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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1987-11-05 - 1987-12-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPP 81-4 (Acute Eye Irritation)
Version / remarks:
revised Nov. 1984
Deviations:
no
GLP compliance:
yes
Remarks:
US FDA (21 CFR 58) and US EPA (40 CFR 160 and 40 CFR 792)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- State of aggregation:fine black powder
- storage conditions: at ambient temperature in the dark in closed container
The test substance was stored in the dark at ambient temperature in the company dispensary at the Elphinstone Research Centre when not in use.


Test animals / tissue source

Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
Six adult female albino rabbits of the New Zealand White strain, SPF quality, were obtained from The Broekman Institute, Someren, The Netherlands. The animals arrived at the animal house August 31, 1987 and were at least 10 week old at the start of the study. The animals were identified by means of ear labels and were individually housed in plastic cages with perforated floors. An acclimatisation period of at least 5 days was allowed. Both eyes were inspected on the day of exposure and found to be intact and normal. The body weights of the animals were measured on the day of treatment. The animals were fed standard laboratory animal diet (100 g per day), obtained from Hope Farms, Woerden (LK-01, pellet diameter 4 mm), and had free access to decalcified tap-water.

ENVIRONMENTAL CONDITIONS
The animal room temperature was 20 - 21°C and the relative humidity 55 - 65 per cent. The artificial light sequence was 12 hours light, 12 hours dark.

The New Zealand White rabbit is the species and strain of choice because it is readily available and easy to handle, house and treat. The absence of pigmentation of the iris facilitates the evaluation of induced eye reactions, and there is a considerable amount of published literature on eye irritation available on this species to assist in the assessment of the significance of the effects observed.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
21 +/- 2 mg (equivalent to 20.1 mL)
Duration of treatment / exposure:
24 hrs
Observation period (in vivo):
10 days
Number of animals or in vitro replicates:
6
Details on study design:
Dose administration
The day of test substance administration was designated as day 0. The test substance was ground to a fine powder using a mortar and pestle. On the cay of dose administration, six portions of 21 + 2 mg (equivalent to 0.1 ml) of the test powder were dispensed in glass containers with screw caps. Each portion of the dispensed amount of test substance was instilled in the conjunctival sac of the left eye of each animal using a spatula. The lids were then neld gently together for two seconds and released. Immediately after treatment, the animals were transferred to their cages. The right eye, remaining untreated, served as a control.

Observations
Immediately after instillation of the test substance, the animals were observed and abnormalities were recorded. In addition, the eyes were examined approximately 60 min, 24, 48 and 72 hours, and 7 and 10 days after instillation of the test substance.

TOOL USED TO ASSESS SCORE: fluorescein (24 h after exposure of the test substance)
2% fluorescein in water (pH adjusted to 7.0 and sterilized) was applied to both eyes of each animal to quantitatively examine the potential for corneal injury. The brightly green staining area indicating epithelial damage was estimated as a percentage of the total corneal area. This procedure was repeated 3 days after dose administration.
Observed local (or systemic) effects other than those covered by the scoring system, were also recorded.

SCORING SYSTEM:
The following scoring system was used for grading of the ocular lesions:

