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Endpoint summary
Administrative data
Description of key information
Not irritant to skin and eye.
Key value for chemical safety assessment
Skin irritation / corrosion
Link to relevant study records
- Endpoint:
- skin irritation: in vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- July from 12 to 15, 2005
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
- Version / remarks:
- adopted 24 April 2002
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
- GLP compliance:
- yes (incl. QA statement)
- Specific details on test material used for the study:
- For the purpose of the study the test material was ground to a powder prior to application.
The pH of the test material (10 % w/w aqueous preparation) was determined prior to commencement of the study and found to be 6.6. - Species:
- rabbit
- Strain:
- New Zealand White
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: David Percival Ltd, Moston, Sandbach, Cheshire, UK.
- Age at study initiation: twelve to twenty weeks old.
- Weight at study initiation: weight range of 2.0 to 3.5 kg.
- Housing: animals were individually housed in suspended cages.
- Diet: certified rabbit diet (Code 5322) supplied by BCM IPS Limited, ad libitum.
- Water: drinking water, ad libitum.
- Acclimation period: at least five days.
ENVIRONMENTAL CONDITIONS
- Temperature: 17 to 23 °C
- Humidity: 30 to 70 %
- Air changes: at least fifteen changes per hour.
- Photoperiod: lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness. - Type of coverage:
- semiocclusive
- Preparation of test site:
- clipped
- Vehicle:
- water
- Controls:
- yes, concurrent no treatment
- Amount / concentration applied:
- 0.5 g of test material, moistened with 0.5 ml of distilled water
- Duration of treatment / exposure:
- 4-hours
- Observation period:
- 72 hours
- Number of animals:
- 3 rabbits
- Details on study design:
- TEST SITE
- Area of exposure: on the day before the test each of a group of three rabbits was clipped free of fur from the dorsal/flank area using veterinary clippers. Only animals with a healthy intact epidermis by gross observation were selected for the study.
- Application: the test material was introduced under a 2.5 cm × 2.5 cm cotton gauze patch and placed in position on the shorn skin.
- Type of wrap if used: the patch was secured in position with a strip of surgical adhesive tape. To prevent the animals interfering with the patches, the trunk of each rabbit was wrapped in an elasticated corset and the animals were returned to their cages for the duration of the exposure period.
REMOVAL OF TEST SUBSTANCE
- Dress removal: any residual test material was removed by gentle swabbing with cotton wool soaked in 74 % Industrial Methylated Spirits.
- Time after start of exposure: 4 hours.
OBSERVATION TIME POINTS
Approximately one hour following the removal of the patches, and 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation.
SCORING SYSTEM
The scores for erythema and oedema at the 24 and 72-hour readings were totalled for the three test rabbits (12 values) and this total was divided by six to give the primary irritation index of the test material. The test material was classified according to the scheme devised by Draize J H (1959) "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.
Primary Irritation Index // Classification of Irritancy
0: Non-irritant
> 0 to 2 Mild irritant
> 2 to 5 Moderate irritant
> 5 to 8 Severe irritant
Erythema and Eschar Formation
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beef redness) to eschar formation preventing grading of erythema 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 millimetre) 3
Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure) 4 - Irritation parameter:
- erythema score
- Basis:
- animal: 3/3
- Time point:
- 24/48/72 h
- Score:
- < 2.3
- Reversibility:
- fully reversible within: 48 hrs
- Irritation parameter:
- edema score
- Basis:
- animal: 3/3
- Time point:
- 24/48/72 h
- Score:
- < 2.3
- Remarks on result:
- no indication of irritation
- Irritant / corrosive response data:
- The test material produced a primary irritation index of 0.2 and was classified as a mild irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.
- Interpretation of results:
- other: )not classified, according to the CLP Regulation (EC 1272/2008)
- Conclusions:
- Not skin irritant.
- Executive summary:
The study was performed to assess the irritancy potential of the test material to the skin of the New Zealand White rabbit. The method was designed to meet the requirements of the OECD guideline and the EU Method B4.
A single 4-hour, semi-occluded application of the test material to the intact skin of three rabbits produced very slight erythema. Two treated skin sites appeared normal at the 24-hour observation and the remaining treated skin site appeared normal at the 48-hour observation. No signs of oedema were noted.
Conclusion
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 does not meet the criteria to be classified as irritating, according to the CLP Regulation (EC 1272/2008).
