3,3'-(1,4-Phenylene)bis(5,6-diphenyl-1,2,4-triazine)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed accpording to GLP and OECD guideline n° 429

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (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:

3-[4-(5,6-diphenyl-1,2,4-triazin-3-yl)phenyl]-5,6-diphenyl-1,2,4-triazine

EC Number:

700-823-1

Cas Number:

55514-22-2

Molecular formula:

C36H24N6

IUPAC Name:

3-[4-(5,6-diphenyl-1,2,4-triazin-3-yl)phenyl]-5,6-diphenyl-1,2,4-triazine

Details on test material:

- Name of test material (as cited in study report): WP30
- Substance type: monoconstituent substance
- Physical state: powder
- Analytical purity: 99.6 % by HPLC
- Purity test date: 13th February 2009
- Lot/batch No.: LP110
- Stability under test conditions: stable
- Storage condition of test material: room temperature, in a dark and dry place

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelman, Germany
- Age at study initiation: 8-9 weeks
- Weight at study initiation:
- Housing: full barrier in an air-conditioned room
- Diet ad libitum
- Water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): at least 10 x hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

other: acetone/olive oil 3/1 (v/v)

Concentration:

6.25 - 12.5 - 25 % (v/v)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: up to 25 % in AOO 3/1
- Irritation: ear thickness was measured in a preliminary test, and cageside observations. No sign of systemic toxicity nor signs of irritation at the application site could be detected in any animal (2 with 25 % WP30, and 1 with 100 % AOO 3/1)

MAIN STUDY
- Criteria used to consider a positive response:
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPMINODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPMNODE values were determined, background values were subtracted. EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation {EC3=c+[(3-d)/(b-d)]x(a-c)}, between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated. A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3 fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the lymph nodes of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).

TREATMENT PREPARATION AND ADMINISTRATION:
Preparation of the Animals
The animals were randomly selected. Identification was ensured by cage number and individual marking (tail).

Clinical Observation
Prior to the application and once a day thereafter all animals were observed in order to detect signs of toxicity, including dermal irritation at site of application.

Weight Assessment
The animals were weighed prior to the application and at the end of the test period (prior to the treatment with 3HTdR).

Dose Groups
3 test groups (3 different concentrations) and 1 negative control group (vehicle) were tested.

Test Regime
Topical Application
Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear. Topical applications were performed once daily over three consecutive days.

Administration of 3H-methyl thymidine
Five days after the first topical application all mice were dosed with 20 µCi 3H_ methyl thymidine by intravenous injection (tail vein) of 250 µL of 3H-methyl thymidine, diluted to a working concentration of 80 µCi/mL.

Preparation of cell suspension
Approximately 5 hours after 3H-methyl thymidine-injection all mice were sacrificed by cervical dislocation. The draining "auricular lymph nodes" were excised, weighed, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated. After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4 °C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.

Determination of incorporated 3H-methyl thymidine
The 3H-methyl thymidine - incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.

Positive control substance(s):

other: p-phenylenediamine

Results and discussion

Positive control results:

Mean value stimulation index: 9.1
Standard deviation: 2.5

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The stimulation index at a concentration of 6.25% was 1.2 (SD 0.2) The stimulation index at a concentration of 12.5% was 0.9 (SD 0.2) The stimulation index at a concentration of 25% was 1.1 (SD 0.2)

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: 6.25 % WP30: mean value = 926.2 dpm (SD 125.9) 12.5 % WP30: mean value =640.1 dpm (SD 161.4) 25 % WP30: mean value = 819.5 dpm (SD 166.8)

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3.

Results of radioactivity determination were supported by the means of the lymph node weights per group, which showed no significant difference compared to the negative control.

Consequently, according to OECD 429 and the criteria given in Annex I of Regulation (EC) 1272/2008, the test item WP 30, as described in this report is expected to have no sensitising properties and therefore, should not be regarded as a dermal sensitiser.

Executive summary:

Based on the results of the preliminary test WP30 was assessed for sensitising properties at concentrations 6.25%, 12.5% and 25% (v/v). At the daily clinical observation the animals did not show any visible clinical symptoms and no case of ilatrtom were observed. From day 2 until day 6, residuals of the test item at the application site were recorded for the animals treated with the 12.5% and the 25% concentration.

Species/strain: Mice, CBAlCaOlaHsd

Number of animals: 20/main test

Vehicle: AOO (3+ 1 (v/v) acetone/olive oil)

The mean weight of the lymph nodes

for the 6.25% test group was 2.6 mg

for the 12.5% test group was 2.3 mg

for the 25% test group was 2.8 mg

for the negative control group was 2.6 mg

None of the three tested concentrations of the test item reached the stimulation index of 3.

The stimulation index at a concentration of 6.25% was 1.2

The stimulation index at a concentration of 12.5% was 0.9

The stimulation index at a concentration of 25% was 1.1