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Administrative data

Description of key information

There is a good quality modern OECD404 GLP compliant skin irritation study in rabbits and an OECD405 GLP compliant eye irritation study in rabbits.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From January 06, 2009 to January 20, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2500 (Acute Dermal Irritation)
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Number of Animals: 3
Sex: Female, nulliparous and non-pregnant
Species/Strain: Rabbit/New Zealand albino
Age: Young adult
Source: Received from Robinson Services Inc., Clemmons, NC on December 31, 2008
Housing: The animals were singly housed in suspended stainless steel caging with mesh floors, which conform to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals DHEW (NIH).
Litter paper was placed beneath the cage and was changed at least three times per week.
Animal Room Temperature and Relative Humidity Ranges: 19-20ºC and 24-51%, respectively. The low humidity occurred during the study due to a temporary malfunction of the environmental control system.
Photoperiod: 12-hour light/dark cycle
Acclimation Period: 6 days
Food: Pelleted Purina Rabbit Chow #5326
Water: Filtered tap water was supplied ad-libitum by an automatic water dispensing system.
Contaminants: There were no known contaminants reasonably expected to be found in the food or water at levels, which would have interfered with the results of this study. Analyses of the food and water are conducted regularly and the records are kept in the test fecility.
Cage: Each cage was identified with a cage card indicating at least the study number and identification and sex of the animal.
Animal: A number was allocated to each rabbit on receipt and a stainless steel ear tag bearing this number was attached to the animal. This number, together with a sequential animal number assigned to study 26614, constituted unique identification.
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
0.5 mL
Duration of treatment / exposure:
4 hours
Observation period:
14 days
Number of animals:
3
Details on study design:
Preparation and Selection of Animals
On the day before application, a group of animals was prepared by clipping the dorsal area and the trunk. On the day of dosing, but prior to application, the animals were examined for health and the skin checked for any abnormalities. Three healthy, naive animals (not previously tested) without pre-existing skin irritation were selected for test.

Application of Test Substance
Five-tenths of a milliliter of the test substance was applied to one 6-cm2 intact dose site on each animal and covered with a 1-inch x-1 inch, 4-ply gauze pad. The pad and entire trunk of each animal were then wrapped with semi-occlusive 3-inch Micropore tape to avoid dislocation of the pad. Elizabethan collars were placed on each rabbit and they were returned to their designated cages.
After 4 hours of exposure to the test substance, the pads and the collars were removed, and the test sites were gently cleansed of any residual test substance.

Evaluation of Test Sites
Individual dose sites were scored according to the Draize scoring system at approximately 30-60 minutes, 24, 48, and 72 hours and at 7, 10 and 14 days after patch removal. Additionally, the average erythema and edema scores across 24, 48 and 72 hours for each animal were calculated for EEC2 classification. The classification of irritancy was obtained by adding the average erythema and edema scores for the 30-60 minute, 24, 48, and 72-hour scoring intervals and dividing by the number of evaluation intervals.

Cage-Side Observations
The animals were observed for signs of gross toxicity and behavioral changes at least once daily during the test period. Observations included gross evaluation of skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behavior pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhea, and coma.
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1.3
Max. score:
4
Reversibility:
not reversible
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.3
Max. score:
4
Reversibility:
not reversible
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
not reversible
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.3
Max. score:
4
Reversibility:
not reversible
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.3
Max. score:
4
Reversibility:
not reversible
Irritation parameter:
edema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
not reversible
Irritant / corrosive response data:
All animals appeared active and healthy during the study. Apart from the dermal irritation noted below, there were no other signs of gross toxicity, adverse pharmacologic effects, or abnormal behavior.
One hour after patch removal, all three treated sites exhibited very slight to well-defined erythema and very slight edema. Desquamation was noted at two or three dose sites between 72 hours and Day 14. Although the overall incidence and severity of irritation decreased gradually with time, dermal irritation (erythema and/or desquamation) persisted for two animals through Day 14 (study termination).
The Primary Dermal Irritation Index for the test substance is 2.3.
Interpretation of results:
Category 3 (mild irritant) based on GHS criteria
Conclusions:
Under the study conditions, the test substance was moderately irritating to the skin.
Executive summary:

