Tetrasodium [2-({4-fluoro-6-[(2-{[4-fluoro-6-({5-(hydroxykappaO)-6-[(2-{[2-(hydroxy-kappaO)-5-sulfophenyl] diazenyl-kappaN1}-4,5-dimethoxyphenyl) diazenyl-kappaN2]-7-sulfo-2-naphthyl} amino)-1,3,5-triazin-2-yl] amino} propyl) amino]-1,3,5-triazin-2-yl} amino) benzene-1,4- disulfonato(6-)] cuprate(4-)

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15-MAR-2006 TO 28-MAR-2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with GLP

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Test system: Mice, CBA/CaHsdRcc(SPF)
Number of animals for the pre-test (non-GLP): 3 females
Number of animals for the main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of negative control group: 1
Age: 8 - 12 weeks (beginning of acclimatization)
Body weight: 16 g - 24 g (ordered)
Identification: Each cage by unique cage card.
Randomization: Randomly selected by computer algorithm at time of delivery.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

HUSBANDRY
Conditions: Standard Laboratory Conditions. Air-conditioned with ranges for room temperature 22 ±3 °C, relative humidity 30 - 70 % and 10 - 15 air changes per hour. There was a 12 hour fluorescent light /12 hour dark cycle with at least 8 hours music during the light period.
Accommodation: Individual in Makrolon type-2 cages with standard softwood bedding.
Diet: Pelleted standard Kliba 3433, batch no. 76/05 mouse maintenance diet available ad libitum.
Water: Community tap water from Itingen, available ad libitum.

Vehicle:

dimethylformamide

Remarks:

N,N-dimethylformamide

Concentration:

5 %, 10 % and 25 %

No. of animals per dose:

4

Details on study design:

Three groups each of four female mice were treated daily with the test item at concentrations of 5 %, 10 % and 25 % in N,N-dimethylformamide (DMF) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. 25 % was the highest technically achievable concentration in the chosen vehicle. A control group of four mice was treated with the vehicle DMF only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine, 3HTdR). Approximately five hours after intravenous injection, the mice were sacrificed, 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 3HTdR measured in a ß-scintillation counter.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables.

Positive control results:

In the study STIMULATION INDICES of 1.8, 2.9 and 6.2 were determined with the test item at concentrations of 5 %, 10 % and 25 %, respectively, in acetone:olive oil, 4:1 (v/v). And Alpha-hexylcinnamaldehyde was therefore found to be a skin sensitizer in the LLNA tests and an EC3 value of 10.5% was derived.

Parameter:

SI

Remarks on result:

other: S.I. of 6.2, 10.1 and 8.1 were determined with the test item at concentrations of 5 %, 10 % and 25 %, respectively, in DMF.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: DPM of 18568, 30345 and 24532 were determined with the test item at concentrations of 5 %, 10 % and 25 %, respectively, in DMF.

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

S.I. of 6.2, 10.1 and 8.1 were determined with the test item at concentrations of 5 %, 10 % and 25 %, respectively, in DMF and the test item was therefore found to be a potential skin sensitizer in the LLNA tests.

Executive summary:

In order to study a possible contact allergenic potential of the test item three groups each of four female mice were treated daily with the test item at concentrations of 5 %, 10 % and 25 % in N,N-dimethylformamide (DMF) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. 25 % was the highest technically achievable concentration in the chosen vehicle. A control group of four mice was treated with the vehicle DMF only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine, 3HTdR). Approximately five hours after intravenous injection, the mice were sacrificed, 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 3HTdR measured in a ß-scintillation counter.

All treated animals survived the scheduled study period.

No clinical / local signs were observed.

After each topical application, the residual test item was found at all the local dosing sites in all mice of Group 3 (10%) and Group 4 (25%). After the second and the third topical application, the residual test item was found at all the local dosing sites in all mice of Group 2 (5%).

In the study, S.I. of 6.2, 10.1 and 8.1 were determined with the test item at concentrations of 5%, 10% and 25%, respectively, in DMF. The test item was therefore found to be a potential skin sensitizer in the LLNA test and it shall be included in the Skin sensitisers Category 1 according to the CLP regulation (Regulation EC No. 1272/2008)

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In order to study a possible contact allergenic potential of the test item three groups each of four female mice were treated daily with the test item at concentrations of 5 %, 10 % and 25 % in N,N-dimethylformamide (DMF) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. 25 % was the highest technically achievable concentration in the chosen vehicle. A control group of four mice was treated with the vehicle DMF only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine, 3HTdR). Approximately five hours after intravenous injection, the mice were sacrificed, 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 3HTdR measured in a ß-scintillation counter.

All treated animals survived the scheduled study period.

No clinical / local signs were observed.

After each topical application, the residual test item was found at all the local dosing sites in all mice of Group 3 (10 %) and Group 4 (25 %). After the second and the third topical application, the residual test item was found at all the local dosing sites in all mice of Group 2 (5 %).

In the study, S.I. of 6.2, 10.1 and 8.1 were determined with the test item at concentrations of 5 %, 10 % and 25 %, respectively, in DMF. The test item was therefore found to be a potential skin sensitizer in the LLNA test.

Migrated from Short description of key information:
The test article was found to be a skin sensitizer in the LLNA.

Justification for selection of skin sensitisation endpoint:
guidance test with GLP compliance

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The test article shall be classified as Category 1 skin sensitiser according to the CLP regulation (Regulation EC No. 1272/2008) and accordingly with Xi, R43 according to DSD (Directive 67/548/EEC). Data on respiratory sensitisation are lacking.