Trisodium 7-(4-(4-fluoro-6-(2-(2-vinylsulfonylethoxy)ethylamino)-1,3,5-triazine- 2-ylamino)-2-ureidophenylazo)naphthalene-1,3,6-trisulfonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 February 2006 to 08 March 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

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:

EPA OPPTS 870.2600 (Skin Sensitisation)

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:

Trisodium 7-(4-(4-fluoro-6-(2-(2-vinylsulfonylethoxy)ethylamino)-1,3,5-triazine- 2-ylamino)-2-ureidophenylazo)naphthalene-1,3,6-trisulfonate

EC Number:

402-170-5

EC Name:

Trisodium 7-(4-(4-fluoro-6-(2-(2-vinylsulfonylethoxy)ethylamino)-1,3,5-triazine- 2-ylamino)-2-ureidophenylazo)naphthalene-1,3,6-trisulfonate

Cas Number:

106359-91-5

Molecular formula:

C26H26FN9O13S4.3Na

IUPAC Name:

trisodium 7-{2-[2-(carbamoylamino)-4-{[4-({2-[2-(ethenesulfonyl)ethoxy]ethyl}amino)-6-fluoro-1,3,5-triazin-2-yl]amino}phenyl]diazen-1-yl}naphthalene-1,3,6-trisulfonate

Details on test material:

Name: FAT 40224/H TE
Batch No.: TZ 2382-BB-503C81
Physical State: powder
Colour: red
Density: 1.46 g/cm3 (20°C)
pH: 6.5 to 7.5 conc.(% w/w): 1%
Melting Point: > 200°C
Purity / Active Components: sum of all coloured substances: 85.4%, main constituents: 50.6%
Date of Analysis: 23.07.2012
Storage Conditions: at room temperature
Expiry Date: 28.01.2017

Specific details on test material used for the study:

Substance name: C.I. Reactive Yellow 174
Item No.: 1897214 from synthesis: 631178/174
Stability: 19.01.2010
Storage: at room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, D-33178 Borchen.
- Age at study initiation: 6-12 weeks
- Housing: The animals were kept in groups in Macrolon-cages on Lignocel bedding
- Diet: Feeding ad libitum, ssniff R/M-H, 10 mm VI534-000 complete diet for rats/mice, totally-pathogen-free (TPF)
- Water: Free access to tap water (drinking water, municipal residue control, microbiol. controlled periodically)
- Acclimation period: 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): Artificial light, sequence being 12 hours light, 12 hours dark

Study design: in vivo (LLNA)

Vehicle:

other: AOO (3+1 (v/v) Acetone/Olive Oil).

No. of animals per dose:

5

Positive control substance(s):

other: P-Phenylenediamine (CAS 106-50-3, Sigma GmbH, purity >98 %) at a concentration of 1 % (w/v) in AOO (3+1 (v/v) Acetone/Olive Oil) served as positive control.

Results and discussion

Positive control results:

Mean ear thickness of the positive control group was 0.19 and 0.20 mm at Day 1 and Day 6, respectively. While the mean weights of the lymph nodes was 5.5 mg. The stimulation of the positive control (Phenylenediamine) at a concentration of 1 % was 9.9.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

7.8

Test group / Remarks:

Concentration level 15 %

Results 2

Parameter:

SI

Value:

5.5

Test group / Remarks:

Concentration level 9 %

Results 3

Parameter:

SI

Value:

3.3

Test group / Remarks:

Concentration level 3 %

Results 4

Parameter:

SI

Value:

4.2

Test group / Remarks:

Concentration level 1 %

Any other information on results incl. tables

Four concentrations were chosen to gain a wide spectrum for the test design: Due to results of a solubility test and in consultation with the sponsor, the test item was assayed at concentrations of 15 %, 9 %, 3 % and 1 % (w/v). Additionally a positive control for verifying the functionality of the current test run was carried along.

The vehicle was AOO (3+1 (v/v) Acetone/Olive Oil). Stability of the test item in the vehicle was proven. Each mouse was treated by topical application of the prepared test item to the entire dorsal surface of each ear once daily over three consecutive days. Five days after the first topical application all mice were injected intravenously with ³H-methyl thymidine. Directly prior to the first application and shortly before excising the lymph nodes the thickness of both ears from all animals was measured. This is to exclude irritating properties of the test item, which may lead to false positive results.

