N-(2-ethylhexyl)-1-[[3-methyl-4-[(3-methylphenyl)azo]phenyl]azo]naphthalen-2-amine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

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:

N-(2-ethylhexyl)-1-[[3-methyl-4-[(3-methylphenyl)azo]phenyl]azo]naphthalen-2-amine

EC Number:

260-125-3

EC Name:

N-(2-ethylhexyl)-1-[[3-methyl-4-[(3-methylphenyl)azo]phenyl]azo]naphthalen-2-amine

Cas Number:

56358-10-2

Molecular formula:

C32H37N5

IUPAC Name:

N-(2-ethylhexyl)-1-[[3-methyl-4-[(3-methylphenyl)azo]phenyl]azo]naphthalen-2-amine

Test material form:

solid

Details on test material:

Batch number ZD 00883/044. Manufactured Oct 1996
91.3% purity. Impurity is residual solvent Shellsol AB

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan UK
- Age at study initiation: Young adults
- Housing: 4 per cage.
- Diet (e.g. ad libitum): R&M No 1 supplied by Special Siet Services Ltd, Witham, Essex UK
- Water (e.g. ad libitum): Mains water
- Acclimation period: 5 days minimum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30-70%
- Air changes (per hr): 15 minimum
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Study design: in vivo (LLNA)

Vehicle:

other:

Remarks:

Acetone

Concentration:

1%, 3% and 10% (w/v)

No. of animals per dose:

4

Details on study design:

TREATMENT PREPARATION AND ADMINISTRATION:

Approximately 25µL of a 1%, 3% of 10% w/v preparation of the test substance in acetone was applied,using a variable volume micropipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using acetone alone. The procedure was repeated daily for 3 consecutive days.

Three days after the third application, the animals were held in a hot box at 37°C for 5-10 mins prior to injection via the tail vain with approx 250µL of phosphate buffered saline (PBS) containing approximately 20µCi of a 2.0Ci/mmol specific activity 3H-methyl thymidine. Approximately 5 hours later the animals were humanely killed. The draining auricular lymph nodes were removed from each animal and together with the nodes from the other animals in the group were place in a container of PBS.

A single cell suspension was prepared by mechanical disaggregation of lymph nodes through a 200-mesh stainless steel gauze. The cell suspensions were then washed 3 times by centrifugation with approx. 10mls of PBS. Approximately 3ml of 5% w/v trichloracetic acid (TCA) was added and after overnight precipitation at 4°C the samples were pelleted by centrifugation and the supernatant was discarded. The cells were then resuspended in 1ml of TCA.

The lymph node suspensions were transferred to scintillation vials and 10ml of scintillant was added prior to Beta-scintillation counting using a Packard Tri-Carb Liquid Scintillation counter.

CLINICAL OBSERVATIONS:
Animals were checked at least once per day for signs of systemic toxicity.

POSTIVE CONTROL STUDY:
The sensitisation potential of the control (Hexylcinnamaldehyde) was assessed using the same method and concentrations (25µL of 1%, 3% and 10% preparations. A vehicle control group was similarly tested using acetone.

Positive control substance(s):

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

Results and discussion

Positive control results:

The application of hexylcinnamaldehyde at contrations of 1%, 3% and 10% w/v in acetone resulted in a greater than 3-fold increase in isotope incorporation at concentrations of 3% and 10%. Therefore hexylcinnamaldehyde was shown to be a skin sensitiser, confirming the validity of the protocol used for the study.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.68

Test group / Remarks:

1% concentration

Results 2

Key result

Parameter:

SI

Value:

2.81

Test group / Remarks:

3% concentration

Results 3

Key result

Parameter:

SI

Value:

5.97

Test group / Remarks:

10% concentration

Any other information on results incl. tables

The application of the test substance at concentrations of 1%, 3%, 10% w/v in acetone resulted in an increase in isotope incorporation which was greater than 3-fold at the 10% w/v concentration. The test substance is therefore a potential skin sensitiser. The full results are tabulated below:

 

Concentration of test substance	Number of lymph nodes assayed	Counts per minute	Cpm per lymph node (x10^-2)	Test:control ratio
Naïve control	8	665	0.83	N/A
0 (Vehicle only)	8	1212	1.52	N/A
1%	8	2040	2.55	1.68
3%	8	3414	4.27	2.81
10%	8	7259	9.07	5.97

 

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

The test substance is a skin sensitiser under the conditions of this test and should be classified as skin sensitier Cat 1B according to EU CLP criteria.

Executive summary:

The test substance was assessed for its skin sensitisation potential using the Local Lymph Node Assay. It was applied at concentrations in acetone of 1%, 3% and 10% to the surface of the ear of male mice for 3 consecutive days.

Primary proliferation of lymphocytes in the lymph node draining the site of application was evaluated using radioactive labelling of proliferating cells. The ratio of the proliferation in treated groups to that in vehicular controls, termed the Stimulation Index, was determined.

The positive control substance Hexylcinnamaldehyde (concentration 1%, 3% and 10% (w/v) elicited a reaction pattern at 3% and 10% confirming the validity of the protocol used.

Comparison of Stimulation Indexes between the treated groups and control vehicle group revealed that the test substance caused a dose dependent increase in radioisotope incorporation into the DNA of dividing lymphocytes. The Stimulation Index of the highest treated group (10% w/v) was 5.97. In accordance with OECD 429, and SI value of >3 in the LLNA test should be regarded as a skin sensitiser. The test substance therefore has the capacity to cause sensitisation and should be classified as a skin sensitiser Cat 1B.