)
- How many animals: all animals
- Parameters checked: Glucose; Urea; Creatinine; Bilirubin, total; Cholesterol, total; Triglycerides; Phospholipids; Aspartate aminotransferase; Alanine aminotransferase; Lactate dehydrogenase
Creatine kinase; Alkaline phosphatase; Gamma-glutamyl transferase; Calcium; Phosphorus; Sodium; Potassium; Chloride; Albumin; Protein, total; Globulin; Albumin/Globulin ratio

URINALYSIS: Yes
- Time schedule for collection of urine: after 4 and 6 weeks
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes: Urine was collected during the 18-hour fasting period into a specimen vial.
- Parameters checked: Volume (18-hour); Specific gravity; Osmolality; Color; Appearance; pH; Protein; Glucose; Ketone; Bilirubin; Blood; Nitrite; Urobilinogen; Urine sediment; Red blood cells; White blood cells; Crystals (Triple phosphate)

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER:
Mortality/viablity: twice daily

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- All animals
- Necropsy after 4 weeks (allocationA); after 6 weeks (allocationB)
- Tissus and organs collected: Adrenal glands; Aorta; Bone (sternum, femur including joint); Bone marrow (femur); Brain (cerebrum, cerebellum, pons); Cecum; Colon; Duodenum; Epididymides (fixed in Bouin's solution); Esophagus; Eyes with optic nerve (fixed in Davidson's solution); Harderian gland (fixed in Davidson’s solution); Heart; Ileum, with Peyer's patches Jejunum with Peyer's patches; Kidneys; Larynx; Lacrimal gland (exorbital); Liver; Lungs (infused with formalin at necropsy); Lymph nodes (mesenteric, mandibular); Mammary gland area Nasal cavity; Ovaries; Pancreas; Pituitary gland; Prostate gland; Rectum; Salivary glands (mandibular, sublingual); Sciatic nerve Seminal vesicles; Skeletal muscle Skin; Spinal cord (cervical, midthoracic, lumbar); Spleen; Stomach; Testes (fixed in Bouin's solution); Thymus; Thyroid (incl. parathyroid gland); Tongue; Trachea; Urinary bladder (infused with formalin at necropsy); Uterus; Vagina; Gross lesions

ABSOLUTE AND RELATIVE ORGAN WEIGHTS
The following organ weights were recorded on the scheduled dates of necropsy: Brain; Heart; Liver; Thymus; Kidneys; Adrenals; Spleen; Testes; Epididymides; Ovaries

HISTOPATHOLOGY: Yes
- Slides of the following organs and tissues which were collected at scheduled sacrifice from the animals of control and high-dose groups were examined by a pathologist: Adrenal glands; Bone marrow (femur); Brain (cerebrum, cerebellum, pons); Cecum; Colon; Duodenum; Epididymides (fixed in Bouin's solution); Heart; Ileum, with Peyer's patches Jejunum with Peyer's patches; Kidneys; Liver; Lungs (infused with formalin at necropsy); Lymph nodes (mesenteric, mandibular); Ovaries; Prostate gland; Rectum; Sciatic nerve Seminal vesicles; Spinal cord (cervical, midthoracic, lumbar); Spleen; Stomach; Testes (fixed in Bouin's solution); Thymus; Thyroid (incl. parathyroid gland); Trachea; Urinary bladder (infused with formalin at necropsy); Uterus; Vagina; Gross lesions.
- If discoloration of the tissues occurred which was attributable to physical properties of the test item (ie a dyestuff), representative additional slides were taken from one male and one female in each of the affected groups.

Other examinations:

DATA CALCULATION

FOOD CONSUMPTION
The food consumption was calculated per rat and per food consumption interval. It expresses the average food consumed per animal and per day over the food consumption interval.
FC = C/AD
where
FC is Food consumption in grams of food per animal and day;
C is measured food consumption in grams per cage over the consumption interval and AD is total consumption days over all animals in the cage during the consumption interval.

RELATIVE FOOD CONSUMPTION
The relative food consumption was calculated according to the following formula:
RFC = [FC/BW(i)]x1000
where
BW(i) is the most ideal body weight in grams or the body weight (of the corresponding rats) recorded on the day most close to the middle of the food consumption interval. In cases of equal "closeness" of two body weight records the later one was chosen;
RFC is relative food consumption in grams of food per kg body weight and day, and FC is food consumption in grams of food per animal and day.

Statistics:

The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, organ weights and ratios, as well as clinical laboratory data :
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution. Student’s t- test was applied to grip strength and locomotor activity.
• Student’s t-test was applied to grip strength and locomotor activity.
• Fisher's exact-test was applied to the macroscopic findings.
• Armitage/Cochran Trend Test was used for non-neoplastic lesions, if appropriate.

