Registration Dossier

Administrative data

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 07 to December 06, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test conducted following internationally accepted testing guidelines and performed according to the GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Justification of Species: the rabbit is preferred by the various regulatory agencies for use in eye irritation testing.
- Source: Ray Nichols Rabbitry; Lumberton, TX.
- Age at study initiation: approx. 4 months.
- Weight at study initiation: males 2.100-2.800 kg; females 2.650-2.850 kg.
- Housing: suspended, wire bottom, stainless steel; one per cage.
- Diet: PMI Feeds, Inc.TM Lab Rabbit Diet #5321, in measured amounts.
- Water: municipal water supply analyzed by TNRCC Water Utilities Division; available ad libitum from automatic water system.
- Quarantine period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3 °C
- Humidity: 30-70 %
- Air changes: 10-12 air change per hour
- Photoperiod: 12 hours cycle dark/light

IN-LIFE DATES
Animlas born in August 01, 2001. They were received by the laboratory at November 07 and were treated with the test substance on December 03. The in-life portion of the study was termined on December 06, 2001.

No contaminants were expected to have been present in the feed or water which would have interfered with or affected the results of the study.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
-Pre-treatment: the test substance was ground to a fine powder prior to dosing.
- Amount applied: 0.04 g (volume: 0.1 ml). The test substance was placed into the conjunctival sac of the left eye of each animal by gently pulling the lower lid away from the eyeball to form a cup into which the test substance was dropped. The lids were gently held together for one second to prevent loss of material. The untreated left eyes served as comparative controls.
Duration of treatment / exposure:
24 hours
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
3 males and 3 females
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: all treated eyes were washed with room temperature deionized water for one minute.
- Time after start of exposure: immediately after recording the 24 hour observation.

TOOL USED TO ASSESS SCORE:
The treated eyes of all animals were examined without magnification under white room lighting provided by daylight-type fluoreseent ceiling fixtures and an additional source of white light present on the examining table.
The grades of ocular reaction were recorded at 1, 24, 48 and 72 hours after treatment. The corneas of all treated eyes were examined immedìately after the 24 hour observation with a fluorescein sodium ophthalmic solution. A Finoff ocular transilluminator with cobalt blue filter (Welch Allyn, Skaneateles Falls, NY) was utilized to enhance visualization of fluorescein staining. Any of the corneas which exhibited fluorescein staining at the 24 hour observation were re-examined with the fluorescein sodium ophthalmic solution at each consecutive observation until fluorescein staining of the cornea no longer occurred.

SCORING SYSTEM
An average irritation score for each scheduled observation was determined, based on the number of animals tested. A maximum average irritation score was used to rate the test substance according to the grading scale reported below, as the scale used to categorized the test substance. Any corneal involvement or iridic irritation with a score of 1 or more is considered positive. any conjunctival irritation (redness or chemosis) with a score of or more is considered positive.

Grading scale
CORNEA:
A Opacity and degree of density (most dense area scored) .
No opacity: 0
Slightly dulling of normal luster: +
Scattered or diffuse area of opacity (other than slightly dulling of normal luster), details of iris clearly visible: 1*
Easily discernible translucent area, details of iris slightly obscured: 2*
Nacreous area, no details of iris visible, size of pupil barely discernible: 3*
Opaque cornea, iris not discernible: 4*

B Area of cornea involved
One quarter (or less) but not zero: 1
Greater than one quarter, but less than half: 2
Greater than half, but less than three quarters: 3
Greater than three quarters, up to whole area: 4

C. Fluoroscein staining - appearance of yellow-green staining of cornea
Cornea not examined with fluoroscein: -
No fluoroscein staining: 0
Positive fluoroscein staining: P
Area of cornea involved
One quarter (or less) but not zero: A
Greater than one quarter, but less than half: B
Greater than half, but less than three quarters: C
Greater than three quarters, up to whole area: D

D. Stippling - appearance of pinpoint roughening
No stippling: 0
Presence of stippling: S
Area of cornea involved
One quarter (or less) but not zero: A
Greater than one quarter, but less than half: B
Greater than half, but less than three quarters: C
Greater than three quarters, up to whole area: D
A x B x 5; Maximum possible score = 80