A study was conducted to determine the skin irritation potential of the test substance in male Himalayan rabbits according to OECD Guideline 404 and EU Method B.4, in compliance with GLP. Five-tenths of a milliliter of the test substance was applied to the skin of three healthy rabbits for 4 h. Following exposure, dermal irritation was evaluated by the method of Draize et al. One hour after patch removal, all three treated sites exhibited very slight to well-defined erythema and very slight edema. Desquamation was noted at two or three dose sites between 72 h and Day 14. Although the overall incidence and severity of irritation decreased gradually with time, dermal irritation (erythema and/or desquamation) persisted for 2 animals through Day 14 (study termination). Under the study conditions, the test substance was moderately irritating to skin (Oley, 2009).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From January 26, 2009 to February 03, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Animals
Number of Animals: 3
Sex: Females, nulliparous and non-pregnant
Species/Strain: Rabbit/New Zealand albino
Age: Young adult
Source: Received from Robinson Services, Inc. Clemmons, NC on January 7, 2009
Housing: The animals were singly housed in suspended stainless steel caging with mesh floors which conform to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals DHEW (NIH). Litter paper was placed beneath the cage and was changed at least three times per week.
Animal Room Temperature and Relative Humidity Ranges: 19-21ºC and 25-59%, respectively. The low humidity reading was due to a temporary malfunction of the environmental control system.
Photoperiod: 12-hour light/dark cycle
Acclimation Period: 19 days
Food: Pelleted Purina Rabbit Chow #5326
Water: Filtered tap water was supplied ad libitum by an automatic water dispensing system.
Contaminants: There were no known contaminants reasonably expected to be found in the food or water at levels which would have interfered with the results of this study.
Analyses of the food and water are conducted regularly and the records are kept at test fecility.
Cage: Each cage was identified with a cage card indicating at least the study number and identification and sex of the animal.
Animal: A number was allocated to each rabbit on receipt and a stainless steel ear tag bearing this number was attached to the animal.
Vehicle:
not specified
Controls:
yes, concurrent no treatment
Amount / concentration applied:
One-tenth of a milliliter of the test substance was instilled into the conjunctival sac of the right eye of each rabbit.
Observation period (in vivo):
Ocular irritation was evaluated at 1, 24, 48, and 72 hours and at 4 and 7 days post-instillation.
Number of animals or in vitro replicates:
3
Details on study design:
Preparation and Selection of Animals
Prior to test initiation, both eyes of a group of animals were examined using a white light source and a fluorescein dye procedure. One drop of 2% ophthalmic fluorescein sodium was instilled into both eyes of each rabbit. The eyes were rinsed with physiological saline (0.9% NaCl) approximately 30 seconds after instillation of the fluorescein and then evaluated for corneal damage using an ultraviolet light source. Prior to instillation, the eyes were re-examined and scored for abnormalities according to the "Scale for Scoring Ocular Lesions". Three healthy naive animals (not previously tested) without pre-existing ocular irritation were selected for test.

Instillation
Prior to instillation, 2-3 drops of ocular anesthetic (Tetracaine Hydrochloride Ophthalmic Solution, 0.5%) were placed into both the treated and control eye of each animal. One-tenth of a milliliter of the test substance was then instilled into the conjunctival sac of the right eye of each rabbit by pulling the lower lid away from the eyeball. The upper and lower lids were then gently held together for about one second before releasing to minimize loss of the test substance. The other eye of each rabbit remained untreated with the test substance and served as a control. The rabbits were then returned to their designated cages.

Ocular Scoring
Ocular irritation was evaluated using a high-intensity white light (Mag Lite) in accordance with Draize et al. at 1, 24, 48, and 72 hours and at 4 and 7 days post-instillation. The fluorescein dye evaluation was done on the treated eye at 24 hours and as needed at subsequent scoring intervals to evaluate the extent of corneal damage or to verify reversal of effects. Individual scores were recorded for each animal. In addition to observations of the cornea, iris and conjunctivae, any other observed lesions were noted. The average score for all rabbits at each scoring period was calculated to aid in data interpretation.