Reference
Individual Skin Reactions
Animal N. | Reaction | 1 hr | 24 hrs | 48 hrs | 72 hrs | Mean 24/48/72 hrs |
Male 152 | Erythema | 1 STA | 0 STA | 0 STA | 0 | 0.00 |
Male 153 | Erythema | 1 STA | 0 STA | 0 STA | 0 STA | 0.00 |
Male 154 | Erythema | 1 STA | 1 STA | 0 STA | 0 | 0.33 |
Male 152 | Oedema | 0 | 0 | 0 | 0 | 0.00 |
Male 153 | Oedema | 0 | 0 | 0 | 0 | 0.00 |
Male 154 | Oedema | 0 | 0 | 0 | 0 | 0.00 |
Sum of 24 and 72-hour Readings (S) | 1 | |||||
Primary Irritation Index (S/6) | 1/6 = 0.2 | |||||
Classification | Mild irritant |
STA = Blue-coloured staining
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Eye irritation
Link to relevant study records
- Endpoint:
- eye irritation: in vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- July from 18 to 29, 2005
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 405 (Acute Eye Irritation / Corrosion)
- Version / remarks:
- adopted 24 April 2002
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
- GLP compliance:
- yes (incl. QA statement)
- Specific details on test material used for the study:
- For the purpose of the study the test material was ground to a powder prior to application.
The pH of the test material (10 % w/w aqueous preparation) was determined prior to commencement of the study and found to be 6.6. - Species:
- rabbit
- Strain:
- New Zealand White
- Details on test animals or tissues and environmental conditions:
- TEST ANIMALS
- Source: David Percival Ltd, Moston, Sandbach, Cheshire, UK.
- Age at study initiation: twelve to twenty weeks old.
- Weight at study initiation: weight range of 2.0 to 3.5 kg.
- Housing: animals were individually housed in suspended cages.
- Diet: certified rabbit diet (Code 5322) supplied by BCM IPS Limited, ad libitum.
- Water: drinking water, ad libitum.
- Acclimation period: at least five days.
- Health check: immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.
ENVIRONMENTAL CONDITIONS
- Temperature: 17 to 23 °C
- Humidity: 30 to 70 %
- Air changes: at least fifteen changes per hour.
- Photoperiod: lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness. - Controls:
- yes, concurrent no treatment
- Amount / concentration applied:
- 0.1 ml of the test material, which was found to weigh approximately 88 mg
- Observation period (in vivo):
- 7 days
- Number of animals or in vitro replicates:
- Three rabbits
- Details on study design:
- APPLICATION
- Schedule: initially, a single rabbit was treated. After consideration of the ocular responses produced in the first treated animal, two additional animals were treated.
- Application: test item was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test material and then released.
TOOL USED TO ASSESS SCORE: examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.
SCORING SYSTEM
Immediately after administration of the test material, an assessment of the initial pain reaction was made according to the six point scale.
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation from Draize J. H. (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington DC pA8 to 49.
The numerical values corresponding to each animal, tissue and observation time were recorded. The data relating to the conjunctivae were designated by the letters A (redness), B (chemosis) and C (discharge), those relating to the iris designated by the letter D and those relating to the cornea by the letters E (degree of opacity) and F (area of cornea involved).
Using the numerical data obtained a modified version of the system described by Kay J Hand Calandra J C (1962), J. Soc. Cosmet. Chern. 13, 281-289 was used to classify the ocular irritancy potential of the test material. This was achieved by adding together the scores for the cornea, iris and conjunctivae for each time point for each rabbit. The group means of the total scores for each observation were calculated. The highest of these group means (the maximum group mean score) together with the persistence of the reactions enabled classification of the eye irritancy potential of the test material.
