Insulin Aspart Ethyl Ester

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000.03.07 - 2000.07.04

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 405 (Acute Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

The nominal temperature of the freezer was kept at < -10oC and not <-20 oC as, and the relative humidity in the animal holding room was up to 15% lower than stated in the protocol. The deviations are not considered to compromise the study integrity

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

The nominal temperature of the freezer was kept at < -10oC and not <-20 oC as, and the relative humidity in the animal holding room was up to 15% lower than stated in the protocol. The deviations are not considered to compromise the study integrity

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

yes

Remarks:

The nominal temperature of the freezer was kept at < -10oC and not <-20 oC as, and the relative humidity in the animal holding room was up to 15% lower than stated in the protocol. The deviations are not considered to compromise the study integrity

GLP compliance:

yes (incl. QA statement)

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 (MI3 dried)
- Source and lot/batch No.of test material: C202483
- Expiration date of the lot/batch: 2001.05.31
- Purity: 98.4%
- Purity test date: 2001.05.31

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: < -10 degrees Celcius
- Stability under test conditions: NA
- Solubility and stability of the test substance in the solvent/vehicle: Soluble in vehicle (water)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: NA

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Purified water (Elgastat purifier used)
- Final preparation of a solid: 100mg/ml MI3 in water, pH of 8.0.

FORM AS APPLIED IN THE TEST (if different from that of starting material): solid dissolved in purified water.

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Rabbit (New Zealand White rabbit)
- Sex: male and female
- Age at study initiation: 11 - 14 weeks.
- Weight at study initiation: 1.97 - 2.54 kg
- Housing: Single housed in floor-pens with wood chips bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: NA

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-22
- Humidity (%): 40 – 80 %
- Air changes (per hr): at least 10 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours

IN-LIFE DATES: From: To: at least 14 - 16 days

Test system

Vehicle:

water

Controls:

other: The unexposed eye of the same animal served as a untreated control.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL

Duration of treatment / exposure:

single instillation dose into the left conjunctival

Observation period (in vivo):

Observations were recorded at: immediately after dosing, 30 min, one hour after dosing and at least twice within the first 24 hours after dosing. Thereafter observations were made once a day up to the third day after exposure.
However, the observation period was extended for the sentinel to five days to allow resulution of minor iridial changes prior to dosing of the remaining animals.

Number of animals or in vitro replicates:

3 animals

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): NA
- Time after start of exposure: NA

SCORING SYSTEM: Standard grading scheme (SGS) graded 0 to 4 (where 0 is no response and 4 is the most severe response).
The following endpoints were evaluated/scored (Sting repsonse; Cornea - degree of opacity; Cornea -area of corneal opacity; Iris; Conjunctivae- redness; Conjunctivae - chemosis, and Conjunctivae -discharge)


TOOL USED TO ASSESS SCORE:fluorescein and ultraviolet light.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

ca. 0.1

Max. score:

0.3

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Results 4

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

ca. 0

Max. score:

0

Remarks on result:

no indication of irritation

Other effects:

- Lesions and clinical observations: No indications of systemic toxicity or ill health were noted for any of the rabbits during the study. MI3 caused practically no initial sting reaction in two animals and a moderate sting in the third.
- Ophthalmoscopic findings: A darkned/red area appeared aorund the iris approximately four hours after instillation in the sentinel animal, but had disappeared by the 24 hour assessment. However, a residual dark speck in the iris appeared from Day 3 to Day 5, and disappeared at Day 6. It is not known if this is a direct result of the indtillation procedure, but it did not apear in the two other animals.
- Histopathological findings: NA
- Effects of rinsing or washing: NA
- Other observations: the eyes of all rabbits were overtly normal by the 48 hour examination with the exception of the persistant dark speck in the iris of the sentinel rabbit until Day 6.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The Study was conducted to determine the irritation caused to the rabbit eye following a single instillation of MI3 into the conjunctival sac. The study was designed in accordance with OECD guideline method 405. One male and two female rabbits received a single instillation of 0.1 mL MI3 into the conjunctival sac of the eye. There was no indicative of systemic toxicity or ill health for any of the rabbits. The instillation caused slight changes in the conjunctivae that resolved within 24 or 48-hours. An exception was a persistent dark speck in the edge of the iris of the sentinel rabbit only. The cornea of each eye was overtly unaffected.

Executive summary:

The Study was conducted to determine the irritation caused to the rabbit eye following a single instillation of MI3 into the conjunctival sac. The study was designed in accordance with OECD guideline method 405, the commission directive 92/69/EEC, Method B5, and the US EPA Health effects test Guidelines OPPTS 870.2400. One male and two female rabbits received a single instillation of 0.1 mL MI3 into the conjunctival sac of the eye. There was no indicative of systemic toxicity or ill health for any of the rabbits. The instillation caused practically no initial sting reaction in two animals and a moderate sting reaction in the third. Only minimal effects were observed after instillation of MI3 to eyes of all three animals. These effects were a slight reddening of the conjunctivae, a slight injection of the conjunctival vasculature, and production of a very slight discharge which all disappeared after 24 or 48-hours. An exception was a persistent dark speck in the edge of the iris of the sentinel rabbit only. The cornea of each eye was overtly unaffected.