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Description of key information

Non skin irritant

Non eye irritant

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 June 11th to 14h, 2007
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:
April 24, 2002
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Version / remarks:
June 16, 2004
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2500 (Acute Dermal Irritation)
Version / remarks:
August 1998
Qualifier:
according to guideline
Guideline:
other: Japan MAFF Testing Guideline of 12 Nousan No. 8147, November 24, 2000
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Centre Lago S. A., 01540 Vonnas, France.
- Age at study initiation: 5 - 8 months.
- Weight at study initiation: 3.60 - 4.80 kg.
- Housing: single housing, stainless steel wire mesh cages with grating, floor area: 3000 cm².
- Diet: Kliba-Labordiät about 130 g/animal per day.
- Water: tap water. ad libitum.
- Acclimation period: at least 5 days before application. Before the beginning of application, the application area was investigated for signs of pre-existing skin irritation or dense patches of hair. Only animals with intact healthy skin were used.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 – 24 °C
- Humidity: 30 - 70 %
- Photoperiod: 12 hrs dark / 12 hrs light
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
water
Remarks:
doubly distilled
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.5 g
- Preoparation: the solid test substance was minimally moistened with a suitable amount of doubly distilled water to guarantee skin contact immediately before test-substance application. Because of the natural moisture of the skin doubly distilled water was used for moistening, so that the test was carried out under conditions as physiological as possible.
Duration of treatment / exposure:
4 hours
Observation period:
72 hours
Number of animals:
3 rabbits: 2 males, 1 female
Details on study design:
TEST SITE
- Area of exposure: at least 24 hours before application clipping of the dorsolateral part of the trunk of the animal(s).
- Type of coverage: the test patch (2.5 x 2.5 cm) was secured in position with a semiocclusive dressing (Idealbinde, Pfälzische Verbandstoff-Fabrik,Kaiserslautern) and Fixomull® stretch (adhesive fleece), Beiersdorf AG).

REMOVAL OF TEST SUBSTANCE
- Washing: the test substance was removed at the end of the exposure period with Lutrol®and Lutrol® / water (1 : 1) (Lutrol® E 400 = Polyethylenglycol, BASF Aktiengesellschaft).
- Time after start of exposure: 4 hours.

OBSERVATIONS
- Readings: Immediately after removal of the patch, approx. 1, 24, 48 and 72 h after removal of the patch and then in weekly intervals maximally up to day 14.
- Illumination used for reading: daylight tubes "Lumilux" (L 58W/860 PLUS ECO 25X1, Osram, Germany).
- Mortality: a check for any dead or moribund animal was made twice each workday (at the beginning and end of work) and once daily at weekends and on public holidays.
- Body weight determination: just before application of the test substance and after the last reading.

SCORING SYSTEM
The evaluation of skin reactions was performed according to the quoted guidelines. For evaluation, the calculation of the mean values of erythema and edema for readings at 24, 48 and 72 hours were quoted. Additionally, the reversibility of the observed findings was taken into account. The evaluation is based on the criteria of the Commission Directive 67/548/EEC and the OECD Harmonized Integrated Classification System that were in place on the date of report signature.

Erythema and eschar formation - Grading
0 No erythema
1 Very slight erythema (barely perceptible)
2 Well defined erythema
3 Moderate to severe erythema
4 Severe erythema (beet redness) to eschar formation preventing grading of erythema

Edema formation - Grading
0 No edema
1 Very slight edema (barely perceptible)
2 Slight edema (edges of area well defined by definite raising)
3 Moderate edema (raised approx. 1 mm)
4 Severe edema (raised more than 1 mm and extending beyond area of exposure)
Irritation parameter:
erythema score
Basis:
animal: 3/3
Time point:
24/48/72 h
Score:
< 2.3
Reversibility:
fully reversible
Irritation parameter:
edema score
Basis:
animal: 3/3
Time point:
24/48/72 h
Score:
< 2.3
Reversibility:
fully reversible
Irritant / corrosive response data:
Moderate erythema (grade 2) was observed in all animals immediately and 1 hour after removal of the patch and persisted in one animal up to 24 hours. Moderate erythema decreased to slight (grade 1) in two animals after 24 hours and in one animal after 48 hours.
Slight erythema persisted in one animal up to 48 hours.
The cutaneous reactions were reversible in one animal within 48 hours and in two animals within 72 hours after removal of the patch.
Mean scores over 24, 48 and 72 hours for each animal were 0.3, 1.0 and 0.7 for erythema and 0.0 for edema.

