Tetrasodium [3-[[8-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-1-hydroxy-3,6-disulpho-2-naphthyl]azo]-4-hydroxynaphthalene-1,5-disulphonato(6-)]cuprate(4-)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October/November 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

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:

Tetrasodium [3-[[8-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-1-hydroxy-3,6-disulpho-2-naphthyl]azo]-4-hydroxynaphthalene-1,5-disulphonato(6-)]cuprate(4-)

EC Number:

238-739-8

EC Name:

Tetrasodium [3-[[8-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-1-hydroxy-3,6-disulpho-2-naphthyl]azo]-4-hydroxynaphthalene-1,5-disulphonato(6-)]cuprate(4-)

Cas Number:

14692-76-3

Molecular formula:

C23H10ClCuN7Na4O14S4

IUPAC Name:

tetrasodium [3-[[8-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-1-hydroxy-3,6-disulpho-2-naphthyl]azo]-4-hydroxynaphthalene-1,5-disulphonato(6-)]cuprate(4-)

Constituent 2

Reference substance name:

tetralithium(1+) 5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-2λ³,25λ³-dioxa-13,14λ⁴-diaza-1-cuprahexacyclo[12.11.0.0³,¹².0⁴,⁹.0¹⁵,²⁴.0¹⁸,²³]pentacosa-3,5,7,9,11,13,15,17,19,21,23-undecaene-1,1-bis(ylium)-2,25-diide-7,11,17,22-tetrasulfonate

Cas Number:

2112848-85-6

Molecular formula:

C23H10ClCuLi4N7O14S4

IUPAC Name:

tetralithium(1+) 5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-2λ³,25λ³-dioxa-13,14λ⁴-diaza-1-cuprahexacyclo[12.11.0.0³,¹².0⁴,⁹.0¹⁵,²⁴.0¹⁸,²³]pentacosa-3,5,7,9,11,13,15,17,19,21,23-undecaene-1,1-bis(ylium)-2,25-diide-7,11,17,22-tetrasulfonate

Test material form:

solid: particulate/powder

Details on test material:

Reactive Blue 13 Na/Li-Salt

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

male

Details on test animals and environmental conditions:

Specification: young adults
Husbandry: A maximum of 4 mice was housed per cage, in cages suitable for animals of this strain and weight range.
Diet: R&M No. 1, supplied by Special Diet Services Limited, UK and mains water, supplied by automatic system were available as libitum.
Water: tap water ad libitum
Acclimatization period: 5 days

Temperature: 21±2 °C
Relative humidity: 30-70%
Air: A minimum of 15 changes per hour
Light cycle: 12 h/ 12 h

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

3%, 10%, 30%

No. of animals per dose:

4

Details on study design:

Approximately 25 µL of a 3, 10 and 30 % w/v preparation of the test substance in propylene glycol was applied, using a variable volume micro-pipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using propylene glycol alone. The procedure was repeated daily for 3 consecutive days.
Three days after the third application, all the animals were injected, via the tail vein, with approximately 250 µL of phosphate buffer saline containing approximately 20 µCi of a 2.0 Ci/mmol specific activity 3H-methyl thymidine. Approximately 5 hours later, the animals were sacrificed by inhalation of halothane vapour followed by cervical dislocation. The draining auricular lymph nodes were removed from each animal and together with the nodes from the other animals in the group were placed 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 three times by centrifugation with approximately 10 mL of PBS. Approximately 3 mL of 5 % w/v trichloroacetic 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 approximately 1 mL of TCA.
The lymph node suspensions were transferred to scintillation vials and 10 mL of scintillant was added prior to β-scintillation counting using a Packard Tri-Card 2500TH liquid Scintillation Counter.
Animals were checked at least once daily for signs of systemic toxicity.

Statistics:

The results are expressed as a count per minute (cpm) value per lymph node for each group. The activity of each test group is then divided by the activity of the vehicle control group to give a test control ratio for each concentration.
The criterion for a positive response is that one or more concentrations of the test substance should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

2.96

Test group / Remarks:

3%

Results 2

Parameter:

SI

Value:

2.97

Test group / Remarks:

10%

Results 3

Key result

Parameter:

SI

Value:

3.07

Test group / Remarks:

30%

Cellular proliferation data / Observations:

The EC3 for this substance was calculated to be 16%

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

The substance was shown to have the capacity to cause a very weak skin sensitization (SI=3.07) when applied as a 30% w/v preparation in propylene glycol.The EC3 for this substance was calculated to be 16%

Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance in CBA/Ca mice according to OECD Guideline 406 (Local Lymph Node Assay), in compliance with GLP. The assay determined the level of T lymphocyte proliferation in the lymph nodes draining the site of chemical application by measuring the amount of radiolabelled thymidine incorporated into the dividing cells. Four male mice were used per test group. Approximately 25 µL of a 3, 10 and 30% w/v solution of test substance in propylene glycol was applied using a variable volume micro-pipette to the dorsal surface of each ear. Propylene glycol served as vehicle control. The procedure was repeated daily for 3 consecutive days. Animals were checked at least once daily for signs of systemic toxicity. Three days after the last application, all animals were injected with 3H-methyl thymidine and, after 5 hours the draining (auricular) lymph nodes were excised and pooled for each animal. After the samples preparation the lymph node suspensions were transferred to scintillation vials and 10 mL of scintillate was added prior to β-scintillation counting using a Packard Tri-Card 2500TH liquid scintillation counter. The substance was shown to have the capacity to cause a very weak skin sensitization when applied as a 30% w/v preparation in propylene glycol. The EC3 for this substance was calculated to be 16%