CONJUNCTIVAE
(A) Redness (refers to palpebral 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 half closed to completely closed 4
(C) 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 a considerable area around the eye 3
THE TOTAL SCORE = (A + B + C) × 2 MAXIMUM TOTAL = 20
IRIS
(D) Values
Normal 0
Folds above normal, congestion, swelling, circumcorneal 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, haemorrhage, gross destruction (any or all of these) 2
THE TOTAL SCORE = D × 5
CORNEA
(E) Degree of Opacity (most dense area used)
No opacity 0
Scattered or diffuse areas, details of iris clearly visible 1
Easily discernible translucent areas, details of iris slightly obscured 2
Opalescent areas, no details of iris visible, size of pupil barely discernible 3
Opaque, iris not discernible through the opacity 4
(F) Area of Cornea Involved
One quarter (or less) but not zero 0
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
THE TOTAL SCORE = (E × F) × 5 MAXIMUM TOTAL = 80
MAXIMUM TOTAL SCORE POSSIBLE = 110 - Irritation parameter:
- cornea opacity score
- Basis:
- animal: 3/3
- Time point:
- 24/48/72 h
- Score:
- < 1
- Remarks on result:
- no indication of irritation
- Irritation parameter:
- iris score
- Basis:
- animal: 3/3
- Time point:
- 24/48/72 h
- Score:
- < 1
- Remarks on result:
- no indication of irritation
- Irritation parameter:
- conjunctivae score
- Basis:
- animal: 3/3
- Time point:
- 24/48/72 h
- Score:
- < 2
- Reversibility:
- fully reversible within: 7 days
- Irritation parameter:
- chemosis score
- Basis:
- animal: 3/3
- Time point:
- 24/48/72 h
- Score:
- < 2
- Reversibility:
- fully reversible within: 72 hrs
- Irritant / corrosive response data:
- The test material produced a maximum group mean score of 8.0 and was classified as a mild irritant (class 4 on 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.
Black staining of the fur was noted around all treated eyes during the study. No corneal or iridial effects were noted during the study. Minimal to moderate conjunctival irritation was noted in all treated eyes one hour after treatment and at the 24-hour observation with minimal conjunctival irritation in all treated eyes at the 48-hour observation and in one treated eye at the 72-hour observation.
Two treated eyes appeared normal at the 72-hour observation and the remaining treated eye appeared normal at the 7-day observation. - Interpretation of results:
- other: not classified, according to the CLP Regulation (EC 1272/2008)
- Conclusions:
- Not eye irritant.
- Executive summary:
The study was performed to assess the irritancy potential of the test material to the eye of the New Zealand White rabbit. The method was designed to meet the requirements of the OECD guideline 405 and the EU Method B5.
A single application of the test material to the non-irrigated eye of three rabbits produced minimal to moderate conjunctival irritation. Two treated eyes appeared normal at the 72-hour observation and the remaining treated eye appeared normal at the 7-day observation.
The test material produced a maximum group mean score of 8.0 and was classified as a mild irritant (class 4 on 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.
Black staining of the fur was noted around all treated eyes during the study. No corneal or iridial effects were noted during the study. Minimal to moderate conjunctival irritation was noted in all treated eyes one hour after treatment and at the 24-hour observation with minimal conjunctival irritation in all treated eyes at the 48-hour observation and in one treated eye at the 72-hour observation.
Two treated eyes appeared normal at the 72-hour observation and the remaining treated eye appeared normal at the 7-day observation.
Conclusion
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 of the treated animals. Alle effects were reversible within 7 days.
Therefore, the substance does not meet the criteria to be classified as eye irritating, according to the CLP Regulation (EC 1272/2008).