IRITIS:
A Values
Normal: 0
Folds above normal, congestion, swelling, circumcornea injection (any or all of these or combination of any thereof) iris still reacting to light (sluggish reaction is positive: 1
No reaction to light, hemorrhage, gross destruction (any or all of these): 2
A x 5; Maximum possible score = 10

CONJUNCTIVAE:
A Redness (refers to lids and bulbar conjunctivae excluding cornea and iris)
Vessels normal: 0
Vessels definitely injected above normal: 1
More diffuse, deeper crimson red, individual vessels not easily discernible: 2
Diffuse beefy red: 3

B Chemosis
No swelling: 0
Any swelling above normal (includes nictitating membrane): 1
Obvious swelling with partial eversion of lids: 2
Swelling with lids about half closed: 3
Swelling with lids about half closed to completely closed: 4

C Discharge
No discharge: 0
Any amount different from normal (does not include small amounts observed in inner cantus of normal animals): 1
Discharge with moistening of the lids and hairs just adjacent to lids: 2
Discharge with moistening of the lids and hairs, and considerable area around the eye: 3

D. Necrosis or Ulceration of the palpebral and bulbar conjunctivae or nictitating membrane
No necrosis or ulceration: 0
Presence of necrosis or ulceration: N
(A + B + C) x 2; Maximum possible score = 20

The total score for the eye is the sum of all scores obtained for the cornea, iris and conjunctivae with the possible maximum total score for the eye being equal to 110.

*: reaction indicates a positive effect

Rating of the test substance based on eye irritation
0.0-0.5 Non irritating: to maintain this rating, all scores at the 24 hours reading must be zero; otherwise, increase increase rating one level.
> 0.5 - 2.5 Pratically non-irritating: to maintain this rating, all scores at the 48 hours reading must be zero; otherwise, increase increase rating one level.
> 2.5 - 15.0 Minimally irritating: to maintain this rating, all scores at the 72 hours reading must be zero; otherwise, increase increase rating one level.
> 15.0 - 25.0 Mildly irritating: to maintain this rating, all scores at the 7 day reading must be zero; otherwise, increase increase rating one level.
> 25.0 - 50.0 Moderately irritating: to maintain this rating, all scores at the 7 day reading must be less than or equal to 10 for 60 % or more of the animals; also, the 7 day mean score must be less than or equal to 20. If the 7 day mean score is less than or equal to 20, but less than 60 % of the animals show scores less than equal to 10, then no animal with a score greater than 10 can exceed a score of 30 if rating is to be maintained; otherwise, increase rating one level.
> 50.0 - 80.0 Moderately irritating: to maintain this rating, all scores at the 7 day reading must be less than or equal to 30 for 60 % or more of the animals; also, the 7 day mean score must be less than or equal to 40. If the 7 day mean score is less than or equal to 40, but less than 60 % of the animals show scores less than equal to 30, then no animal with a score greater than 30 can exceed a score of 60 if rating is to be maintained; otherwise, increase rating one level.
> 80.0 - 110.0 Extremely irritating.

The rating of the test substance is not to be increased more than one level above its maximum average score.

Criteria of eye irritation for classification into toxicity criteries (per proposed Rule, FR Vol. 49, No. 188)
Caregory I: corrosive (irreversible destruction of ocular tissue) or corneal involved or "possible" conjunctival irritation persisting through Day 21.
Caregory II: corneal involved or "positive" conjunctival irritation clearing in 8-21 days.
Caregory III: corneal involved or "positive" conjunctival irritation clearing in 7 days or less.
Caregory IV: minimal effects clearing in less than 24 hours. No "positive" effects at 24 hours.

Results and discussion

In vivo

Irritant / corrosive response data:
Maximum average irritation score was 3.2 obtained 1 hour after treatment, thus the substance can be rate as minimal irritating.
The toxicity category IV was assigned, since all "positive" and minimal effects had cleared by 24 hours.