 

Mean Ear thickness at:                                 day 1                          day 6

of the 15 % group was                                 0.21 mm                     0.22 mm

of the 9 % group was                                   0.21 mm                     0.21 mm

of the 3 % group was                                   0.20 mm                     0.21 mm

of the 1 % group was                                   0.22 mm                     0.22 mm

of the negative control group was                     0.20 mm                     0.20 mm

of the positive control group was                      0.19 mm                     0.20 mm

 

Approximately 5 hours after ³H-methyl thymidine-injection all mice were sacrificed and the draining "auricular lymph nodes" were excised and weighed individually.

The mean weights of the lymph nodes

for the 15 % group was 4.7 mg

for the 9 % group was 4.5 mg

for the 3 % group was 3.7 mg

for the 1 % group was 3.9 mg

for the negative control-group was 2.6 mg

for the positive control-group was 5.5 mg

 

A single cell suspension of the lymph node cells for each animal was prepared. The ³H-methyl thymidine - incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Determination of radioactivity was performed individually for each animal.

 

The proliferative response of lymph node cells was calculated as the ratio of ³H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals. A stimulation index, ratio of test item / negative control, was calculated for each concentration.

The stimulation index at a concentration 15, 9, 3 and 1 % was 7.8, 5.5, 3.3 and 4.2 respectively.

The stimulation of the positive control (Phenylenediamine) at a concentration of 1 % was 9.9.

All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study. At the daily clinical observation the animals did not show any visible clinical symptoms.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

FAT 40224 (C.I. Reactive Yellow 174) has shown skin sensitizing properties in LLNA study in mice.

Executive summary:

The LLNA study in CBA female mice was carried out with FAT 40224 according to OECD guideline 429. Five female mice were used per concentration. Four concentrations were chosen to gain a wide spectrum for the test design: Due to results of a solubility test and in consultation with the sponsor, the test item was assayed at concentrations of 15 %, 9 %, 3 % and 1 % (w/v). Additionally a positive control for verifying the functionality of the current test run was carried along. The vehicle was AOO (3+1 (v/v) Acetone/Olive Oil). Stability of the test item in the vehicle was proven. Each mouse was treated by topical application of the prepared test item to the entire dorsal surface of each ear once daily over three consecutive days. Five days after the first topical application all mice were injected intravenously with ³H-methyl thymidine. Directly prior to the first application and shortly before excising the lymph nodes the thickness of both ears from all animals was measured. This is to exclude irritating properties of the test item, which may lead to false positive results.

 

Mean Ear thickness at:                                   day 1                          day 6

of the 15 % group was                                       0.21 mm                     0.22 mm

of the 9 % group was                                         0.21 mm                     0.21 mm

of the 3 % group was                                         0.20 mm                     0.21 mm

of the 1 % group was                                         0.22 mm                     0.22 mm

of the negative control group was                     0.20 mm                     0.20 mm

of the positive control group was                      0.19 mm                     0.20 mm

 

Approximately 5 hours after ³H-methyl thymidine-injection all mice were sacrificed and the draining "auricular lymph nodes" were excised and weighed individually.

The mean weights of the lymph nodes

for the 15 % group was 4.7 mg

for the 9 % group was 4.5 mg

for the 3 % group was 3.7 mg

for the 1 % group was 3.9 mg

for the negative control-group was 2.6 mg

for the positive control-group was 5.5 mg

 

A single cell suspension of the lymph node cells for each animal was prepared. The ³H-methyl thymidine - incorporation was measured in a ßcounter and expressed as the number of disintegrations per minute (DPM). Determination of radioactivity was performed individually for each animal. The proliferative response of lymph node cells was calculated as the ratio of ³H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals. A stimulation index, ratio of test item / negative control, was calculated for each concentration.

The stimulation index at a concentration 15, 9, 3 and 1 % was 7.8, 5.5, 3.3 and 4.2 respectively.

The stimulation of the positive control (Phenylenediamine) at a concentration of 1 % was 9.9.

All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study. At the daily clinical observation the animals did not show any visible clinical symptoms.

The EC3 value could not be calculated as the stimulation indices of all concentrations were above 3. This finding was confirmed by the second endpoint, the weight of the lymph nodes, as all of the test groups showed increased lymph node weights compared to the control group. Based on the study results, FAT 40224 (C.I. Reactive Yellow 174) has shown skin sensitizing properties in LLNA study in mice under the given experimental conditions.