Reference
Individual scores and individual total scores for ocular irritation
Animal no. | Reaction | 1 hr | 24 hrs | 48 hrs | 72 hrs | 7 days | Mean 24/48/72 hrs |
Male 21 | Cornea | 0 | 0 | 0 | 0 | 0 | 0.00 |
Male 71 | Cornea | 0 | 0 | 0 | 0 | 0 | 0.00 |
Male 72 | Cornea | 0 | 0 | 0 | 0 | 0 | 0.00 |
Male 21 | Iris | 0 | 0 | 0 | 0 | 0 | 0.00 |
Male 71 | Iris | 0 | 0 | 0 | 0 | 0 | 0.00 |
Male 72 | Iris | 0 | 0 | 0 | 0 | 0 | 0.00 |
Male 21 | Conjunctival redness | 2 | 2 | 1 | 1 | 0 | 1.33 |
Male 71 | Conjunctival redness | 1 | 1 | 1 | 0 | 0 | 0.67 |
Male 72 | Conjunctival redness | 1 | 1 | 1 | 0 | 0 | 0.67 |
Male 21 | Conjunctival oedema | 2 | 2 | 1 | 0 | 0 | 1.00 |
Male 71 | Conjunctival oedema | 1 | 1 | 0 | 0 | 0 | 0.33 |
Male 72 | Conjunctival oedema | 1 | 0 | 0 | 0 | 0 | 0.00 |
21 Male, IPR=2 | 71 Male, IPR=2 | 72 Male, IPR=2 | |||||||||||
Time after treatment | 1 hrs | 24 hrs | 48 hrs | 72 hrs | 7 d | 1 hrs | 24 hrs | 48 hrs | 72 hrs | 1 hrs | 24 hrs | 48 hrs | 72 hrs |
Cornea |
|||||||||||||
E = degree of opacity | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
F = area of cornea involved | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Score (E × F) × 5 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Iris |
|||||||||||||
D | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Score (D × 5) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Conjunctival |
|||||||||||||
A = Redness | 2 | 2 | 1 | 1 | 0 | 1 | 1 | 1 | 0 | 1 | 1 | 1 | 0 |
B = Chemosis | 2 | 2 | 1 | 0 | 0 | 1 | 1 | 0 | 0 | 1 | 0 | 0 | 0 |
C = Discharge | 2Sf | 1Sf | 0Sf | 0Sf | 0Sf | 1Sf | 1Sf | 1Sf | 0Sf | 1Sf | 1Sf | 1Sf | 0 |
Score (A + B + C) × 2 | 12 | 10 | 4 | 2 | 0 | 6 | 6 | 4 | 0 | 6 | 4 | 4 | 0 |
Total Score | 12 | 10 | 4 | 2 | 0 | 6 | 6 | 4 | 0 | 6 | 4 | 4 | 0 |
IPR = Initial pain reaction; 2 means that rabbit blinks and tries to open eye, but reflex closes it; slight initial pain
Sf= Black staining of the fur around the treated eye
Individual total scores and group mean scores for ocular irritation
Individual total scores at | |||||
Rabbit no. | 1 hrs | 24 hrs | 48 hrs | 72 hrs | 7 days |
Male 21 | 12 | 10 | 4 | 2 | 0 |
Male 71 | 6 | 6 | 4 | 0 | - |
Male 72 | 6 | 4 | 4 | 0 | - |
Group total |
24 | 20 | 12 | 2 | 0 |
Group Mean Score |
8.0 | 6.7 | 4.0 | 0.7 | 0.0 |
- = Observation not required
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Respiratory irritation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
SKIN IRRITATION
The skin irritancy potential of Direct Blue 267 was assessed in New Zealand White rabbit, following the method and procedures described into the OECD guideline 404 and the EU Method B4. Animals were treated with 0.5 g of the test material, moistened with 0.5 ml of distilled water, over a period of 4-hour, by semi-occluded application. Very slight erythema was observed. Two treated skin sites appeared normal at the 24-hour observation and the remaining treated skin site appeared normal at the 48-hour observation. The test material produced a primary irritation index of 0.2 and was classified as a mild irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted (Mullaney, 2005).
EYE IRRITATION
The eye irritancy potential of Direct Blue 267 was assayed in New Zealand White rabbit. The method was designed to meet the requirements of the OECD guideline 405 and the EU Method B5. A single application of 0.1 ml of the test material was applied to the non-irrigated eye of three rabbits; minimal to moderate conjunctival irritation was observed. Two treated eyes appeared normal at the 72-hour observation and the remaining treated eye appeared normal at the 7-day observation.
The test material produced a maximum group mean score of 8.0 and was classified as a mild irritant (class 4 on 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system. Black staining of the fur was noted around all treated eyes during the study. No corneal or iridial effects were noted during the study. Minimal to moderate conjunctival irritation was noted in all treated eyes one hour after treatment and at the 24-hour observation with minimal conjunctival irritation in all treated eyes at the 48-hour observation and in one treated eye at the 72-hour observation. Two treated eyes appeared normal at the 72-hour observation and the remaining treated eye appeared normal at the 7-day observation (Mullaney, 2005).
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.
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.
According to the CLP Regulation (EC 1272/2008), serious eye damage means the production of tissue damage in the eye, or serious physical decay of vision, following application of a test substance, which is not fully reversible within 21 days of application.
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 of the treated animals. All the reactions recorded were fully reversible within 7 days.
In conclusion, the substance does not meet the criteria to be classified either for the skin, neither for the eye irritation, according to the CLP Regulation (EC 1272/2008).
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