Reaciton Readings (hours) Mean 24, 48 and 72 hrs
Animal 0 1 24 48 72
Erythema Animal 01 (M) 2 2 1 0 0 0.3
Animal 02 (M) 2 2 2 1 0 1.0
Animal 03 (F) 2 2 1 1 0 0.7
Oedema Animal 01 (M) 0 0 0 0 0 0.0
Animal 02 (M) 0 0 0 0 0 0.0
Animal 03 (F) 0 0 0 0 0 0.0
Interpretation of results:
other: not classified, according to the CLP Regulation (EC 1272/2008)
Conclusions:
Not irritating
Executive summary:

The potential of the test substance to cause acute skin irritation was assessed according to the OECD guideline 404. Intact skin of 3 White New Zealand rabbits was exposed to 0.5 g of the test substance in a single topical application for 4 hours. A patch of 2.5 cm x 2.5 cm, covered with semiocclusive dressing was used. After removal of the patch, the application area was washed off. The cutaneous reactions were assessed immediately after removal of the patch, approximately 1, 24, 48 and 72 hours after removal of the patch. Slight or moderate erythema were observed in all animals during the course of the study. The cutaneous reactions were reversible in all animals within 72 hours after removal of the patch.

The average score (24 to 72 hours) for irritation was calculated to be 0.7 for erythema and 0.0 for edema. Considering the described cutaneous reactions as well as the average score for irritation, the test substance does not show a skin irritation potential under the test conditions chosen.

Discussion and 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).

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October from 21st to 22th, 2015
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 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
adopted 26th July, 2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
other: Bovine
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: breeding service CHOVSERVIS a.s., division TORO® Hlavečník, Hradec Králové, Czech Republic.
- Collection: eyes were collected by slaughterhouse employees. The eyes were enucleated as soon as possible after death.
- Deterget: no detergent was used.
- Animals: only healthy animals (12 to 60 months old) considered suitable for entry into the human food chain were used as a source of corneas for use in the BCOP test.
- Storage: the risk of contamination was minimized (e.g., by keeping the container containing the eyes on ice, by adding antibiotics to the HBSS used to store the eyes during transport (e.g., penicillin at 100 IU/ml and streptomycin at 100 μg/ml).
The time interval between collection of the eyes and use of corneas in the BCOP was minimized (typically collected and used on the same day). All eyes used in the assay were from the same group of eyes collected on a specific day.
Vehicle:
physiological saline
Controls:
yes
Amount / concentration applied:
Application form preparation: the test substance was tested as suspension prepared from test substance at 20 % concentration in a 0.9 % sodium chloride solution. 2 g of the test substance was suspended in 10 ml of 0.9 % sodium chloride solution.
Open-chamber method was used, because the test substance at 20 % concentration was a viscous paste. The test substance (enough test substance to completely cover the cornea) was applied directly to the epithelial surface of the cornea using the micropipette. After dosing, the glass window was replaced on the anterior chamber to recreate a closed system.
Number of animals or in vitro replicates:
Exposed group (test substance) - 3 corneas
Details on study design:
EYE SELECTION AND EXANMINATION
The eyes, once they arrive at the laboratory, were carefully examined for defects including scratched, and neovascularisation. Only corneas from eyes free of such defects were used. The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity > 7 opacity units were discarded. From 20 eyes the 3 eyes were eliminated after inductive incubation, because the baseline opacity values were > 7.

EYE PREPARATION
Corneas free of defects were dissected with a 2 to 3 mm rim of sclera remaining to assist in subsequent handling, with care taken to avoid damage to the corneal epithelium an endothelium. Isolated corneas were mounted in specially designed corneal holders that consisted of anterior and posterior compartments, which interfaced with the epithelial and endothelial sides of the cornea, respectively. Both chambers were filled to excess with pre-warmed Eagle's Minimum Essential Medium (EMEM). The device was then equilibrated at 32 ± 1°C for at least one hour in water bath to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity, to the extent possible. Following the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Any corneas that showed macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity > 7 opacity units were discarded. Each test group (test substance, concurrent negative and positive controls) consisted of the three eyes. The three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment and positive control groups.

