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Description of key information

Not skin irritating
Not eye irritating

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 November 28 to December 07, 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.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2500 (Acute Dermal Irritation)
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Justification of Species: the rabbit is preferred by the various regulatory agencies for use in primary dermal irritation testing.
- Source: Ray Nichols Rabbitry; Lumberton, TX.
- Age at study initiation: approx. 2.5-3 months.
- Weight at study initiation: males 2.300-2.525 kg; females 2.250-2.675 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 September 09, 2001. They were received by the laboratory at November 28 and were treated with the test substance on December 04. The in-life portion of the study was termined on December 07, 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.
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
other: corn oil
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.5 g of the test substance were moistened with 0.5 ml of com oil
Duration of treatment / exposure:
4 hours
Observation period:
72 hours
Number of animals:
3 males and 3 females
Details on study design:
TEST SITE
- Area of exposure: 8 x 8 cm
- Type of wrap if used: the test substance was applied to each test site and covered with 4 ply, 2.5 x 2.5 cm surgical gauze patch. Each patch was secured in place with a strip of non-irritating adhesive tape. The entire trunk of each animai was loosely wrapped with a semi-permeable dressing (orthopedic stockinette) which was secured an both edges with strips of tape to retard evaporation of volatile substances and to prevent possible ingestion of the test substance.

REMOVAL OF TEST SUBSTANCE
- Washing: the test site was gently washed with room temperature tap water and a clean cloth to remove as much residual test substance as possible.
- Time after start of exposure: 4 hours.

SCORING SYSTEM
The test sites were observed for erythema and edema formation, and any other dermal defects or irritation at 1, 24, 48 and 72 hours after unwrap.
The scoring scale used to rate dermal irritation is reported below.
For each animal, all of the erythema and oedema scores throught 7 hours were added, and the sum was divided by 4 to obtain an individual irritation score. The primary irritation index was determined by calculating the mean of the irritation scores for all the animals and was used to obtain a rating for the test substance.
Evaluation of the skin reactions
Erythema Formation
No erythema: 0
Very slight erythema (barely perceptible): 1
Well defined erythema: 2
Moderate to severe erythema: 3
Severe erythema (beef redness) to slight eschar formation (injuries in depth): 4
Maximum possible: 4

Oedema Formation
No oedema: 0
Very slight oedema (barely perceptible): 1
Slight oedema (edges of area well defined by definite raising): 2
Moderate oedema (raised approximately 1 mm): 3
Severe oedema (raised more than 1 mm and extending beyond area of exposure): 4
Maximum possible: 4

Classification of the test substance
Primary irritation index
0.0: non-irritant
0.1 - 1.9: slightly irritant
2.0 - 5.0: moderately irritant
5.1 - 8.0: severe irritant

Dermal irritation toxicity categories (per proposed rule, FR Vol. 49, No 188)
Toxicity category I: corrosive
Toxicity category II: severe irritation at 72 hours
Toxicity category III: moderate irritation at 72 hours
Toxicity category IV: non-irritating, mild, or slight irritation at 72 hours
Irritant / corrosive response data:
Primary irritation index: 0.1; based on the 72 hours observations the toxicity category IV was assigned.
Very slight erythema was present in three animals only at the 1 hour observation.
Oedema was not detected at any throughout the study.
No other signs of irritation were observed during the study.

Reaction Observation time Animal number-Sex
3590 -M 3592 -M 3594 -M 3591-F 3593-F 3595-F
Erythema 1-h 1 1 0 0 1 0
24-h 0 0 0 0 0 0
48-h 0 0 0 0 0 0
72-h 0 0 0 0 0 0

Mean 24, 48 and 72 hrs

0.0 0.0 0.0 0.0 0.0 0.0
Oedema  1-h 0 0 0 0 0 0
24-h 0 0 0 0 0 0
48-h 0 0 0 0 0 0
72-h 0 0 0 0 0 0

Mean 24, 48 and 72 hrs

0.0 0.0 0.0 0.0 0.0 0.0
Primary irritation scores 0.25 0.25 0.0 0.0 0.25 0.0
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:

A primary dermal irritation study was conducted on six albino rabbits. There was one intact test site per animal. Each test site was treated with 0.5 g of test substance moistened with 0.5 ml of corn oil and covered with a semi-permeable dressing. The test substance was maintained in contact with the skin for 4 hours. Observations for dermal irritation and defects were made at 1, 24, 48 and 72 hours after removal of the dressings.

Very slight erythema was present in three animals only at the 1 hour observation; on the contrary, oedema was not detected at any throughout the study. No other signs of irritation were observed during the study.

The primary irritation index: was stated at 0.1 and, based on the 72 hours observations, the toxicity category IV was assigned.

Conclusion

Not irritating.

The scoring system criteria used to record the hardness of the skin reactions followed in the current test are the same as those reported into the OECD guideline (404), therefore it is possible to re-evaluate the results according to the CLP Regulation (EC 1272/2008), because the raw tables are included into the study report.

Mean values from gradings at 24, 48 and 72 hours after patch removal were lower than 2.3 in all animals for both erythema/eschar and oedema reactions, thus the test item is out of the classification criteria for skin irritation potential, according to the CLP Regulation (EC 1272/2008).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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 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.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
GLP compliance:
yes
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.
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.
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.

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
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).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

SKIN IRRITATION

A primary dermal irritation study was conducted on six albino rabbits. There was one intact test site per animal. Each test site was treated with 0.5 g of test substance moistened with 0.5 ml of corn oil and covered with a semi-permeable dressing. The test substance was maintained in contact with the skin for 4 hours. Observations for dermal irritation and defects were made at 1, 24, 48 and 72 hours after removal of the dressings.

Very slight erythema was present in three animals only at the 1 hour observation; on the contrary, oedema was not detected at any throughout the study. No other signs of irritation were observed during the study.

EYE IRRITATION

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.


Justification for selection of skin irritation / corrosion endpoint:
Test conducted according to internationally accepted testing guidelines.

Justification for selection of eye irritation endpoint:
Test conducted according to internationally accepted testing guidelines.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), 3.2 Skin corrosion/irritation section, skin Irritation means the production of reversible damage to the skin following the application of a test substance for up to 4 hours.

The mean values from gradings at 24, 48 and 72 hours after patch removal were lower than 2.3 in at least 2 of 3 animals for both erythema/eschar and oedema reactions.

According to the CLP Regulation (EC 1272/2008) substances that have the potential to induce reversible eye irritation are classified in Category 2 (irritating to eyes) if, when applied to the eye of an animal, a substance produces at least in 2 of 3 tested animals, a positive response of corneal opacity ≥ 1 and/or iritis ≥ 1, and/or conjunctival redness ≥ 2 and/or conjunctival oedema (chemosis) ≥ 2, calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material, and which fully reverses within an observation period of 21 days.

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 assayed.

In conclusion, the substance is not classified for the eye/skin irritation, according to the CLP Regulation (EC 1272/2008).