Blue MGi 1037-Na (notification presscake)

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:

5 June 2000 - 2 August 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

27 July 1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Directive 69/54/EEC, B.7

Version / remarks:

30 September 1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test animals

Species:

rat

Strain:

other: HanRcc:WIST (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 Füllinsdorf/Switzerland
- Age at study initiation: 6 weeks
- Weight at study initiation: males: 118-162 g; females: 104-143 g
- Fasting period before study: yes
- Housing: in groups of five in Makrolon type-4 cages
- Diet: ad libitum, pelleted standard Provimi Kliba 3433 rat maintenance diet
- Water: ad libitum), tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

bi-distilled water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item formulations were prepared weekly. BLUE MGi 1037 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). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

Analytical verification of doses or concentrations:

yes

Remarks:

HPLC

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 during acclimatization. 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 a HPLC method supplied by the Sponsor.

Duration of treatment / exposure:

28 days

Frequency of treatment:

7 days/week

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:

10 (groups 1 and 4 ) or 5 (groups 2 and 3)

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 in which the substance was administered by gavage to 2 rats per group and sex. Other than dark feces at the highest dose level, animals treated with 200, 600 or 1000 mg/kg bw/day showed no reaction to treatment.
- Fasting period before blood sampling for clinical biochemistry: yes
- 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: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION
- recorded once during the pre-test period and weekly thereafter

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of exposure period (4 weeks)
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: all
- Parameters checked: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count PLATELETS, Reticulocyte count, Reticulocyte fluorescence ratios Nucleated erythrocytes (normoblasts), Heinz bodies, Methemoglobin, Total leukocyte count, Differential leukocyte count, Red blood cell morphology, thromboplastin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of exposure period (4 weeks)
- Animals fasted: Yes
- How many animals: all
- Parameters checked: Glucose, Urea, Creatinine, Uric Acid, Bilirubin, total, Cholesterol, total, Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Creatine, 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 weeks and after 6 weeks
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters checked: osmolarity, pH, protein, glucose, ketone, bilirubin, blood and urobilinogen, volume, specific gravity, color appearance, ketone, bilirubin, blood nitrite, sediment, red blood cells, white blood cells, crystals

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4
- Dose groups that were examined: all animals
- Battery of functions tested: grip strength / locomotor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Adrenal glands, Aorta, Bone (sternum, femur including joint), Bone marrow (femur), Brain (telecephalon, 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

HISTOPATHOLOGY: Yes
Bone marrow (femur), brain, cecum, colon, duodenum, epididymes, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes, ovaries, prostate gland, rectum sciatic nerve, seminal vesicles, spindal cord, spleen, stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus, vagina, gross lesions

Slides of these organs were collected at scheduled sacrifice from the animals of control and high-dose groups were examined by a pathologist. As test item-related morphologic changes were detected in the kidney and stomach of animals treated with 1000 mg/kg/day, these organs from animals of the mid- and low-dose groups were examined.

Statistics:

The following statistical methods were used to analyze grip strength, locomotor activity, body weights, organ weights and all 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 locomotor activity and grip strength.
• Fisher's exact-test was applied to macroscopic findings.
• Armitage/Cochran Trend Test for non-neoplastic lesions.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinical signs of toxicity were noted at any dose level. Dark feces were noted during daily observations in males and females treated with 200 mg/kg bw/day and 1000 mg/kg bw/day. These findings were considered to be passive test item-related findings which commonly occur following oral administration of a dyestuff, rather than an indication of toxicity.

