N-(5-chloro-2,4-dimethoxyphenyl)-4-[[5-[(diethylamino)sulphonyl]-2-methoxyphenyl]azo]-3-hydroxynaphthalene-2-carboxamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 October 2007 - 25 January 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: performed in accordance with OECD and GLP guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

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)

Remarks:

according to German Chemikaliengesetzt and OECD Principles of Good Laboratory Practice

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

3-hydroxy-N-(o-tolyl)-4-[(2,4,5-trichlorophenyl)azo]naphthalene-2-carboxamide

EC Number:

229-440-3

EC Name:

3-hydroxy-N-(o-tolyl)-4-[(2,4,5-trichlorophenyl)azo]naphthalene-2-carboxamide

Cas Number:

6535-46-2

Molecular formula:

C24H16Cl3N3O2

IUPAC Name:

3-hydroxy-N-(2-methylphenyl)-4-[(2,4,5-trichlorophenyl)diazenyl]-2-naphthamide

Test material form:

solid: nanoform, no surface treatment

Details on test material:

Name of test material (as cited in study report): (no report title): Pigment Red 112
Analytical purity: 99.8% P.R. 112

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
-strain: CBA/CaOlaHsd
- Source: Harlan Netherlands
- Age at study initiation: 8-9 weeks (beginning of treatment)
- Weight at study initiation: 18.6 g to 22.0 g
- Housing: single caging, Makrolon Type I, with wire mesh top, granulated soft wood bedding
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30 - 70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

0, 0.5, 10 and 20% (w/v)

No. of animals per dose:

5 animals per treatment group, 5 animals in the control group

Details on study design:

RANGE FINDING TESTS:
- Compound solubility:
The highest test item concentration, which could be technically used was a 20 % suspension in DMSO. In other vehicles a higher concentration could not be suspended or the test substance was not considered stable.
- Irritation:
No irritation was observed in a pretest of an earlier LLNA with Pigment Red 112.
- Lymph node proliferation response: not evaluated


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Individual (5 animals/group) LLNA
- Criteria used to consider a positive response:
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.


TREATMENT PREPARATION AND ADMINISTRATION:
Five female mice were treated with different concentrations of the test item and vehicle alone by topical application at the dorsum of each ear lobe (left and right) on three consecutive days. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Five hours after intravenous injection, the mice were sacrificed and the draining auricular lymph nodes excised and pooled per animal. 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 ß-scintillation counter.

Positive control substance(s):

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

Statistics:

not required

Results and discussion

Positive control results:

Test item concentration %(w/v) Group Measurement DPM DPM-BG a) Number of lymph nodes DPM per lymph node b) S.I.
- BG I 51 - - - -
- BG II 24 - - - -
- CG 1 5421 5384 8 672.9
5 TG 1 6985 6948 8 868.4 1.29
10 TG 2 16340 16303 8 2037.8 3.03
25 TG 3 34048 34011 8 4251.3 6.32
BG = Background (1 ml 5% trichloroacetic acid) in duplicate
CG = Control Group (Vehicle: acetone:olive oil (4+1))
TG = 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

EC3 = (a-c) [(3-d)/(b-d)] + c = 9.9%(w/v)

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Results expressed as mean per group Group -- DPM per lymph node -- SI Vehicle -- 1715.4 -- 1.00 0.5% -- 981.4 -- 1.16 10% -- 1974.1 -- 1.15 20% -- 1164.8 -- 0.68

Any other information on results incl. tables

No deaths occurred during the study period.

Due to the intense violett colour of the test item local irritation reactions such as ear redness could not be detected. However, no swelling of the ears was observed. Body weights were within the normal range.

Applicant's summary and conclusion

Conclusions:

67/548/EEC: Skin irritation: no classification warranted
1272/2008/EC: Skin irritation: no classification warranted
In an OECD guideline and GLP compliant LLNA, Pigment Red 112 (containing <0.01% Naphtol AS-D) was found to be not a skin sensitiser when tested at 0, 0.5, 10 and 20% in DMSO.

Executive summary:

In the study the test item suspended in dimethylsulfoxide was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay was performed using test item concentrations of 0.5, 10, 20%. The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. Redness of the ear skin could not be observed due to the colour of the test item. In this study Stimulation Indices (S.I.) of 1.16, 1.15, 0.68 were determined with the test item at concentrations of 0.5, 10, 20% in dimethylsulfoxide, respectively. The test item was not a skin sensitiser in this assay.