Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-11-03 till 2009-12-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- public name of test material : Coupling reaction on diazotized (aminonaphthalen)sulfonyl)ethanol polysulfanate with 6-chloro-N-ethyl-N-(3-(ethylsulfonyl)phenyl)-N'-naphthalen-1-yl-1,3,5-triazine polysulfonate, polyhydroxide, polyamine, sodium and potassium salts
- Substance type: textile dyestuff
- Physical state: red powder
- Analytical purity: ca. 63 % of all colored components
- Lot/batch No.: BOP 07-09
- Expiration date of the lot/batch: 2014-07-31
- Storage condition of test material: At room temperature at about 20 °C

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS: Mice, CBA/CaOlaHsd
- Source: Harlan Laboratories B.V. Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks (beginning of treatment)
- Weight at study initiation: 18.3 - 23.4 g
- Housing: Single caging.
- Diet : pelleted standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 + 2°C
- Humidity (%): relative humidity 45-65%
- Photoperiod (hrs dark / hrs light): 12/12
- bedding: granulated soft wood bedding

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
The highest test item concentration, which can be technically used was a 20 % solution in dimethylformamide.
First pre-test two mice were treated with concentrations of 10 and 20 %
Second pre-test two mice were treated with concentrations of 2.5 and 5 %
The test item in the main study was assayed at 1, 2.5, and 5%.
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration, which can be technically used was a 20 % solution in dimethylformamide.
In the first pre-test two mice were treated with concentrations of 10 and 20 % each on three consecutive days. Within 1 hour after the first and second and 24 hours after the third application both animals showed reduced spontaneous activity and eyelid closure.
In the second pre-test two mice were treated with concentrations of 2.5 and 5 % each on three consecutive days. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity.

MAIN STUDY

TOPICAL APPLICATION
Each of the three groups of four female 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, 2.5, and 5% (w/v) in dimethylformamide. 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).

ADMINISTRATION OF 3H-METHYL THYMIDINE AND DETERMINATION OF INCORPORATED 3H-METHYL THYMIDINE
Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed and 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 subsequently washed 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.

INTERPRETATION OF RAW DATA
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 lymph nodes relative to that recorded for control lymph nodes (stimulation index). 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 sensitiser 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 concentrations) for either local toxicity or immunological suppression.

OBSERVATIONS
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: once daily (week day) from experimental start to necropsy.
Body weights: prior to the first application and prior to treatment with 3HTdR.
Clinical signs (local / systemic): In the pre-test clinical signs were recorded within 1 hour and 24 ± 4 hours after each application as well as on day 7. In the main experiment clinical signs were recorded within 1 hour after each application, and 24 ± 4 hours after the first and second application as well as on the day of preparation. Especially the treatment sites were observed carefully.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
The mean values and standard deviations were calculated in the body weight tables.

Results and discussion

Positive control results:

In the study with the positive control substance alpha-hexyl cinnamic aldehyde, Stimulation Indices of 1.79, 2.09, and 6.84 were determined with test item concentrations of 5, 10, and 25% in acetone:olive oil (4+1). The EC3 value was calculated to be 12.9%.

The test item positive control substance alpha-hexyl cinnamic aldehyde was found to be a skin sensitiser under the described conditions, demonstrating the validity of the study.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: In this study Stimulation Indices of 3.40, 8.79, and 11.62 were determined with the test item at concentrations of 1, 2.5, and 5% in dimethylformamide. The EC3 value could not be calculated, since all S.I. are above 3.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: As depicted in the table below.

Any other information on results incl. tables

Calculation and Results of Individual Data; Vehicle: dimethylformamide

Test item concentration

Group

Measured DPM

Calculation

Result

DPM – BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

-

BG 1

23

-

-

-

-

-

BG 2

32

-

-

-

-

-

1

3917

3890

8

486.2

 

1

2

13267

13240

8

1654.9

3.40

2.5

3

34209

34182

8

4272.7

8.79

5

4

45221

45194

8

5649.2

11.62

BG = Background (1 ml 5% trichloroacetic acid) in duplicate

1 = Control Group

2-4 = Test Group

S.I. = Stimulation Index

a)= The mean value was taken from the figures BG I and BG II

b)= 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

 

The EC3 value could not be calculated, since all S.I. are above 3.

 

VIABILITY / MORTALITY

No deaths occurred during the study period.

 

CLINICAL SIGNS

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. Due to the colour of the test item, redness of the ear skin could not be observed.

 

BODY WEIGHTS

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, 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
Conclusions:
The test material was found to be a skin sensitiser under the described conditions.
Executive summary:

In the study the test material dissolved in dimethylformamide was assessed for its possible contact allergenic potential.

For this purpose a local lymph node assay according to OECD guideline 429 was performed using test item concentrations of 1, 2.5, and 5%.

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. Due to the colour of the test item, redness of the ear skin could not be observed.

In this study Stimulation Indices (S.I.) of 3.40, 8.79, and 11.62 were determined with the test item at concentrations of 1, 2.5, and 5% in dimethylformamide, respectively. The EC3 value could not be calculated, since all S.I. are above 3.

In conclusion, the test material was found to be a skin sensitiser.

According to the referred classification (Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008), the test material has to be classified with respect to skin sensitisation.