POST EXPOSURE
After the exposure period, the negative control and the positive control substance was removed from the anterior chamber with EMEM (containing phenol red - the effectiveness of rinsing acidic or alkaline materials). The corneas were given a final rinse with EMEM (without phenol red). The EMEM (without phenol red) was used as a final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded. The test substance was removed from the anterior chamber with EMEM (containing phenol red). The corneas (applied the test substance) were also repeatedly rinsed with EMEM (without phenol red), because the test substance is coloured. Rinsing was finalized after complete removal of the test substance. The EMEM (without phenol red) was used as a final rinse to the opacity measurement. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded.

CONTROLS
- Positive control group: 20 % Imidazole in 0.9 % NaCl; 3 corneas
- Negative control group: 0.9 % NaCl; 3 corneas

MEASUREMENTS
Opacity: the amount of light transmission through the cornea. Corneal opacity was measured quantitatively with the aid of an opacitometer resulting in opacity values measured on a continuous scale. Permeability: the amount of sodium fluorescein dye that penetrates all corneal cell layers (i.e., the epithelium on the outer cornea surface through the endothelium on the inner cornea surface) measured indirectly using visible light spectrophotometry. 1 ml sodium fluorescein solution (5 mg/ml) was added to the anterior chamber of the corneal holder, which interfaced with the epithelial side of the cornea, while the posterior chamber, which interfaced with the endothelial side of the cornea, is filled with fresh EMEM. The holder was incubated in horizontal position for 1.5 hours at 32 ± 1 °C. The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured with the aid of UV/VIS spectrophotometry. The values of absorbance measured at 490 nm were recorded as optical density (OD490) values. This term was used because the measuring is performed with visible light spectrophotometer using a standard 1 cm path length.

STUDY ACCEPTANCE CRITERIA
A test is considered acceptable if the positive control gives IVIS that falls within one standard deviations of the current historical mean, which is to be updated at least every three months, or each time an acceptable test is conducted in laboratories where tests are conducted infrequently. The negative or solvent/vehicle control responses should result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control.

SCORING SYSTEM
The IVIS cut-off value for identifying the test substance as including serious eye damage (UN GHS Category 1) and the test substance not requiring classification for eye irritation or serious damage (UN GHS No Category) will be given hereafter:
≤ 3: no category
≤ 55: no prediction can be made
≥ 55: category 1
Irritation parameter:
in vitro irritation score
Value:
< 3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
IVIS: 2.69 not irritating
No macroscopic damage was observed on corneas before application. Corneal opacity was observed on the corneas treated by positive control. The corneas treated by negative control were without macroscopic damage. The corneas treated by the test substance were without macroscopic damage.

NEGATIVE CONTROL
The value of opacity for negative control (0.9 % NaCl) obtained during the study was 1.00 and value of permeability was 0.032. The values obtained during this study not exceeded upper limits, so the study is considered acceptable.

POSITIVE CONTROL
The value of IVIS for positive control (20 % imidazole in 0.9 % NaCl) obtained during the study was 75.45. This value is within the acceptance limit (one standard deviations of the current historical mean), so the study is considered acceptable.

IVIS values

Group IVIS
Calculation Result
NC
(0.9 % NaCl)
1.00 + 15 x 0.032  1.48
PC
(20 % Imidazole in 0.9 % NaCl)
48.00 + 15 x 1.830   75.45
EXP
(test item)
2.67 + 15 x 0.001 2.69

NC- negative control; PC- positive control; EXP – test substance application form

Appearance of Corneas after the Test Substance Exposure

Group Cornea No. Appearance after exposure 
Negative control 1 Without macroscopic damage
4 Without macroscopic damage
5 Without macroscopic damage
Positive control 11 Corneal opacity 
12 Corneal opacity 
14 Corneal opacity 
Test substance 8 Without macroscopic damage
9 Without macroscopic damage
10 Without macroscopic damage

Opacity

Group Cornea No. Baseline opacity Opacity after treatment Opacity difference Mean opacity difference Mean Opacity(corrected)
NC
(0.9 % NaCl)
1 5 7 2 1.00 -
4 5 6 1
5 6 6 0
PC
(20 % Imidazole in 0.9 % NaCl)
11 3 48 45 49.00 (49.00 – 1.00)
48.00
12 6 57 51
14 5 56 51
EXP
(test item)
8 6 10 4 3.67 (3.67 – 1.00)
2.67
9 4 7 3
10 5 9 4