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:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Significantly lower red blood cell count (p<0.05), hemoglobin levels (p<0.01) and hematocrit (p<0.01) were noted in males treated with 1000 mg/kg/day for four weeks. Significantly higher absolute and relative reticulocyte counts (p<0.01) were also recorded in the males treated with 1000 mg/kg/day. Although these differences were within the 95% tolerance limits of the historical control data, they were close to the lower or upper limits of the ranges. These changes were considered to be evidence of very slight anemia with compensatory reticulocytosis, but these findings were reversible after two weeks' recovery.
Males treated with 50 mg/kg/day or 200 mg/kg/day for four weeks were not affected.
Females treated with 50 mg/kg/day, 200 mg/kg/day or 1000 mg/kg/day for four weeks had slightly lower mean cell hemoglobin when compared with the controls. Although these differences attained statistical significance at 50 mg/kg/day (p<0.01) and 1000 mg/kg/day (p<0.05), they were not dose-dependant and therefore considered to be incidental changes. The mean prothrombin time of the test item-treated females was abbreviated when compared with the controls, and the differences attained statistical significance at 50 mg/kg/day (p<0.01) and 1000 mg/kg/day (p<0.05). These differences were well within the 95% tolerance limits of the historical control data and therefore considered to be incidental.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related changes included significantly lower total protein levels (p<0.01) and significantly lower globulin levels (p<0.01) in males and females treated with 1000 mg/kg/day when compared with the control values. The albumin levels were significantly lower in females treated with 1000 mg/kg/day when compared with the control values, whereas the males were unaffected. These findings were largely reverted after the two-week recovery period, although some differences remained. The remaining differences from the control values (higher glucose levels in males treated with 50 mg/kg/day or 1000 mg/kg/day; higher uric acid levels in males and females at 1000 mg/kg/day; lower levels of total bilirubin in females treated at 1000 mg/kg/day, higher levels of total cholesterol in males treated with 1000 mg/kg/day; higher phospholipids in males at 1000 mg/kg/day; lower gamma glutamyl transferase activity in males treated at 1000 mg/kg/day; higher phosphorus levels in males treated with 1000 mg/kg/day; lower chloride levels in males treated with 1000 mg/kg/day) were either well within the 95% tolerance limits of the historical control data, not supported by concomitant differences in related parameters or not observed in the opposite sex and therefore considered to be fortuitous.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

After four weeks' treatment, males treated with 1000 mg/kg/day had significantly higher specific gravity (p<0.05), higher osmolality (p<0.05) and higher pH (p<0.05) when compared with the controls. These changes were considered to be test item-related effects upon the ability of the kidney to concentrate urine. In females treated with 1000 mg/kg/day, a slight but statistically significant increase in the urinary pH was noted, when compared with controls. Ketone, considered likely to be a metabolic byproduct, was also present in the urine of males treated with 1000 mg/kg/day. All other differences from the control values noted after four week's treatment were considered to be fortuitous. All values compared favorably after two weeks' recovery.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

