Insulin DesB30

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:

march to december 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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

other: Cream powder (84% w/w)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: C202483
- Expiration date of the lot/batch: 31.05.2001
- Purity: 98.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: < -10 degrees C

- Solubility and stability of the test substance in the solvent/vehicle:
MI3 was homogeneous and stable in purified water for up to 6 hours after dose preparation.

Test animals

Species:

rat

Strain:

other: Han Wistar rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
- Age at study initiation: 8 weeks
- Weight at study initiation: males: 193,8-228,9 g; females: 122,5-175 g
- Fasting period before study: not specified
- Housing: The animals were housed in a single, exclusive room, airconditioned (15 air changes/hour)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 4 weeks

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purified water

Details on oral exposure:

Oral exposure by gavage

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of the formulations for achieved concentration were performed in week 1. The analysis was performed by Covance and demonstrated that the formulations were accurately performed.

Duration of treatment / exposure:

29 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Controls

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

low dose

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

intermediate dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High dose

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

N/A

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily observation for signs of ill health

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

Post dosing observations were performed daily during the first week of treatment (excluding Day 2, as functional obervational battery was being performed) at the following time intervals: immediately after dosing, 30 minutes, 1, 2 and 4 hours after dosing. For the remainder of the study, post dosing observations were recorded twice weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Before treatment in the first day of dosing, at weekly intervals and before necropsy

FOOD CONSUMPTION:
- Food consumption was determined weekly and calculated as g food/animal/week

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of week 4
- Anaesthetic used for blood collection: Yes (fluothane)
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood:
- Animals fasted: Yes overnight
- How many animals: all animals
- Parameters examined:
haemoglobin concentration
packed cell volume
mean cell haemoglobin
reticulocytes
platelet couunt
red blood cell count
mean cell volume
mean cell haemoglobin concentration
total and differential white cell count
and prothromnin time (in trisodium citrate anticoagulant)

URINALYSIS: Yes / No / Not specified
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes: FUNCTIONAL OBSERVATIONAL BATTERY (FOB):
- Time schedule for examinations: before initiation of treatment and once weekly thereafter.
- Dose groups that were examined: All animals
- Battery of functions tested: sensory activity / grip strength / motor activity (week 4)

IMMUNOLOGY:No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes on control and high dose animals


HISTOPATHOLOGY: Yes on control and high dose animals

Statistics:

Statistical analysis were perforned where appropriate.
Body weight gains, necropsy body weight, haematology, clinical chemistry, locomotor and FOB data were analysed using two-way analysis of variance (ANOVA). Pairwise comparison with control for each sex separately, were made using Dunnett’s test. For each sex separately, a regression test was performed to determine whether there was a dose response relationship. Non-parametric methods were used for clinical parameters with values above or below limit of detection. Levene’s test for equality of variance across groups, sexes and any interactions was also performed.
Organ weights were analysed using analysis of covariance (ANCOVA) and Dunnett’s test.
Significance level of p>0.05 was used. (p<0.01 for Levene's test)

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:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Total group mean motor activity for females given 1000 mg/kg/day was slightly lower than controls. Landing foot display for males given 300 or 1000 mg/kg/day were slightly higher than controls.
Individual results for both landing footsplay and motor activity for the control and the treated groups were generally variable. This slight changes in footsplay and motor activity are therefore not considered to be related to treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Liver weight for males given 1000 mg/kg/day and brain weight for females given 1000 mg/kg/day were slightly lower than controls, however, there were no clear sex or dosage-relationships. These findings are therefore considered to be fortuitous.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The administration of MI3 at doses of 100, 300 or 1000 mg/kg/day to Han Wistar rats for 28 days produced no evidence of toxicity and no effects which could be confidently attributed to treatment.

Executive summary:

MI3 was administered to Han Wistar rats at doses of 100, 300 or 1000 mg/kg/day for 28 days. No evidence of toxicity and no effects which could be confidently attributed to treatment was observed.