Classification of Eye Scores
The time interval with the highest mean score (Maximum Mean Total Score - MMTS) for all rabbits was used to classify the test substance by the system of Kay and Calandra. The average individually-determined irritation scores for cornea, iris and conjunctiva (redness and chemosis) across 24, 48 and 72 hours were calculated for EEC classification.

Cage-Side Observations
The animals were observed for signs of gross toxicity and behavioral changes at least once daily during the test period. Observations included gross evaluation of skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behavior pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhea, and coma.
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1.7
Max. score:
4
Reversibility:
not reversible
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.7
Max. score:
4
Reversibility:
not reversible
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1.7
Max. score:
4
Reversibility:
not reversible
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
1.7
Max. score:
2
Reversibility:
not reversible
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
not reversible
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1.7
Max. score:
2
Reversibility:
not reversible
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2.7
Max. score:
3
Reversibility:
not reversible
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
2
Max. score:
3
Reversibility:
not reversible
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
2.3
Max. score:
3
Reversibility:
not reversible
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
3
Max. score:
4
Reversibility:
not reversible
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.7
Max. score:
4
Reversibility:
not reversible
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
3
Max. score:
4
Reversibility:
not reversible
Irritant / corrosive response data:
All animals appeared active and healthy during the study. Apart from the eye irritation there were no other signs of gross toxicity, adverse pharmacologic effects, or abnormal behavior.
One hour after test substance instillation, all three treated eyes exhibited corneal opacity, iritis and ‘positive’ conjunctivitis. The overall incidence and severity of irritation appeared to be severe. A brownish red discharge and pannus was noted for all three treated eyes between Days 1 and 7.
Therefore following the Day 7 evaluation, the study was terminated and all animals were euthanized for humane reasons. The maximum mean total score for the test substance is 49.0.
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Under the study conditions, the test substancewas severely irritating to the eye.
Executive summary:

A study was conducted to determine the eye irritation potential of the test substance in female New Zealand albino rabbits according to OECD Guideline 405, EU Method B.5 and EPA OPPTS Method 870.2400, in compliance with GLP. A dose of 0.1 mL of the test substance was administered into the conjunctival sac of the right eye of the rabbits. The upper and lower lids were then gently held together for about one second in order to prevent loss of test substance. The left eye, which remained untreated, served as a control. Ocular irritation was evaluated using a high-intensity white light (Mag Lite) in accordance with Draize et al.1 at 1, 24, 48, and 72 h and at 4 and 7 d post-instillation. The fluorescein dye evaluation was done on the treated eye at 24 h and as needed at subsequent scoring intervals to evaluate the extent of corneal damage or to verify reversal of effects. All animals appeared active and healthy during the study. Apart from the eye irritation there were no other signs of gross toxicity, adverse pharmacologic effects or abnormal behavior. One hour after test substance instillation, all three treated eyes exhibited corneal opacity, iritis and ‘positive’ conjunctivitis. The overall incidence and severity of irritation appeared to be severe. A brownish red discharge and pannus was noted for all three treated eyes between Days 1 and 7. Therefore, following the Day 7 evaluation, the study was terminated and all animals were euthanized for humane reasons. The maximum mean total score for the test substance was 49.0. Under the study conditions, the substance was severely irritating to the eye (Oley, 2009).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

The OECD404 skin irritation study showed moderate irritation to the rabbit’s skin, the mean scores at 24/48/72 hours for the three rabbits corresponded to GHS category 3 for mild skin irritation, but were not classified as irritating to skin based on the EU CLP criteria.

The OECD405 eye irritation study showed severe irritation in the eyes of all three the rabbits such that the study was terminated on day 7 for humane reasons. The severity of the effects resulted in a classification of category 1 for serious irreversible eye damage by both GHS and EU CLP criteria.