Sodium 4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]toluene-3-sulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 3 February 2004 to 16 February 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 24th April 2002

Deviations:

yes

Remarks:

The storage temperature of the test item was above +4 deg Celsius on one occasion for a total of 6 hours. The deviation did not compromise the validity and integrity of the study

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]toluene-3-sulphonate

EC Number:

224-618-7

EC Name:

Sodium 4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]toluene-3-sulphonate

Cas Number:

4430-18-6

Molecular formula:

C21H15NO6S.Na

IUPAC Name:

sodium 2-[(4-hydroxy-9,10-dioxo-9,10-dihydroanthracen-1-yl)amino]-5-methylbenzenesulfonate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Supplied by l'Oreal / Batch no. 10130
- Expiration date of the lot/batch: March 2005
- Purity test date: 31 March 2004

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: not specified
- Specific activity: 25 Ci/mmol
- Locations of the label: 3H-Tdr / 3H methyl-thymidine, provided by Amersham (France)
- Expiration date of radiochemical substance: not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at +4 deg celsius, protected from light and under nitrogen gas
- Stability under test conditions: Not specified
- Solubility and stability of the test substance in the solvent/vehicle: water : <1g/100ml / Ethanol : <1g/100ml / DMSO : <1g/100ml

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was prepared as a solution in the vehicle at the chosen concentrations. The test item dosage forms were prepared extemporaneously under nitrogen atmosphere and were stored protected from light and under nitrogen gas before use. They were used within the 4 hours following the preparation according to the known stability results (CIT/Study No. 26931 AHS).

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier (France)
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: no information
- Age at study initiation: 9 weeks old
- Weight at study initiation: 19.3±0.8g
- Housing: Individually housed individually in disposable crystal polystyrene cages (22 x 8.5 x 8.0 cm)
- Diet (e.g. ad libitum): free access to A04 C pelleted diet (SAFE, France)
- Water (e.g. ad libitum): tap water (filtered using a 0.22 micron filter) contained in bottles.
Each batch of diet is analyzed for composition and contaminant level by the supplier.
- Acclimation period: at least 5 days before the beginning of the study
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 ± 2°C
- Humidity (%): 30 to 70%
- Air changes (per hr): 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12 h/12 h

- IN-LIFE DATES: Preliminary study From: 3 February 2004 To: 11 February 2004
Main study 11 February 2004 To: 16 February 2004

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Preliminary study : 1.5, 3, 6 and 15% pure dye (w/v)
Main study :0, 0.6, 1.5, 3, 6, 15, 25% pure dye (w/v)

No. of animals per dose:

Main study : 4 mice per group P
Preliminary study : four mice for the study

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: 15% in vehicle : maximum practicable concentration
- Ear thickness measurements: using a micrometer
- Erythema scores: not performed

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA Local Lymph Node Assay
- Criteria used to consider a positive response:

TREATMENT PREPARATION AND ADMINISTRATION:
On days 1, 2 and 3, a dose-volume of 25 μL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip. In order to avoid licking and to ensure an optimized application of the test material, the animals were placed under light isoflurane anesthezia during the administration. No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed between each application.
Bodyweight and Clinical signs were releved during the study.
Ear thickness measurements and recording of local reactions were performed in order to assess any possible irritant effect of the test item, as possible irritancy may be involved in false positive lymphoproliferative responses.
On days 1, 2 and 3 (before application) as well as on day 6 (after sacrifice), the thickness of the left ear of each animal of groups 1 to 6 was measured.

AURICULAR NODES SAMPLES
On day 6, all animals of all groups received a single intravenous injection of 250 μL of 0.9% NaCl containing 20 μCi of 3H-TdR via the tail vein. Approximately 5 hours later, the animals were killed by cervical dislocation and the auricular lymph nodes were excised. The lymph nodes were pooled for each experimental group. Cell suspensions were prepared by mechanical dissagregation thereafter resuspended in NaCl solution and viability (by trypan blue) was performed. Each suspension was centrifugated. 3H-Tdr incorporation was measured by Beta-scintillation counted.

