Methyl 4-cyano-5-[[5-cyano-2,6-bis[(3-methoxypropyl)amino]-4-methyl-3-pyridyl]azo]-3-methyl-2-thenoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

no quantitatative data is given for the effects in the pre-test;

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2002

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2004

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 4-cyano-5-[[5-cyano-2,6-bis[(3-methoxypropyl)amino]-4-methyl-3-pyridyl]azo]-3-methyl-2-thenoate

EC Number:

277-146-9

EC Name:

Methyl 4-cyano-5-[[5-cyano-2,6-bis[(3-methoxypropyl)amino]-4-methyl-3-pyridyl]azo]-3-methyl-2-thenoate

Cas Number:

72968-71-9

Molecular formula:

C23H29N7O4S

IUPAC Name:

methyl 4-cyano-5-[[5-cyano-2,6-bis[(3-methoxypropyl)amino]-4-methyl-3-pyridyl]azo]-3-methyl-2-thenoate

Specific details on test material used for the study:

Name of test material given in the report: Sandoplast Red BB
appearance: red solid
purity: 98.5%

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands, B.V. Postbus 6174, NL-5960 AD Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known:
- Age at beginning of acclimatization: 8-12 weeks
- Weight: 16 - 24 g
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C (plus/minus 3°C)
- Humidity (%): 30-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12h
- IN-LIFE DATES: From: 15.09.2004 To: 29.09.2004

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

non-GLP pre-test: 1, 2.5, 5, and 10%
GLP main test: 1, 2.5, and 5%

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
- Irritation: in a non-GLP pretest in two mice test item concentrations of 1, 2.5, 5, and 10% (w/v) were tested on one ear each; after a single application no irritation effects were observed up to the concentration of 5%; slight ear swelling was noted 24 hours after treatment at the local site dosed at 10%
- Ear thickness measurements: slight ear swelling was noted 24 hours after treatment at the local site dosed at 10%; thus, 5% was determined as the highest applicable concentration while avoiding systemic toxicity and excessive local irritation in the chosen vehicle.

MAIN STUDY
Test item preparation: The weight/volume dilutions were prepared individually using a magnetic stirrer as homogenizer. The test item formulation were made freshly before each dosing occasion and no more than 4 hours prior to application to the ears. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: radioactive LLNA
- Criteria used to consider a positive response:
A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- first, exposure to at least one concentration results in ³HTdR incorporation of at least 3-fold over control mice as indicated by the Stimulation Index (S.I.)
- second, 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:
Treamtent: Mice were treated topically on the dorsal surface of each ear lobe with the test item concentrations 1, 2.5 and 5% (w/v) in acetone/olive oil (4:1). The application volume (25 µL) was spread over the entire dorsal surface of the ear lobes once daily for 3 consecutive days. A hair dryer was passed briefly over the ear's surface to prevent the loss of any test item applied.
Administration of ³H-Methly Thymidine: (specific activity 2Ci/mmol, concentration 1 mCi/mL) five days after the first topical application all mice were administered with 250 µL of 76.6 µCi/mL ³HTdR (equal to 19.1 µCi) by intravenous injection via a tail vein.
Determination of ³HTdR: approximately 5 hours after treatment with ³HTdR all mice were euthanized by inhalation of CO2. Draining lymph nodes were excised and pooled for each experimental group (8 nodes per group). The level of ³hThR incorporation and background activity were measured on a beta-scintillation counter (disintegration per minute, DPM).

Positive control substance(s):

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

Statistics:

mean values and standard deviations were calculated for the body weights

Results and discussion

Positive control results:

Stimulation indices of 2.7, 3.4, and 12.4 were determined for the positive control in concentrations of 5, 10, and 25%. The positive control was thus found to be a skin sensitizer and an EC3 value of 7.1% (w/v) was derived.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.2

Test group / Remarks:

1%

Results 2

Parameter:

SI

Value:

1.4

Test group / Remarks:

2.5%

Results 3

Parameter:

SI

Value:

1.4

Test group / Remarks:

5%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
no data given on cellular proliferation

DETAILS ON STIMULATION INDEX CALCULATION
DPM were calculated for pooled lymph nodes (8 per group) and the DPM per lymph node were calculated by dividing the measured value by the number of lymph nodes pooled.
The DPM per lymph node is given with 351 for the control and 417, 484, and 508 for the treatment groups 1%, 2.5%, and 5%.

EC3 CALCULATION
Calculation of EC3 could not be done because no test concentration produced a S.I. of 3 or higher

CLINICAL OBSERVATIONS:
No symptoms of local toxicity at the ears and no systemic findings were observed during the study.

BODY WEIGHTS
Body weights were within the range of the control with the exception of two mice (one in the 1% and 5% group each) that lost weight during the study. This was considered to be incidental.

Applicant's summary and conclusion

Executive summary:

A radioactive mouse LLNA (OECD 429) was performed with the test item at concentrations of 1%, 2.5%, and 5% (w/v) in acetone/olive oil (4:1, v/v) on 4 mice per group. 5% was the highest applicable concentration avoiding local irritation determined in pre-test in two mice. Treatment occurred on 3 consecutive days. ³H-methyl thymidine was injected intravenously on day 5.

No clinical signs or indications of local effects were seen after treatment. Lymph nodes were pooled and ³H-methyl thymidine activity was measured by beta-scintillation. Stimulation indices (S.I.) of 1.2, 1.4, and 1.4 were determined with the test item at the concentrations 1, 2.5 and 5%. The test item did thus not show an allergenic potential when tested up to the concentration of 5%.