References :
- C.W. Dunnett: A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
- R.G. Miller: Simultaneous Statistical Inference, Springer Verlag, New York (1981).
- R.A. Fisher: Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).
- P. Armitage: Test for linear trends in proportions and frequencies. Biometrics 11, 375-386 (1955).

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

GENERAL CAGESIDE OBSERVATIONS (DAILY)
Slight to moderate dark feces was noted in all test item treated animals from the second until the last day of treatment or until the second day of the recovery period. This was due to a passive effect of the test item, a dyestuff

DETAILED CLINICAL OBSERVATIONS (WEEKLY)
Mydriasis in the right eye was observed in one female treated with 200 mg/kg/day of the test item during the first week of treatment. This effect was considered to be incidental because it was seen only in one female of one dose group.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weights and mean body weight gain of females treated with 200 mg/kg/day of the test item were significantly increased compared with controls in treatment week three. This effect was considered to be incidental because it was not dose dependent, only seen in females and reversible in week four.
No changes in mean body weights or mean body weight gain were observed in males compared with controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean daily and relative food consumption of the test item-treated animals were comparable with these of the control animals.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes in parameters of hematology were observed.
The thromboplastin time was significantly (p<0.01) decreased in females treated with 50 mg/kg/day after four weeks compared with controls. This decrease was considered to be incidental, because it was not dose dependent.
A significant decrease (p<0.05) of absolute and relative segmented neutrophils was measured in males treated with 200 mg/kg/day of the test item compared with controls. This was considered to be incidental because no dose response-relationship was noted and it was not seen in females.
The high reticulocyte fluorescence ratio was significantly (p<0.01) increased in males treated with 1000 mg/kg/day compared with the control. The low reticulocyte fluorescence ratio was significantly (p<0,05) decreased in males of the same dose group. This left-shift was considered to be incidental because these values were within the range of the historical control data and no changes were seen in females.
No further changes in parameters of hematology were observed after four weeks in males or females compared with control animals.
All differences in parameters of hematology observed during the recovery period (after 6 weeks) were considered to be incidental because the values were within the range of the historical control data.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes in parameters of clinical biochemistry were noted.
A significantly (p<0.01) increased level of creatinine was observed in females treated with 1000 mg/kg/day after 4 weeks. This was considered to be incidental because this value lies within the range of the historical control data.
The chloride value was significantly (p<0.05) increased in males treated wrr 200 mg/kg/day, after 4 weeks compared with control animals. This was considered to be nc rest item-related because the value lies within the range of the historical data and it was not seen in females.
The value of protein was significantly (p<0.05) decreased in males treated with 200 mg/kg/day after 4 weeks compared with controls. This was considered to be also incidental because the value lies within the range of historical data.
The level of globulin was significantly decreased in males treated with 200 (p<0.01) and 1000 (p<0.05) mg/kg/day after 4 weeks compared with control animals. This was considered to be incidental because the values lie within the range of the historical data. The albumin to globulin ratio was significantly (p<0.05) increased in males treated with 200 mg/kg/day and in males treated with 1000 m/kg/day after 4 weeks compared with controls. This calculated value was considered to be incidental because the values lie within the range of the historical data and were due to the differences in globulin levels.
The lactate dehydrogenase was significantly increased in females treated with 50 (p<0.01), 200 (p<0.05) and 1000 (p<0.01) mg/kg/day after 4 weeks compared with control animals. This was considered to be not test item related because the values are within the range of the historical data.
The creatin kinase was significantly (p<0.05) increased in females treated with 1000 mg/kg/day compared with controls after 4 weeks. This was considered to be incidental because the value lies within the range of the historical data.
The level of phosphorus was significantly (p<0.01) decreased in females treated with 1000 mg/kg/day after 4 weeks compared with control animals. This was considered to be incidental because the value lies within the range of the historical data. Sodium was significantly (p<0.05) decreased in females of the same dose group after 4 weeks compared with controls. This was considered to be also incidental because the value lies within the range of the historical data. Potassium was significantly increased in females treated with 50 (p<0,05), 200 (p<0.01) and 1000 (p<0,01) after 4 weeks compared with controls. This was considered to be not test item related because these values lie within the range of the historical data.
All differences in parameters of clinical biochemistry observed during the recovery period (after 6 weeks) were considered to be incidental because the values lie within the range of the historical control data.
No further changes in parameters of clinical biochemistry were observed in males or females.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes in parameters of urinalysis were noted.
The 18h urine-volume was significantly (p<0.05) increased in males treated with 1000 mg/kg/day after 4 weeks compared with control animals. The specific gravity and the osmolality were significantly (p<0.01) decreased in the same dose group after 4 weeks compared with controls. These effects were considered to be not test item-related because all lie within the range of the historical data.
The pH was significantly (p<0.05) increased in females treated with 1000 mg/kg/day after 4 weeks compared with controls. This was considered to be incidental because the value lies within the range of the historical data.
Female no. 56 was excluded from the group mean calculation and statistical analysis because it was considered as an outlier concerning the recorded urine volume/18 h after 4 weeks of treatment. The outlier was considered to be the result of dilution with drinking water during the time of urine collection.
No further changes in parameters of urinalysis were observed.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No clinical signs were observed during functional observation battery (week 4).