CORNEA:
Opacity: degree of density (area most dense taken for reading) - No ulceration or opacity 0
Scattered or diffuse areas of opacity (other than slight dulling of normal lustre),details of iris clearly visible 1*
Easily discernible translucent areas, details of iris slightly obscured 2*
Nacreous areas, no details of iris visible, size of pupil barely discernible 3*
Opaque cornea, iris not discernible through the opacity 4*
CORNEA:
Area of cornea involved:
No ulceration or opacity 0
One quarter or less but not zero 1*
Greater than one quater, but less than half 2*
Greater than half, but less than three quarters 3*
Greater than three quarters, up to whole area 4*
IRIS:
Normal 0
Markedly deepened rugae, congestion, swelling,
moderate circumcorneal hyperaemia or injection,
any of these or any combination thereof, iris still
reacting to light (sluggish reaction is positive) 1*
No reaction to light, haemorrhage, gross
destruction (any or all of these) 2*
CONJUNCTIVAE:
Redness:(refers to palpebral and bulbar conjunctivae, excl. cornea and iris)
Blood vessels normal 0
Some blood vessels definitely hyperaemic or injected 1
Diffuse, crimson colour, individual vessels not easily discernible 2*
Diffuse beefy red 3*
Chemosis: lids and/or nictating membranes
No swelling 0
Any swelling above normal (includes nictating membranes) 1
Obvious swelling with partial eversion of lids 2*
Swelling with lids about half closed 3*
Swelling with lids more than half closed 4*
Discharge:
No discharge 0
Any amount different from normal (does not include small
amounts observed in inner canthus 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
* Starred figures indicate positive effect.

Interpretation of the results The results of the study were expressed according to the Draize scoring system. This score was calculated with the following formula: (5 x corneal opacity grade x area of opacity grade) + (5 x iridial injury grade) + 2 x (conjunctival redness grade + chemosis grade + di scharge grade).
The calculated Draize score was interpreted according to the scheme of Kay and Calandra (see below).
Draize score Classification according to Kay and Calandra
0 - 0.5 not irritating
0.5 - 2.5 practically not irritating
2.5 - 15 minimally irritating
15 - 25 mildly irritating
25 - 50 moderately irritating
50 - 80 severely irritating
80 - 100 extremely irritating
100 - 110 maximally irritating

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24/48/72 h
Score:
>= 0 - <= 3
Max. score:
3
Reversibility:
fully reversible within:
Remarks:
7 days
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
>= 1 - <= 2
Max. score:
2
Reversibility:
fully reversible within:
Remarks:
7 days
Irritation parameter:
other: Draize caclulation
Basis:
mean
Time point:
other: 1 h
Score:
5.7
Reversibility:
fully reversible
Irritation parameter:
other: Draize calculation
Basis:
mean
Time point:
24 h
Score:
6.3
Reversibility:
fully reversible
Irritation parameter:
other: Draize calculation
Basis:
mean
Time point:
48 h
Score:
4.7
Reversibility:
fully reversible
Irritation parameter:
other: Draize calculation
Basis:
mean
Time point:
72 h
Score:
5.3
Reversibility:
fully reversible
Irritation parameter:
other: Draize calculation
Basis:
mean
Time point:
7 d
Score:
0.7
Reversibility:
fully reversible
Irritation parameter:
other: Draize calculation
Basis:
mean
Time point:
10 d
Score:
0
Irritant / corrosive response data:
Instillation of the test substance into one of the eyes of each of six female rabbits affected the conjunctivae only. All six animals showed very slight conjunctival redness and very slight to obvious chemosis 60 minutes after exposure. The capillary injection of the scleral conjunctivae in animal 2298 and of the nictating membrane in animal 2308 increased briefly to diffuse beefy red on day 3. The conjunctival redness had disappeared in four animals by day 7 and in the others by day 10. The obvious chemosis decreased within 48 hours to very slight and had disappeared completely in all animals between study day 3 and day 7. Adverse effects on the cornea were not observed in any of the rabbits during the entire observation period. Treatment of the eyes with fluorescein 24 and 72 hours after instillation of the test substance revealed no epithelial damage. Twenty-four hours after instillation of the test substance all six rabbits showed a considerable amount of tne test material in the eyes and therefore the eyes of all six animals were washed out with tepid tap-water. It proved impractical to remove all the test material due to its adhesion to the eyebols.
Four animals showed some lacrimation or an increased amount of discharge during study days 1 and 2.
Other effects:
No signs of systemic intoxication were observed in any of the rabbits during the entire observation period.