Stimulation index (SI) was calculated as follows : SI = dpm of treated group / dpm of control group

Positive control substance(s):

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

Results and discussion

Positive control results:

In the positive control group given HCA at the concentration of 25%, a moderate increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI = 6.55) were noted. The study was therefore considered valid.

In vivo (LLNA)

Results

Key result

Parameter:

SI

Value:

1.55

Test group / Remarks:

15% dose group

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
No lymphoproliferation was noted at any tested concentrations, while significant lymphoproliferation was observed with HCA at 25%.

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation Index SI = dpm of treated group / dpm of control group

EC3 CALCULATION
EC3 value = theorical concentration resulting in a SI value of 3

CLINICAL OBSERVATIONS:
No mortality and no clinical signs were observed during the study

BODY WEIGHTS
The body weight changes were similar for control and treated animals in both experiments

LOCAL IRRITATION
No cutaneous reactions and no increases in ear thickness were observed in the animals of the treated groups.
A black coloration of the skin of the ears which could have masked a possible discrete erythema was noted in all treated animals on days 2 and 3 or from day 2 up to day 6.

Any other information on results incl. tables

Table 1: Study results

 

 

 

Groups	Treatment and concentrations	Cell count		viability(%)	Amount of cells (x 106cells)	cellularity index	Number of nodes pergroup	dpmper group	dpmper node	Stimulation index (SI)	Increase in ear thickness (% between day 1 and day 6)	EC3 value	Irritationclasse
		viable	dead
1	Acetone / olive oil 0	141       14     116       15     124       26     100       30     100       23     151       19     220       43		90.97     88.55     82.67     76.92     81.30     88.82     83.65	7.05     5.80     6.20     5.00     5.00     7.55     22.00		8     8     8     8     8     8     8	569.60     492.27     689.54     486.58     546.17     885.48     3730.37	71.20     61.53     86.19     60.82     68.27     110.69     466.30		3.00     0.00     6.19     4.12     2.04     4.90
2	Acid Violet 43 (C063) 0.6%*					0.82     0.88     0.71     0.71     1.07     3.12				0.86     1.21     0.85     0.96     1.55     6.55		NA	I
3	Acid Violet 43 (C063) 1.5%*
4	Acid Violet 43 (C063) 3%*
5	Acid Violet 43 (C063) 6%*
6	Acid Violet 43 (C063) 15%*
7	HCA 25%

NA = not applicable

dpm= disintegrations per minute

 

 

Viability = Viable cells / Viable cells + dead cells

 

Cellular index= amountof cells (x10E6cells) in the treated / amount of cells (x10E6cells) in the vehicle group

* = expressed in % active dye (w/v)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the registered substance Jarocol Violet 43 did not induced delayed contact hypersensitivity in murine LLNA.

Executive summary:

This GLP compliant study was performed to assess the potential sensitisation property of the test item Jarocol Violet 43 by contact in mice during a Local Lymph Node Assay (LLNA) according OECD guideline 429 method.

Animals were separated in 7 groups (4 mice/group) consisting of 5 treated groups receiving Jarocol Violet 43, a negative control group receiving the vehicle (AOO) alone and a positive control group receiving alpha-hexylcinnamaldehyde (HCA), at 25% (v/v) in AOO. The vehicle was selected in a previous solubility study showing that 15% (w/v) active Jarocol Violet 43 was the maximal practicable concentration, and that this concentration was non-irritant in a preliminary irritation test. During induction period test substances were applied over the ears (25 μL per ear) for three consecutive days. After 2 days of resting, the proliferation of lymphocytes in the lymph nodes draining the application sites was measured by incorporation of tritiated methyl thymidine (day 6). The values obtained were used to calculate stimulation indices (SI). The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.

No cutaneous reactions and no increases in ear thickness were observed in animals treated with the test substance. No lymphoproliferation was observed at any tested concentration (SI ranging from 0.9 for 0.6% to 1.6 for 15% active dye).