Disodium 3-[[2,4-bis(2-methylphenoxy)phenyl]azo]-4-hydroxy-5-[[(p-tolyl)sulphonyl]amino]naphthalene-2,7-disulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment. GLP compliant

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according 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:

Disodium 3-[[2,4-bis(2-methylphenoxy)phenyl]azo]-4-hydroxy-5-[[(p-tolyl)sulphonyl]amino]naphthalene-2,7-disulphonate

EC Number:

274-402-1

EC Name:

Disodium 3-[[2,4-bis(2-methylphenoxy)phenyl]azo]-4-hydroxy-5-[[(p-tolyl)sulphonyl]amino]naphthalene-2,7-disulphonate

Cas Number:

70210-05-8

Molecular formula:

C37H31N3O11S3.2Na

IUPAC Name:

sodium (E)-3-((2,4-bis(o-tolyloxy)phenyl)diazenyl)-4-hydroxy-5-(4-methylphenylsulfonamido)naphthalene-2,7-disulfonate

Test material form:

other: solid

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

male/female

Details on test animals and environmental conditions:

Source: Recognised as the recommended test system Harlan Netherlands B.V. Postbus 6174 NL - 5960 AD Horst / The NetherlandsNumber of animals for the pre-test (non-GLP): 2 females Number of animals for the main study: 16 females (experiment 1), 8 females (experiment 2) Number of animals per group: 4 females (nulliparous and non-pregnant)Number of test groups: 3 (experiment 1) 1 (experiment 2)Number of control (vehicle) group: 1 (experiment 1 and 2, each) Age: 7 - 8 weeks (beginning of acclimatization) Identification: Single caging. The animals will be distributed into the test groups at random and identified by cage number. Acclimatisation : Under test conditions after health examination. Only animals without any visible signs of illness will be used for the study.The animals were kept conventionally. The experiment was conducted under standard laboratory conditions. Housing: single Cage Type: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen) Bedding: granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178 Borchen) Feed: pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen) Water: tap water, ad libitum, (Gemeindewerke, D-64380 Rossdorf) Environment: temperature 22 + 3°C; relative humidity 24-70%; artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

4, 8, and 16.0 % (w/v).

No. of animals per dose:

4 animals per dose

Details on study design:

The main experiment was repeated with the high dose of 16% (experiment 2), since in the first experiment on the first day of application accidentally 12% was used for the high dose. Topical Application Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 4,.8 and 16% (w/v) in propylene glycol. The application volume, 25 ul, was spread over the entire dorsal surface (0 - 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 Amersham International (Amersham product code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/m1). Five days after the first topical application, all mice were administered with 250 ul of 79.1 (experiment 1) and 80.1 (experiment 2) uCi/m1 3HTdR (corresponds to 19.775 (experiment 1) and 20.025 (experiment 2) uCi 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 pm 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 glass scintillation vials with 10 ml of 'Ultima Gold' scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured on a 3-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1m1-aliquots of 5 % trichloroacetic acid. The 13-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

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

In Experiment 1, Stimulation Indices of 0.7 and 0.8 were determined with the test item at concentrations of 4 and 8 % (w/v) in propylene glycol, while for the group treated with 12/16 % an S.I. of 0.9 was determined. In Experiment 2 an S.I. of 2.1 was obtained with the test item at a concentration of 16 % (w/v). The difference between the values obtained for the high dose in experiments 1 and 2 was considered to reflect the normal variation range of the LLNA. The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.

Results 2

Key result

Parameter:

SI

Value:

ca. 0.7

Test group / Remarks:

TG2

Results 3

Key result

Parameter:

SI

Value:

ca. 0.8

Test group / Remarks:

TG3

Results 4

Key result

Parameter:

SI

Value:

ca. 0.9

Test group / Remarks:

TG4

Any other information on results incl. tables

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 and no cases of mortality were observed. Due to the intense red colour of the test item local irritation reactions such as ear redness could not be detected.

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

Remarks:

Migrated information

Conclusions:

The substance is considered to be a non-sensitizer substance.

Executive summary:

In this study the test item suspended in propylene glycol was assessed for its possible contact allergenic potential.

For this purpose a local lymph node assay was performed on three groups each of four female mice, treated with different concentrations of the test item by topical application at the dorsum of each ear lobe (left and right) on three consecutive days. One control group of four mice were treated with the vehicle only.

The test item concentrations were 4, 8 and 16%. Accidentally, animals of the high dose group were treated with a concentration of 12 % instead of 16 % on the first day (the high dose group in Experiment 1 is therefore designated as “12/16 %”). Consequently, the high dose group (16 %) was repeated in Experiment 2.

 

Approximately five hours after intravenous injection, the mice were sacrificed and 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 ß-scintillation counter.

 

 The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. Due to the intense red colour of the test item local irritation reactions such as ear redness could not be detected.

In Experiment 1, Stimulation Indices of 0.7 and 0.8 were determined with the test item at concentrations of 4 and 8 % (w/v) in propylene glycol, while for the group treated with 12/16 % an S.I. of 0.9 was determined. In Experiment 2 an S.I. of 2.1 was obtained with the test item at a concentration of 16% (w/v). The difference between the values obtained for the high dose in experiments 1 and 2 was considered to reflect the normal variation range of the LLNA.

Therefore the substance is classified as non-sensitizer substance.