The mean locomotor activity of the males and females treated with 1000 mg/kg bw/day was reduced when compared with the control animals. The difference from the controls attained statistical significance in males during the first two measurement intervals (p<0.01: 0-15 minutes; p<0.05: 15-30 minutes), and in females during the first measurement interval (p<0.01, 0-15 minutes). The overall mean locomotor activity of the males was reduced significantly (p<0.01) when compared with the controls. These differences were considered to be test item related. The animals treated with 50 mg/kg bw/day or 200 mg/kg bw/day were unaffected.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Changes in the kidney weights of males treated with 200 mg/kg/day and 1000 mg/kg/day for four weeks were considered to be test item-related. All weights and ratios exceeded those of the control males, and in several cases, attained statistical significance (absolute kidney weights at 200 mg/kg/day, p<0.01; kidney-to-body weight ratio at 200 mg/kg/day and 1000 mg/kg/day, p<0.05), and were considered to be indications of slight toxicity. After the two-week recovery period, the differences in absolute and relative kidney weights were partly reversible in the males previously treated with 1000 mg/kg/day when compared with the controls. The absolute and relative kidney weights of the females treated with 1000 mg/kg/day were generally similar to those of the control females after four weeks' treatment and two weeks' recovery. Females treated with 200 mg/kg/day had slightly elevated kidney weights and ratios (absolute kidney weight was statistically significant, p<0.05) when compared with the controls. This finding was considered to be incidental, as similar differences were not seen at the higher dose level. The remaining statistically significant changes noted in males (higher absolute spleen weight at 50 mg/kg/day, p<0.05; higher absolute brain and liver weights at 200 mg/kg/day, p<0.05) and females (higher absolute heart weight at 200 mg/kg/day, p<0.05; higher absolute and relative spleen weights at 200 mg/kg/day, p<0.05) after four week's treatment were considered to be incidental. After the two-week recovery period, the absolute and relative liver weights of females previously treated with 1000 mg/kg/day were lower than those of the control females. This finding was considered to be incidental.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In animals treated for four weeks, discolored kidneys were deemed to be test item-related findings.
The remaining findings were considered to be within the range of background lesions which may be recorded in animals of this strain and age. They consisted of renal pelvic dilation, seminal vesicles and thyroid glands reduced in size, uterus horn dilation, and discoloration or discolored foci in various organs.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the kidneys of animals treated for four weeks, tubular basophilia and lymphoid cell focus/foci increased slightly in incidence in animals treated with 1000 mg/kg/day. These findings were accompanied by minor degrees of tubular cell swelling consisting of small vacuolated tubular cells in some females. Such findings were not recorded in recovery animals.
Hyaline droplets were recorded in the glandular mucosa of one male and one female treated with 50 mg/kg/day, five males and four females treated with 200 mg/kg/day and all rats treated with 1000 mg/kg/day. This finding was also noted after recovery in two control females, as well as in two males and three females previously treated with 1000 mg/kg/day. Vacuolation of the squamous epithelium at the limiting ridge was recorded after four weeks' treatment in one control male and one control female, in four males and four females treated with 200 mg/kg/day, as well as four males and five females treated with 1000 mg/kg/day, and persisted after two weeks' recovery in one control female and two females previously treated with 1000 mg/kg/day.
The severity of these findings was increased slightly after the four week treatment period with dose dependence. Furthermore, increased inflammatory cell focus/foci in the glandular submucosa was recorded after four weeks treatment in all animals treated with 1000 mg/kg/day and after two weeks' recovery in one animal previously treated with 1000 mg/kg/day.

Histopathological findings: neoplastic:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

200 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, 50 mg/kg body weight/day of BLUE MGi 1037 was established as the NOEL and NOAEL.

Executive summary:

Oral administration of BLUE MGi 1037 to Wistar rats at doses of 50, 200 and 1000 mg/kg/day, for 28 days resulted no toxic effects upon mortality, daily or weekly clinical signs, functional observational battery, grip strength, food consumption or body weights. Treatment-related findings were generally restricted to males and females treated with 1000 mg/kg/day, manifested by transient reductions of locomotor activity, changes in hematology parameters (indicative of very slight anemia with compensatory reticulocytosis, males only), changes in clinical biochemistry parameters (lower total protein and lower globulin levels in both sexes, and lower albumin levels in females only), and increased absolute and relative kidney weights (males only). These findings were noted after four week's treatment, but were reversible after two weeks' recovery.

Test item-related discoloration was noted macroscopically in the kidneys of animals treated with 1000 mg/kg/day after four weeks. Histologically, the incidence of lymphoid cell focus/foci increased slightly in animals treated with 1000 mg/kg/day after four weeks' treatment and two weeks' recovery. These findings were accompanied by minor degrees of tubular cell swelling, consisting of small vacuolated tubular cells in two females after four weeks of treatment.

In the stomach, the findings consisted of a higher incidence and severity of hyaline droplets in the glandular mucosa accompanied by an increased incidence and severity of epithelial vacuolation at the limiting ridge at 200 mg/kg/day and 1000 mg/kg/day, as well as increased inflammatory cell focus/foci at 1000 mg/kg/day. These findings are indicative of a minor irritant potential of the test article. These findings were mostly recovered after the two week recovery period.