[[4-[[4-(anilino)phenyl][4-(phenylimino)-2,5-cyclohexadien-1-ylidene]methyl]phenyl]amino]benzenesulphonic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from July 09, 2012 to November 15, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline test with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Pigment blue 61

IUPAC Name:

Pigment blue 61

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

colour:dark blue
consistency:solid
form:crystalline powder
Designation Pigment blue 61
Product name Print Alkali Blue 61DT6101
CAS no. #1324-76-1
Receipt no. 51074
Date of receipt: June 28, 2012
Characteristics Powder
Storage conditions In a cool dry place, isolated from incompatible materials. Kept away from heat, sparks, flame and from direct sunlight.

In vivo test system

Test animals

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

4.1 Animals / Animal maintenance
NMRI mice supplied by Charles River Deutschland GmbH were used in this experiment.

Species Mice (non-pregnant, nulliparous, healthy)

Strain / Stock NMRI / Crl:NMRI

Breeder Charles River Laboratories,
Research Models and Services,
Germany GmbH
Sandhofer Weg 7
97633 Sulzfeld, Germany

Selection of species The mouse is a rodent commonly used for such a study.

Sex Female

Number of animals 36 (6 groups of 6 female animals each)

Body weight (on test day 1) 28 - 32 g

Age (on test day 1) 62 days

Identification of animals By cage label; ear tags were not used for identification of animals

Adaptation period At least 5 days
Housing
Before application the animals were housed in groups in MAKROLON cages (type III) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 15 cm at a room temperature of 22°C  3°C (maximum range) and a relative humidity of 55%  15% (maximum range). After application the animals were housed singly in order to prevent their licking off the test item from the ears of the other animals.
Deviations from the maximum range caused for example during cleaning procedures and change of cages are dealt with in SOPs.
The rooms were alternately lit (150 lux at approx. 1.50 m room height) and darkened for periods of 12 hours each. The air in the rooms was changed 12 - 18 times per hour.
Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. The cages were changed and cleaned once a week.
Periodic analysis of the bedding material for contaminants based on EPA/USA is conducted at least once a year by LUFA-ITL (see Appendix 2: Limitation for contaminants in the bedding material).
Drinking water
Tap water was offered ad libitum.
This food was offered ad libitum. Food residue was removed.

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

5%, 10% and 25%, w/w) dissolved in dimethyl sulfoxide (DMSO)

No. of animals per dose:

6

Details on study design:

Preliminary experiment
A preliminary experiment was carried out in 3 animals to examine the irritating potential and handling/application of the test item in order to select the appropriate concentra-tions. Three concentrations of 5, 10 and 25% of Pigment blue 61 in DMSO were exam-ined. Doses were selected according to OECD guideline from the concentration series 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5% etc.
Pigment blue 61 was a brown powder. A 25% solution was the maximum applicable concentration of Pigment blue 61 in DMSO.
The experimental schedule of the assay was as follows:
- Day 1:
The weight of each animal was individually identified and recorded. In addition, ear swelling measurements were carried out at the helical edge of both ears using an Oditest micrometer.
Open application of 25 µL of the appropriate dilution of the test item, the vehicle alone or the positive control (as appropriate) were administered to the dorsum of each ear.
- Days 2 and 3:
The application procedure carried out on day 1 was repeated.
- Day 4 (24 hours after the last application the animals were sacrificed under ether anaesthesia by cutting the aorta abdominalis):
Ear swelling measurements (immediately before sacrificing the mice) were carried out at the helical edge of both ears using an Oditest micrometer.
Punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and immediately weighed on an analytical balance.
Lateral pairs of auricular lymph nodes draining the ear tissue were excised, carefully separated from remaining fatty tissue and weighed on an analytical balance immediately following preparation. The lymph nodes were then stored on ice in PBS /0.5% BSA and subjected to the preparation of single cell suspensions by mechanical tissue disaggregation. The cells were counted automatically in a cell counter.

In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitising properties.

Observations
Dated and signed records of all activities relating to the day by day running and maintenance of the study within the animal unit as well as to the group observations and examinations outlined in the Study Plan were recorded in the appropriate documentation. In addition, observations related to individual animals were made throughout the study and were recorded.
The following observations were made during the course of the study:

Clinical signs
Animals were observed once daily for any clinical signs of local systemic irritation at the application site or of systemic toxicity. Observations were recorded for each individual animal. Cageside observations included skin/fur, eyes, mucous mem¬branes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.
In addition, animals were checked regularly throughout the working day from 7:30 a.m. to 4:30 p.m. On Saturdays and Sundays animals were checked regularly from 8:00 a.m. to 12:00 noon with a final check performed at approximately 4:00 p.m., if applicable.
Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.

Body weight
The weight of each mouse was recorded at the time of allocation of animals to groups (test day 1) and at the time of necropsy (test day 4).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the vehicle treated ones. Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the inter-laboratory validation of this method (Ehling et al. 2005a and 2005b).

Results and discussion

Positive control results:

The positive control group caused the expected increases in lymph node cell count and lymph node weight (statistically significant at p ≤ 0.01). Therefore, the study can be regarded as valid.The vehicle of the positive control (acetone/olive oil (3+1 v/v)) showed neither sensitising nor irritating properties.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

In the main study treatment with Pigment blue 61 at concentrations of 5%, 10% or 25% did not reveal statistical significantly increased values for lymph node cell count. The stimulation indices of the lymph node cell count did not exceed the threshold level of 1.4 (details see table in the field for other information).

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: no data

Any other information on results incl. tables

In a preliminary experiment, concentrations of 5%, 10% and 25%ofPigment blue 61, employing 1 animal per concentration, were examined. No pronounced irritating properties were observed in this preliminary experiment at concentrations of 5%, 10% or 25%, no differences in ear weight and ear thickness were noted.

No signs of local or systemic intolerance were recorded. The animal body weight was not affected by the treatment.

Stimulation indices (SI):

Parameter	Group 1, negative control	Group 2, 5%	Group 3, 10%	Group 4, 25%	Group 5, positive control	Group 6, vehicle of positive control
Lymph node cell count	1.000	1.072	1.306	0.905	2.234	1.204
Lymph node weight	1.000	0.950	1.050	1.083	1.460	0.983
Ear weight	1.000	1.031	0.985	1.056	1.072	0.959
Difference of ear thickness	1.000	1.077	1.032	1.044	1.177	0.929

____ significantly different from control at p ≤ 0.01

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the present test conditions, test article at concentrations of 5%, 10% or 25% (w/w) in DMSO did not reveal any sensitising properties in the local lymph node assay and therefore should not be classified according to (EC) No.: 1272/2008.

Executive summary:

This study was performed to determine the sensitizing potential of test article in the local lymph node assay in mice. The study employed the alternative method of the lymph node weight and lymph node cell count to assess proliferation. Treatment with test article atconcentrations of 5%, 10% or 25% did not reveal statistical significantly increased values for lymph nodecell count. The stimulation indices of the lymph node cell count did not exceed the threshold level of 1.4. Hence, the test item is classified as not sensitizing. No signs of local or systemic intolerance were recorded. The animal body weight was not affected by the treatment. Based on the results of this study, test article at concentrations of 5%, 10% or 25% (w/w) did not reveal any sensitising properties in the local lymph node assay and therefore should not be classified according to (EC) No.: 1272/2008.