Any other information on results incl. tables

Mean values from gradings at 24, 48 and 72 hours

Animan N-Sex Hour Corneal Opacity Mean at 24, 48 and 72 hrs Iritis Mean at 24, 48 and 72 hrs Conjunctival redness Mean at 24, 48 and 72 hrs Conjunctival chemosis Mean at 24, 48 and 72 hrs
3560 -M 1 0 0.0 0 0.0 0 0.0 0 0.0
24 0 0 0 0
48 0 0 0 0
72 0 0 0 0
3562 -M 1 0 0.0 0 0.0 1 0.0 0 0.0
24 0 0 0 0
48 0 0 0 0
72 0 0 0 0
3556 -M 1 0 0.0 0 0.0 1 0.0 0 0.0
24 0 0 0 0
48 0 0 0 0
72 0 0 0 0
3555 -F 1 1 0.0 0 0.0 1 0.0 1 0.0
24 0 0 0 0
48 0 0 0 0
72 0 0 0 0
3561 -F 1 + 0.0 0 0.0 1 0.0 0 0.0
24 0 0 0 0
48 0 0 0 0
72 0 0 0 0
3563 -F 1 + 0.0 0 0.0 1 0.0 0 0.0
24 0 0 0 0
48 0 0 0 0
72 0 0 0 0

Scores and score summary

Time after treatment Animal N-sex Average score
3560 -M 3562 -M 3556 -M 3555 -F 3561 -F 3563 -F
1 0 2 2 9 4 2 3.2
24 0 0 0 0 0 0 0.0
48 0 0 0 0 0 0 0.0
72 0 0 0 0 0 0 0.0
Maximum average score 3.2
Toxicity category IV

Ocular reactions

Reaction Animal number -sex
3560 -M 3562 -M 3556 -M 3555 -F 3561 -F 3563 -F
Observation time 1 24 48 72 1 24 48 72 1 24 48 72 1 24 48 72 1 24 48 72 1 24 48 72
Cornea A. Opacity 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 + 0 0 0 + 0 0 0
B. Area 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 4 0 0 0 4 0 0 0
C. Fluorescein staining - 0 - - - 0 - - - 0 - - - 0 - - - 0 - - - 0 - -
D. Stippling 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
SCORE 0 0 0 0 0 0 0 0 0 0 0 0 5 0 0 0 0 0 0 0 5 0 0 0
Iritis A. Grade 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
SCORE 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Conjunctivae A. Redness 0 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0
B. Chemosis 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
D. Discarge 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
D. Necrosis or ulceration 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
SCORE 0 0 0 0 2 0 0 0 2 0 0 0 4 0 0 0 4 0 0 0 2 0 0 0

TOTAL SCORE

0 0 0 0 2 0 0 0 2 0 0 0 9 0 0 0 4 0 0 0 2 0 0 0

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information according to the CLP Regulation Criteria used for interpretation of results: EU
Conclusions:
Not irritating
Executive summary:

An acute eye irritation study was conducted on six albino rabbits. The test substance was ground to a fine powder before dosing, and 0.1 ml by volume (0.04 g) was placed into the conjunctival sac of the right eye of each animal selected for testing. All treated eyes were washed with room temperature deionized water for one minute immediately after recording the 24 hour observation.

There were no "positive" effects exhibited in any eyes at 24 hours after treatment. The maximum average irritation score was 3.2 obtained 1 hour after treatment, thus the substance can be rate as minimal irritating. Furthermore, the toxicity category IV was assigned, since all "positive" and minimal effects had cleared by 24 hours.

Conclusion

The criteria for the evaluation of the eye irritation potential followed in the test are different from those reported into the OECD guideline (405), nevertheless, the scoring system used for the cornea opacity and degree of density, for the iris and conjunctivae redness and chemosis is the same of that reported into the OECD. Because the raw tables are included into the study report, it is possible to re-evaluate the results according to the CLP Regulation (EC 1272/2008).

The mean values from gradings at 24, 48 and 72 hours were lower than 1 for corneal opacity, lower than 1 for iritis, lower than 2 for both conjunctival redness and oedema, in all animals.

Therefore the substance does not meet the criteria to be classified as eye irritating, according to the CLP Regulation (EC 1272/2008).