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Description of key information

No severe irritation or corrosion was detected in an in vitro eye corrosion test (BCOP).
Instillation of the test substance in the eyes of 3 rabbits resulted in slight irritation of the conjunctivae, which consisted of redness and discharge. The irritation had completely resolved within 48 hours in one animal and within 72 hours in the other two animals.
In a n accepted in vitro test system (EPISKIN-SM) the test material did not show irritant effects.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation / corrosion
Remarks:
other: in vitro human model
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05-Jun-2012 to 11 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: EU method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): C.I. Leuco Sulphur Brown 37 was applied and incubated with a cotton swab directly on top of the skin tissue

NEGATIVE CONTOL:
- Amount(s) applied (volume or weight with unit): 25 µl Phosphate buffered saline

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 25 µl
- Concentration (if solution): 5% (aq) Sodium dodecyl sulphate
Duration of treatment / exposure:
Exposure:15 minutes
Post incubation period: 42 hours
Details on study design:
TEST SITE
- Area of exposure: human epidermis model
- % coverage: 0.38 cm2

REMOVAL OF TEST SUBSTANCE
- Washing (if done): phosphate buffered saline
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
- 42 hours

SCORING SYSTEM:
- After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.
Irritation parameter:
other: percentage viability
Basis:
other: percentage of control
Time point:
other: 15 minutes
Score:
102
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
C.I. Leuco Sulphur Brown 37 is non-irritant in the in vitro skin irritation test
Executive summary:

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with C.I. Leuco Sulphur Brown 37 compared to the negative control tissues was 102%. Since the mean relative tissue viability for C.I. Leuco Sulphur Brown 37 was above 50% after 15 minutes treatment C.I. Leuco Sulphur Brown 37 is considered to be non-irritant.

 

The positive control had a mean cell viability of 4% after 15 minutes exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 5%, indicating that the test system functioned properly.

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:
14-Aug-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: OECD guideline 437 “Bovine corneal opacity and permeability (BCOP) test method for identifying ocular corrosives and severe irritants”
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method B.47 “Bovine corneal opacity and permeability method for identifying ocular corrosives and severe irritants
Deviations:
no
GLP compliance:
yes
Test material information:
Composition 1
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. Due to the consistency of the test substance, dark brown paste, the test substance was applied directly on the corneas with a cotton swab in such a way that the cornea was completely covered

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µl of physiological saline per cornea

POSITIVE CONTROL
Amount(s) applied (volume or weight with unit): 750 µl per cornea
Concentration (if solution): 10% (w/v) Benzalkonium Chloride


Duration of treatment / exposure:
- Exposure: 10 minutes
- Post incubation period: 120 minutes
Details on study design:
TEST SITE
- Isolated bovine cornea

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 10 minutes

SCORING SYSTEM:
- After the 10 minutes exposure and washing step, the corneas were incubated for 120 +/- 10 minutes at 32 °C. After the completion of the incubation period opacity determination was performed.

- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).


TOOL USED TO ASSESS SCORE:
- opacitymeter and microplate reader

DATA EVALUATION:
A test substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant
Irritation parameter:
other: In vitro irritancy score (IVIS)
Basis:
mean
Time point:
other: 10 minutes
Score:
0
Interpretation of results:
other: not severe irritant or corrosive
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
C.I. Leuco Sulphur Brown 37 is not irritant or corrosive in the Bovine Corneal Opacity and Permeability test

Executive summary:

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical rangeindicating that the negative control did not induce irritancy on the corneas,except the response of one cornea. However since the opacity and permeability value were just above the upper limit of the range and all other values were less than the upper limits of the laboratory historical range,this deviation in the opacity and the premeabilityscorehad no effect on the results of the study.

 

The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 117 and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

 

C.I. Leuco Sulphur Brown 37 did not induce ocular irritation through both endpoints, resulting in a meanin vitroirritancy score of 0 after 10 minutes of treatment.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No adverse effects were detected in an in vitro human skin model (EPISKIN-SM) after application for 15 minutes. Viability of cells was not reduced. Positive as well as negative controls gave the expected results.

No signs of irritation or corrosion were detectet in an in vitro eye corrosion test (BCOP).

Instillation of the test substance into the eyes of 3 rabbits resulted in slight irritation of the conjunctivae, which consisted of redness and discharge. Corneae as well as irises were not affected. The irritation had completely resolved within 48 hours in one animal and within 72 hours in the other two animals.


Justification for selection of skin irritation / corrosion endpoint:
only study

Justification for selection of eye irritation endpoint:
only study

Justification for classification or non-classification

not classified; no relevant adverse effects in in vitro skin test as well as in vitro eye corrosion and in vivo eye irritation studies.