NC- negative control; PC- positive control; EXP – test substance application form

Permeability

Group Cornea No. Values of Permeability (Optical density at 490nm) Mean Permeability Mean Permeability(corrected)
NC
(0.9 % NaCl)
1 0.038 0.032 -
4 0.026
5 0.032
PC
(20 % Imidazole in 0.9 % NaCl)
11 2.135 1.862 (1.862 – 0.032)
1.830
12 1.722
14 1.729
EXP
(test item)
8 0.035 0.033 (0.033 – 0.032)
0.001
9 0.031
10 0.034

NC- negative control; PC- positive control; EXP – test substance application form

Interpretation of results:
other: not classified, according to the CLP Regulation (EC 1272/2008)
Conclusions:
Not irritating
Executive summary:

The test substance was tested for the evaluation the potential ocular corrosivity or severe irritancy as measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The test was performed according to the OECD Test Guideline No. 437, Adopted 26th July 2013. 

The test was performed using nine isolated bovine corneas. The testing was performed on three groups of corneas: test substance treatment group, positive control group and negative control group. Three corneas per group were used. Open-chamber method was used, because the test substance was higly viscous. The opacity and permeability of each cornea were measured. The In Vitro Irritancy Score (IVIS) was calculated from the values of opacity and permeability.

No macroscopic damage was observed on corneas before application. Corneal opacity was observed on the corneas treated by positive control. The corneas treated by negative control were without macroscopic damage. The corneas treated by the test substance were without macroscopic damage.

The In Vitro Irritancy Score (IVIS) for test item was 2.69. This value of IVIS is lower than 3 therefore the classification according to UN GHS criteria for eye irritation or serious eye damage is: no category.

Conclusion

Not irritating

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

SKIN IRRITATION

The potential of the Basic Blue 140 to cause acute skin irritation was assessed according to the OECD guideline 404. Intact skin of 3 White New Zealand rabbits was exposed to 0.5 g of the test substance in a single topical application for 4 hours. Slight or moderate erythema were observed in all animals during the course of the study. The cutaneous reactions were reversible in all animals within 72 hours after removal of the patch. The 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).

EYE IRRITATION

Basic Blue 140 was tested for the evaluation the potential ocular corrosivity or severe irritancy as measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The test was performed according to the OECD Test Guideline No. 437, Adopted 26th July 2013.  The test was performed using nine isolated bovine corneas. Three corneas per group were used. Open-chamber method was used, because the test substance was highly viscous. No macroscopic damage was observed on corneas before application. Corneal opacity was observed on the corneas treated by positive control. The corneas treated by negative control were without macroscopic damage. The corneas treated by the test substance were without macroscopic damage. The In Vitro Irritancy Score (IVIS) for test item was 2.69. This value of IVIS is lower than 3, therefore the classification according to UN GHS criteria for eye irritation or serious eye damage is: no category.

An older test is also available on Basic Blue 140; it was conducted according to the OECD guideline 405. Single ocular application of 0.1 ml bulk volume (about 30 mg) of the test substance was done to one eye of a White New Zealand rabbit. The conclusion stated into the study report indicates the substance as potentially able to cause serious eye damage, on the basis of not reversible ocular reactions (i.e. discouloured part of the cornea and conjunctiva). Nevertheless, the tables included into the study report, in which the ocular reactions are reported with the relative scores, indicates that all the reactions observed were recovered within 21 days; in fact, the reading at the 21th day are all equal to 0. The conclusion declared into the study report is inconsistent with the results indicated in tables. In light of this discrepancy, a clear conclusion cannot be reached, thus the test has been disregarded and the above mentioned in vitro experiment performed.

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 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. Eye irritation means the production of changes in the eye, which are fully reversible within 21 days of application. In vitro alternatives that have been validated and accepted can be used to make classification decisions.

The test was performed according to the OECD Guideline No. 437, Adopted 26th July 2013; the BCOP test method is recommended to identify chemicals inducing serious eye damage (i.e. chemicals to be classified as UN GHS Category 1) and to identify chemicals that do not require classification for eye irritation or serious eye damage (i.e. UN GHS No Category).

The IVIS value resulted lower than 3, therefore the classification according to UN GHS criteria for eye irritation or serious eye damage is no category.

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