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Skin irritation / corrosion

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skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July from 12 to 15, 2005
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference Type:
study report

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
adopted 24 April 2002
according to guideline
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Direct Blue 267
Direct Blue 267
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.

Test animals

New Zealand White
Details on test animals or test system and environmental conditions:
- 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.

- 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.

Test system

Type of coverage:
Preparation of test site:
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:
Observation period:
72 hours
Number of animals:
3 rabbits
Details on study design:
- 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.

- 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.

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.

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

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
erythema score
animal: 3/3
Time point:
24/48/72 h
< 2.3
fully reversible within: 48 hrs
Irritation parameter:
edema score
animal: 3/3
Time point:
24/48/72 h
< 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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

Interpretation of results:
other: )not classified, according to the CLP Regulation (EC 1272/2008)
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.


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).