Grip Strength
Slightly decreased (p<0.05) hind limb grip strength was noted in males treated with 200 or 1000 mg/kg/day of the test item. Slightly decreased fore limb grip strength was observed in females treated with 200 mg/kg/day of the test item. These effects were considered to be incidental because they were seen either in one dose group or in one sex only.

Locomotor Activity
Slightly decreased locomotor activity (p<0.05) recorded from 15 to 30 minutes was noted in males treated with 1000 mg/kg/day. This was reversible when observed over 45 or 60 minutes. Slight decreased locomotor activity (p<0.05) recorded from 0 to 15, 15 to 30 or 45 to 60 minutes was observed in females of the same dose group. This effect was considered to be not test item related because it was not dose dependent, similar values were seen in females treated with 50 mg/kg/day.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

A significantly (p<0.05) decreased absolute liver weight was noted in males treated with 1000 mg/kg/day after 6 weeks compared with the control group. This was considered to be incidental because no changes were observed in liver to body- or liver to brain-weights.
A significantly (p<0.01) increased liver to brain weight ratio was observed in females of the same dose group after 6 weeks compared with the control animals. This was due to a relative high absolute liver weight of females of this group and considered to be not test item- related because no significant differences were observed in the absolute liver weight and in the liver to body weight ratio in females of this dose group and this effect was not seen in males.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At necropsy, bluish discoloration of the mucosa, of the stomach and/or small and large intestine was recorded in a few males of males treated with 200 or 1000 mg/kg/day, and females treated with 1000 mg/kg/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

A small number of microscopic findings were noted in the organs and tissues examined at the termination of the 28-day treatment period and after the recovery period. The type, incidence and severity of these findings did not distinguish test item treated rats from control rats.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

None of the observed effects has been considered as adverse.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Oral administration of BLUE GS 5664.80 to Wistar rats at doses of 50, 200 and 1000 mg/kg/day, performed according to OECD 407/method EC B7, for 28 days resulted in the following:
Based on the results of this study, 1000 mg/kg body weight/day of BLUE GS 5664.80 was established as the no-observed-effect-level (NOEL) as well as the no-observed-adverse- effect-level (NOAEL).

Executive summary:

In this subacute toxicity study performed according to OECD 407/method EC B7, BLUE GS 5664.80 was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 50, 200 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, PEG 300, only.

The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4.

At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals.

MORTALITY / VIABILITY

All animals survived until scheduled necropsy.

CLINICAL SIGNS

No test item-related clinical signs of relevance for the toxicological evaluation were observed during daily or weekly observations (week 1-3).

FUNCTIONAL OBSERVATIONAL BATTERY

No test item-related findings of relevance for the toxicological evaluation were observed during functional observational battery (week 4).

Grip Strength

No test item-related changes in fore- or hind-limb grip strength were observed.

Locomotor Activity

No test item-related differences in locomotor activity were noted.

FOOD CONSUMPTION

The mean daily and relative food consumption of the test item-treated animals were comparable with these of the control animals.

BODY WEIGHT

No test item related changes in mean body weights or mean body weight gain were observed compared with controls.

CLINICAL LABORATORY INVESTIGATIONS Hematology

No test item-related changes in the hematology parameters were observed.

Clinical Biochemistry

No test item-reiated differences in parameters of clinical biochemistry were evident.Urinalysis

No test item-related changes in parameters of urinalysis were observed.

ORGAN WEIGHTS

No test item-related differences in organ weights were noted when compared with the control animals.

MACROSCOPIC / MICROSCOPIC FINDINGS

At necropsy, bluish discoloration of the mucosa, of the stomach and/or small and large intestine was recorded in a few males of males treated with 200 or 1000 mg/kg/day, and females treated with 1000 mg/kg/day.

No microscopic findings, considered to be treatment related, were noted in this study. No findings, corresponding to the aforementioned bluish discoloration, were noted.

All findings noted in this study were considered to be spontaneous findings commonly observed in rats of this breed and age.

ASSESSMENT

Oral administration of BLUE GS 5664.80 to Wistar rats at doses of 50, 200 and 1000 mg/kg/day, for 28 days resulted in the following:

Based on the results of this study, 1000 mg/kg body weight/day of BLUE GS 5664.80 was established as the no-observed-effect-level (NOEL) as well as the no-observed-adverse- effect-level (NOAEL).