N-(4-{[3-(dimethylamino)propyl]sulfamoyl}phenyl)-2-[(2-methoxy-4-nitrophenyl)diazenyl]-3-oxobutanamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

N-(4-{[3-(dimethylamino)propyl]sulfamoyl}phenyl)-2-[(2-methoxy-4-nitrophenyl)diazenyl]-3-oxobutanamide

EC Number:

700-222-4

Cas Number:

1065519-44-9

Molecular formula:

C22H28N6O7S

IUPAC Name:

N-(4-{[3-(dimethylamino)propyl]sulfamoyl}phenyl)-2-[(2-methoxy-4-nitrophenyl)diazenyl]-3-oxobutanamide

Constituent 2

Reference substance name:

No. 408 Yellow

IUPAC Name:

No. 408 Yellow

Details on test material:

- Name of test material (as cited in study report): No. 408 Yellow
- Physical state: yellow powder
- Analytical purity: 98.6%
- Purity test date: 07 November 2008
- Lot/batch No.: 081105-1
- Expiration date of the lot/batch: 04 November 2009
- Stability under test conditions:
- Storage condition of test material: room temperature, dark, sealed container
- Other: Source: TOYO INK MFG. CO. LTD.; pH of 33% w/v dispersion was 9.3

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Limited, Bicester.
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: 18 - 23 g
- Housing: individual
- Diet (e.g. ad libitum): SQC(E) Rat and Mouse Maintenance Diet No 1, Special Diets Services Ltd, Witham, UK
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 - 15 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 40 - 70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0.5, 1.0, 2.5 % (w/v)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Formulation trials were carried out with the test article using 80% v/v acetone in olive oil, dimethyl formamide, methyl ethyl ketone and dimethylsulphoxide. At the start of each trial, 500 mg of test article was added to 1 mL of vehicle. At this concentration, all formulations were thick suspensions. A further 1 mL of vehicle was added to each formulation and left stirring overnight. The resulting suspensions were then further diluted with 0.5 mL of vehicle. A visual inspection confirmed that the formulation in dimethyl formamide appeared to be the most suitable, therefore a series of dilutions were carried out until a solution was obtained. This occurred at approximately 3%.

As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test article, the preliminary screening test was performed with one mouse. The mouse was treated by daily application of 25 μL of the test article at the maximum attainable concentration (2.5% w/v in dimethyl formamide) to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The mouse was observed daily for five days from the initiation of treatment. Any signs of toxicity or irritation during this period were recorded.

- Irritation: Death or signs of systemic toxicity/excessive irritation were not noted.
- Lymph node proliferation response: Not reported


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Groups of five female mice were assigned to study according to the table given below. Doses were selected from the concentration series 2.5%, 1.0%, 0.5% etc. Three consecutive concentrations were selected based on the preliminary screening test so that the highest concentration maximised exposure whilst avoiding systemic toxicity and excessive local irritation.

- Name of test method: Individual Method
- Criteria used to consider a positive response: The test article is regarded as a sensitiser when the maximum value of the SI is 3.0 or above.


TREATMENT PREPARATION AND ADMINISTRATION:
Doses were selected from the concentration series 2.5%, 1.0%, 0.5% etc. Three consecutive concentrations were selected based on the preliminary screening test so that the highest concentration maximised exposure whilst avoiding systemic toxicity and excessive local irritation.Formulations were freshly prepared as required, on Days 1, 2 and 3. The formulations were mixed by multiple inversion of the containers prior to administration to ensure homogeneity. The formulations were stored at room temperature, in sealed, air-tight containers prior to dosing. All formulations were used within two hours of preparation.

The five groups of five female mice were subjected to application of the vehicle control, positive control or one of the test formulations to the outer aspect of the auditory pinnae, once daily on Days 1, 2 and 3. On Day 6 0.25 mL phosphate buffered saline incorporating 20 μCi of 3HTdR were administered into a tail vein of each mouse by slow bolus injection. Approximately five hours after intravenous injection of the 3HTdR, all mice were killed by exposure to a rising concentration of carbon dioxide. Killing was organised to minimise the interval between death and the recovery of the auricular lymph nodes to no more than fifteen minutes.

The isolated lymphocytes of individual animals were subsequently subject to scintillation counting after the preparation procedure.

Positive control substance(s):

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

Statistics:

The scintillation counter data included the DPM value (disintegrations per minute during a ten minute period) for each individual animal. The DPM value was transformed into a mean DPM value for each group. The mean DPM value for each test group was divided by the mean DPM for the control group to provide the Stimulation Index (SI) value for each test group.

Results and discussion

Positive control results:

The positive control article produced a Stimulation Index of 9.60 demonstrating adequate performance of the assay.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 0.5%: 1.55 1.0%: 1.10 2.5%: 0.77

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal ± Standard Deviation: Vehicle: 217 ± 60.1 0.5%: 338 ± 149.8 1.0%: 238 ± 133.1 2.5%: 168 ± 42.1 Positive control: 2084 ± 745.9

Any other information on results incl. tables

Conclusion:

The Local Lymph Node Assay demonstrated that No.408 Yellow does not have the potential to cause skin sensitisation.

The test article did not meet the criteria for classification as a sensitiser according to EU labelling regulations Commission Directive 2001/59/EC. No symbol and risk phrase are required.