Reaction mass of 3-[(4-amino-6-bromo-5,8-dihydro-1-hydroxy-8-imino-5-oxo-2-naphtyl)amino]-N,N,N-trimethylanilinium chloride and 3-[(8-amino-dibromo-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzenaminium chloride and 3-[(bromo-1,4-dihydroxy-8-imino-5-oxo-5,8-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzenaminium chloride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 3-[(4-amino-6-bromo-5,8-dihydro-1-hydroxy-8-imino-5-oxo-2-naphtyl)amino]-N,N,N-trimethylanilinium chloride and 3-[(8-amino-dibromo-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzenaminium chloride and 3-[(bromo-1,4-dihydroxy-8-imino-5-oxo-5,8-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzenaminium chloride

EC Number:

916-466-1

IUPAC Name:

Reaction mass of 3-[(4-amino-6-bromo-5,8-dihydro-1-hydroxy-8-imino-5-oxo-2-naphtyl)amino]-N,N,N-trimethylanilinium chloride and 3-[(8-amino-dibromo-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzenaminium chloride and 3-[(bromo-1,4-dihydroxy-8-imino-5-oxo-5,8-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzenaminium chloride

Test material form:

solid: particulate/powder

Details on test material:

Identification : Basic Blue 99
Batch number : RS27988101
3-[(Bromo-8-amino-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (main) : 50.2a/a%
3-[(Dibromo-8-amino-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (E) : 14.6a/a% 3-[(Bromo-8-amino-5-hydroxy-1,4-dioxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (F) : 16.9a/a%
3-[(Bromo-5,8-dihydroxy-1,4-dioxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (J) : 4.8a/a% 3-[(8-Amino-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (A) : 1.0a/a% 3-[(Sulpho-8-amino-5-hydroxy-1,4-dioxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (B) : 2.0a/a%
3-[(Dibromo-8-amino-5-hydroxy-1,4-dioxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (G): 2.8a/a%
3-[(Sulpho-8-amino-5-hydroxy-4-imino-1-oxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (O) :<0.3a/a%
3-[(Bromo-sulpho-8-amino-5-hydroxy-1,4-dioxo-1,4-dihydronaphthalenyl)amino]-N,N,N-trimethylbenzeneaminium cation (N) : 2.1a/a%
Tribromo-8-amino-5-hydroxy-1,4-naphthoquinone (L) : 1.3a/a%
Chloride ion : 14.5%
Sulphate ion : 0.1%
Acetate ion : 0.2%
Zinc ion : 5.5%
Water : 4.1%

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/JNCRJBR

Sex:

female

Study design: in vivo (LLNA)

Vehicle:

other: distilled water

Concentration:

The test item was prepared as a solution in the vehicle at the following concentrations : 1%, 5%, 25%

No. of animals per dose:

Four females per dose

Details on study design:

To determine the highest non-irritant test item concentration, a non-GLP pretest was performed in 2 mice. The test item was assayed at three consecutive concentrations which were selected upon irritant characteristics or solubility factors of the test item. No severe irritant effects were tolerated choosing the test concentrations.

Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 1%, 5% and 25% in distilled water.

Positive control substance(s):

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

Results and discussion

Positive control results:

The positive control is regarded as a sensitiser if the exposure to at least one concentration resulted in at least a 3 fold increase in isotope incorporation relative to the vehicle. The mean stimulation indices were 2.4, 3.7 and 7.0 at the concentration of 5%, 10% and 25% respectively. Hexyl cinnamic aldehyde was regarded as a sensitiser when tested at concentrations of 10% and 25%.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

The mean stimulation indices were 0.7, 1.1 and 1.1 at the concentration of 1%, 5% and 25% respectively. The treatment of mice with the test item induced a negative response as it did not elicit at least a 3 fold increase in isotope incorporation relative to the vehicle.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The mean DPM values were 2103, 3509 and 3232 at the concentration of 1%, 5% and 25% respectively. The mean DPM value for the vehicle is 3060.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In this study stimulation indices of 0.7, 1.1 and 1.1 were determined with the test item at concentrations of 1%, 5% and 25% in distilled water. A test item is regarded as a sensitiser in the LLNA if the exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index. The test item was found to be a non-sensitiser.

Executive summary:

In order to study a possible allergenic potential of Basic Blue 99 three groups of four female mice each were treated with the test item at concentrations of 1%, 5% and 25% in twice distilled water by topical application to the dorsum of each ear lobe on three consecutive days. A control group of four mice was treated with the vehicle only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were washed subsequently and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a beta-scintillation counter. No test item related clinical signs were observed. All treated animals survived the scheduled study period. A test item is regarded as a sensitiser in the LLNA if the exposure resulted in an incorporation of 3HTdR at least 3 fold or greater than that recorded in control mice, as indicated by the stimulation index. In this study stimulation indices of 0.7, 1.1 and 1.1 were determined with the test item at concentrations of 1%, 5% and 25% in distilled water. Basic Blue 99 is regarded as a non-sensitiser.