Sodium 5-({4-chloro-6-[ethyl(3-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)amino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(E)-(1-sulfonato-2-naphthyl)diazenyl]naphthalene-2,7-disulfonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

RCC Ltd, 4452 ltingen Switzerland

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): FAT 40'817/A
- Description: Red powder
- Purity: 93.3%
- Lot/batch No.: WIA 260/2
- Expiration date of the lot/batch: 20 february 2009
- Stability of test item: Stable under storage conditions
- Storage: ln the original container at room temperature (20 C° ± 3 °C), away from direct sunlight.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Test system: Mice, CBA/CaOlaHsd
- Source: Harlan Netherlands B.V., Postbus 6174, NL - 5960 AD Horst, The Netherlands
- Age at acclimatization: 8 - 12 weeks
- Weight at study initiation: 16 g-24 g (ordered)
- Housing: lndividual in Makrolon type-2 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet (e.g. ad libitum): Pelleted standard Kliba 3433, batch no. 78/03 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst), ad libitum.
- Water (e.g. ad libitum): Community tap water from ltingen, available ad libitum.
- Acclimation period: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12 with at least 8 hours music during the light period.

Room temperature and humidity were monitored continuously and values outside of these ranges occasionally occurred, usually following room cleaning. These transient variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at RCC.

Study design: in vivo (LLNA)

Vehicle:

other: ethanol:water, 7:3 (v/v)

Concentration:

2.5, 5, 10% (w/v) in ethanol:water, 7:3 (v/v)

No. of animals per dose:

4 females/dose

Number of animals for the pre-test (non-GLP): 2 females

Details on study design:

PRE-TEST
To determine the highest non-iritant and technically applicable test item concentration, a non-GLP pretest was performed in two mice with concentrations of 1 %, 2.5%, 5% and 10 % (w/v) in ethanol:water, 7:3 (v/v). No irritation effects were observed at these concentrations after single application.

MAIN TEST
The test item in the main study was assayed at three consecutive concentrations. The top dose is the highest technically applicable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations. Concentrations were in terms of material as supplied.

The test item was placed into a volumetric flask on a tared Mettler balance and the vehicle (ethanol:water, 7:3 (v/v)) was quantitatively added. The weight/volume dilutions were prepared individually using a magnetic stirrer as homogenizer. Test item formulations were made freshly before each dosing occasion and no more than 4 hours prior to application to the ears. Homogeneity of the lest item in the vehicle was maintained during treatment with the magnetic stirrer.

TOPICAL APPLICATION
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 2.5%, 5% and 10% (w/v) in ethanol:water, 7:3 (v/v). The application volume, 25 µl, was spread over the entire dorsal surface (Ø - 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was passed briefly over the ear’s surface to prevent the loss of any of the test item applied.

ADMINISTRATION OF ³H-METHYL THYIMIDINE'
³H-methyl thymidine (³HTdR) was purchased from Amersham International (Amersham product code no, TRA 310: specific activity, 2 Ci/mmol; concentration, 1 mCi/ml). Five days after the first topical application, all mice were administered with 250 µl of 82.7 µCi/ml ³HTdR (equal to 20.7 µCi ³HTdR) by intravenous injection via a tail vein.

DETERMINATION OF INCORPORATED ³HTDR
Approximately five hours after treatment with ³HTdR all mice were euthanized by inhalation of CO2(dry ice). The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4°C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of 'Ultima Gold' scintillation liquid and thoroughly mixed. The level of ³HTdR incorporation was then measured on a β-scintillation counter. Similarly, background ³HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses ³HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

INTERPRETATION
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of ³HTdR incorporated into lymph node cells of test group relative to that recorded for control group (STIMULATION INDEX) (S.I.). Before DPM/node values were determined, mean scintillation {background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- First, that 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.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentration) for either local toxicity or immunological suppression.

Positive control substance(s):

other: Alpha-Hexylcinnamaldehyde

Results and discussion

Positive control results:

ln this study (RCC study number 852080) STIMULATION INDICES of 1.5, 2.3 and 8.4 were determined with Alpha-Hexylcinnamaldehyde at concontrations of 5 %, l0% and 25% (w/v), respectively, in acetone:olive oil, 4:1 (v/v).
The test item ALPHA-HEXYLCINNAMALDEHYDE was found to be a skin sensitizer and an EC3 value of 11.7 % (w/v) was derived.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Test substance concentration 2.5% (w/v): 4.2 Test substance concentration 5% (w/v): 2.6 Test substance concentration 10% (w/v): 5.3

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Control group: 173 DPM/node Test substance concentration 2.5% (w/v): 718 DPM/node Test substance concentration 5% (w/v): 446 DPM/node Test substance concentration 10% (w/v): 907 DPM/node Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled.

Any other information on results incl. tables

VIABILITY / MORTALITY

No death occurred during the study period.

CLINICAL SIGNS

No symptoms of local toxicity at the ears of the animals end no systemic findings were observed during the study period.

BODY WEIGHTS

The body weight of the animals, recorded prior to the 1st application and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substance was found to be a skin sensitizer.

Executive summary:

In a GLP compliant Local Lymph Node Assay, tested according to OECD guideline 429, 4 female CBA mice per dose were treated daily with the test item at concentrations of 2.5, 5 and 10% (w/v) in ethanol:water, 7:3 (v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. 10% was the highest technically applicable concentration in the vehicle. A control group of four mice was treated with the vehicle (ethanol:water, 7:3 (v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio labelled thymidine (³H-methyl thymidine), five hours thereafter the mice were sacrificed and the draining auricular lymph nodes were excised and pooled per group. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine. Stimulation Indices of 4.2, 2.6 and 5.3 were determined with the test item at concentrations of 2.5, 5 and 10% (w/v), respectively, in ethanol:water, 7:3 (v/v). Based on the described study and under the conditions reported, it is concluded that the test substance was found to be a skin sensitizer.