Hydrogen [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

other: read across from analogue substance

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

only 1 concentration tested, however far above the solubility in water

GLP compliance:

no

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Hydrogen [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

EC Number:

276-857-1

EC Name:

Hydrogen [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

Cas Number:

72812-34-1

Molecular formula:

C32H18CrN6O8.C11H25NO.H

IUPAC Name:

hydrogen [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Rationale: Recognised as the recommended test system
- Source: Harlan Netherlands; B.V. Postbus 6174L; NL - 5960 AD Horst / The Netherlands
- Age at study initiation: 7 - 8 weeks
- Weight at study initiation: 17,7 -19,6 g
- Housing: single
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

main study: 25%
Pre-test 3.13, 6.25, 12.5, and 25 %

No. of animals per dose:

Number of animals for the pre-test: 2 females
Number of animals for the main study: 8 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 1
Number of control (vehicle) groups: 1

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The test item was placed into a volumetric flask on a tared balance and the vehicle (dimethyl sulfoxide) was quantitatively added. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer.
The preparations were made freshly before each dosing occasion.
The data showed that the highest test item concentration, which could be technically used, was a 25 % suspension in DMSO. In other vehicles tested a higher concentration could not be suspended.
In a pre-test in two mice, test item concentrations of 3.13, 6.25, 12.5, and 25 % were tested on one ear each. No irritation effects were observed at these concentrations after a single application.

- Irritation: Due to the intense colour of the test item local irritation reactions such as ear redness could not be detected.
- Systemic toxicity: At the 25 % suspension the treated mouse did not show any signs of systemic toxicity.
- Ear thickness measurements: not swelling of the ears was observed
- Erythema scores: not applicable

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
- Criteria used to consider a positive response:

TREATMENT PREPARATION AND ADMINISTRATION:
The test item in the main study was, therefore, assayed at 25%.
Concentrations were in terms of material as supplied.

group concentration animals/group
control (vehicle) 0 4
high dose 25% 4

Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with test item concentrations of 25% (w/v) in dimethyl sulfoxide. The application volume, 25 microl, was spread over the entire dorsal surface (size, 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
3H-methyl thymidine (3HTdR) was purchased from GE Healthcare (GE Healthcare 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 microl of 78.8 MCi/ml 3HTdR (corresponds to 19.7 MCi 3HTdR per mouse) by intravenous injection via a tail vein.

Determination of Incorporated 3HTdR
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Na-thiopental (Trapanal, Altana, D-78467 Konstanz).
The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 Mm 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 plastic scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a beta-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Positive control substance(s):

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

Results and discussion

Positive control results:

SI 4.07 at 10%

In vivo (LLNA)

Results

Parameter:

SI

Value:

ca. 2.2

Test group / Remarks:

25%

Any other information on results incl. tables

est item Concentration	Group	DPM		Calculation		Result
			DPM-BG	Lymph nodes (LN)	DPM/LN	SI
	BG I	23,72
	BG II	19,95
	1	6360,36	6338,5	8	792,3	1
25	2	14125,80	14104,0	8	1763,0	2,2

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

The animals did not show any clinical signs during the course of the study. Due to the intense colour of the test item reddening of the ear skin could not be evaluated.

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:

GHS criteria not met

Conclusions:

The substance was tested for in vivo skin sensitization followign OECD 429, LLNA. Under the experimental conditions the substance is not considered a skin sensitizer.

Executive summary:

The substance was tested for in vivo skin sensitization followign OECD 429, LLNA.

In order to study a possible contact allergenic potential of test item, one group each of four female mice was treated daily with the test item at a concentration of 25% (w/v) in dimethyl sulfoxide by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (dimethyl sulfoxide) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, 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.

All treated animals survived the scheduled study period and no signs of toxicity were observed.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Index of 2.2 was determined with the test item at concentrations of 25% in dimethyl sulfoxide.