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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:
14 - 18 Mar 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Qualifier:
according to
Guideline:
other: JMAFF 12-Nousan-8147 (2000)
Qualifier:
according to
Guideline:
other: CounciI Regulation (EC) No. 440/2004
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:
Animals: New Zealand White Rabbits
Source: Inhouse Bred (Outbred), Toxicology, Department of Safety Assessment, Advinus Therapeutics Limited, Bangalore 560 058, INDIA
No. of rabbits: 3 male rabbits
Age at treatment: 7 - 8 months
Date of birth: 26 July 2010 to 30 July 2010
Body weight range at treatment: 2.61 to 2.94 kg
Accl imatization: One rabbit (RB8767) was acclimatized for five days while the other two (RB8768 and RB8769) rabbits were acclimatized for six days under laboratory conditions after physical examination.

Conditions
Animals were housed under standard laboratory conditions, air conditioned with adequate fresh air supply (12-15 air changes/hour). Environment: temperature 2 1-23°C, relative humidity 59-67%, with a 12 hour light and 12 hour dark cycle. The maximum and minimum temperature and relative humidity in the experimental room was recorded once dail y. The relative humidity in the experimental room was calculated from dry and wet bulb temperature recordings.

Housing
The rabbits were housed individually in rabbit cages (approx. size: L 65 x B 65 x H 45 cm) with noryl shallow cage body and faci lities for pelleted food (stainless steel feed hopper) and drinking water (750 mL markrolon bottle fitted with sipper tube). The litter collection tray (noryl waste tray) was changed daily (except on Sunday). The feed hoppers were changed once a week.

Diet ad libitum
The animals were offered rabbit feed manufactured by Pranav agro Industries Ltd, Sangli Maharashtra, India.

Water ad libitum
Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier, manufactured by Eureka Forbes Ltd., Mumbai-40000 I, India was provided to animals in Markrolon bottles with stainless steel sipper tubes. The water bottles were replenished once daily and the water bottles were changed once a week.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: The second eye of an animal remained untreated serving as control
Amount / concentration applied:
0.1 g
Duration of treatment / exposure:
24 hours (until eyes were flushed following fluorescein administration)
Observation period (in vivo):
Toxic Signs and Pre-Terminal Deaths
The rabbits were observed for toxic signs and pre-terminal deaths four times (at hourly intervals) on the day of test substance instillation and once daily till 72 hours post instillation.

Body Weights
Body weights were recorded at the start of acclimatization, Day 1 of treatment (just before test substance instillation) and at study termination.

Eye Scoring
Ocular lesions were evaluated with the illumination of a white light source at intervals of approximately 1, 24, 48 and 72 hours post-instillation of the test substance according to the "Scale for Scoring Ocular Lesions" (Draize, 1977). The fluorescein dye evaluation procedure was used to examine the treated eye of one rabbit (# RB8767, which showed corneal opacity at 24 hours) at the 24- and 48-hour observation. At the 72-hour observation, this rabbit had no opacity and all its eye scores were 0. Hence, fluorescein was not used and the experiment was terminated. The other two rabbits had no corneal opacity, so their eyes were not observed using ophthalmic fluorescein.

Individual scores were recorded for each rabbit. All scores were calculated as described in the "Scale for Scoring Ocular Lesions", and the total possible points (out of 110) were tallied. In addition to the observation of the cornea, iris and conjunctivae, any other lesions that were observed were noted. To aid in the interpretation of data, the average score for all rabbits at each scoring period was calculated.

Reference:
Draize. J.H 1977. Dermal Toxicity In: Appraisal of the safety of chemicals in Foods, Drugs and Cosmetics. Association of Food and Drug Officials of the United States. Austin, Texas. p. 47.
Number of animals or in vitro replicates:
3
Details on study design:
Test Substance Instillation
Prior to instillation, both eyes of each rabbit were examined with the aid of a pen light for irritation and ocular defects. The eyes were then checked for opacity using ophthalmic fluorescein sodium strips. The eyes were then irrigated with enough physiological saline (0.9% NaCI) to remove any excess fluorescein (approximately 30 seconds). Using an ultraviolet lamp, the eyes were checked and evaluated for pre-existing corneal damage. No corneal defects were observed in any of the animals.

One hundred milligrams (0.1 g) of the finely ground test substance was placed in the everted lower lid (conjunctival sac) of the left eye of each rabbit.

The eye lids were gently held together for about one second, in order to minimize loss of the test substance. The right eye of each rabbit remained untreated and served as the reference control.

Animal Restraint
Each rabbit was restrained using an Elizabethan collar fixed around the neck. One rabbit RB8767 was restrained for 72 hours and the other two rabbits (RB8768 and RB8769) were restrained for 48 hours post-instillation.

Stepwise exposure of Animals
A single rabbit was initially exposed to the test substance. Since the test substance was not corrosive, the test was completed using two additional
animals.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
other: mean over 24, 48 and 72 h
Score:
1.3
Max. score:
3
Reversibility:
not fully reversible within: 72 h
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
other: mean over 24, 48 and 72 h
Score:
0.3
Max. score:
3
Reversibility:
fully reversible within: 72 h
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
other: mean over 24, 48 and 72 h
Score:
0.3
Max. score:
3
Reversibility:
fully reversible within: 72 h
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
other: mean over 24, 48 and 72 h
Score:
0.7
Max. score:
4
Reversibility:
fully reversible within: 72 h
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
out of animal #2 and animal #3
Time point:
other: mean over 24, 48 and 72 hrs
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
other: mean over 24, 48 and 72 hrs
Score:
0.7
Max. score:
4
Reversibility:
fully reversible within: 72 h
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
out of animal #2 and animal #3
Time point:
other: mean over 24, 48 and 72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
iris score
Basis:
mean
Remarks:
out of all 3 animals
Time point:
other: mean over 24, 48 and 72 h
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Irritant / corrosive response data:
The test material caused conjunctival irritation and corneal opacity in the rabbit eye below GHS and CLP C&L thresholds, which cleared by 72 hours.
Other effects:
There were no toxic signs or pre-terminal deaths. The body weights of all rabbits increased slightly through the observation period.

Any other information on results incl. tables

The individually determined mean irritation scores for each animal for corneal opacity, iris lesion, conjunctival redness and conjunctival chemosis for the 24, 48 and 72-hour intervals are:

 Animal No. (Sex)  Corneal Opacity  Iris Lesion  Conjunctival Redness  Conjunctival Chemosis
 RB8767 (M)  0.7  0  1.3  0.7
 RB8768 (M)  0  0  0.3  0
 RB8769 (M)  0  0  0.3  0

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
CLP: not classified
DSD: not classified