Any other information on results incl. tables

Table 1: Eye irritation Scores in the rabbit
Rabbit #, Sex and Body Weight Obser-vations Cornea Iris Conjunctivae Draize total score Fluorescence area % Remarks
opacity area redness chemosis discharge
(0-4) (0-4) (0-2) (0-3) (0-4) (0-3)
2296
F
3560 g
1 hr 0 0 0 1 2 0 6 chemosis score 2 for lower eyelid only
1 day 0 0 0 1 2 0 6 0 chemosis score 2 for lower eyelid only
2 day 0 0 0 1 1 0 4
3 day 0 0 0 1 1 0 4 0
7 day 0 0 0 0 0 0 0
10 day 0 0 0 0 0 0 0
2298
F
3057 g
1 hr 0 0 0 1 2 0 6

chemosis score 2 for lower eyelid only

1 day 0 0 0 1 1 2 8 0
2 day 0 0 0 1 1 1 6
3 day 0 0 0 3 1 0 8 0

redness score 3 for scleral conjunctivae only

7 day 0 0 0 1 0 0 2

redness score 1 for scleral conjunctivae only

10 day 0 0 0 0 0 0 0
2300
F
3212 g
1 hr 0 0 0 1 2 0 6 chemosis score 2 for lower eyelid only
1 day 0 0 0 1 1 1 6 0
2 day 0 0 0 1 1 0 4
3 day 0 0 0 1 1 0 4 0
7 day 0 0 0 0 0 0 0
10 day 0 0 0 0 0 0 0
2306
F
3112 g
1 hr 0 0 0 1 1 0 4
1 day 0 0 0 1 1 0 4 0
2 day 0 0 0 1 1 0 4
3 day 0 0 0 1 1 0 4 0
7 day 0 0 0 1 0 0 2 redness score 1 for scleral conjunctivae only
10 day 0 0 0 0 0 0 0
2308
F
3462 g
1 hr 0 0 0 1 2 0 6 chemosis score 2 for lower eyelid only
1 day 0 0 0 1 2 1 8 0
2 day 0 0 0 1 1 1 6
3 day 0 0 0 3 1 0 8 0 redness score 3 for nictating membrane only
7 day 0 0 0 0 0 0 0
10 day 0 0 0 0 0 0 0
2310
F
3229 g
1 hr 0 0 0 1 2 0 6 chemosis score 2 for nictating membrane only
1 day 0 0 0 1 1 1 6 0
2 day 0 0 0 1 1 1 4
3 day 0 0 0 1 1 0 4 0 chemosis score 1 for eyelids only
7 day 0 0 0 0 0 0 0
10 day 0 0 0 0 0 0 0
Draize calculation 1 hr 1 day 2 day 3 day 7 day 10 day
Mean total score 5.7 6.3 4.7 5.3 0.7 0.0
Kay and Calandra interpretation: mildly irritating

Applicant's summary and conclusion

Interpretation of results:
other: Category 2 (irritating to eyes) based on EU GHS criteria
Conclusions:
Based on the calculated Draize score of 6.3 (24 hours) the test substance should be classified as mildly irritationg according to the scheme of Kay and Calandra.
Executive summary:

A sample of the test material was tested in the in vivo rabbit primary eye irritation/corrosion test according to EPA OPP 81-4 (Acute Eye Irritation) and in compliance with GLP to determine its possible irritating or corrosive effects. A single instillation of approximately 21 mg (equivalent to 0.1 ml) of the test substance in one of the eyes of each of six female rabbits resulted in effects on the conjunctivae only. Very slight conjunctival redness was observed in all six animals with a short increasing to diffuse redness in two animals on day 3. The redness had disappeared on day 10. Five of the six animals showed obvious chemosis, the sixth showed very slight chemosis. Chemosis was not noted after day 7 in any rabbit.

Some lacrimation or an increased amount of discharge was observed during days 1 and 2 in four of the six animals. No signs of systemic intoxication were observed in any of the rabbits.

Based on the calculated Draize score of 6.3 (24 hour) the test substance should be classified as mildly irritating according to the scheme of Kay and Calandra.