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Description of key information

A modified local lymph node assay (IMDS) was performed on 24 female NMRI mice of the strain HsdWin:NMRI (6 animals/test item group and 6 control animals) to determine if there is any specific (sensitising) or non-specific (irritant) stimulating potential of the test item Astra Blau6GLL HSP.

A concurrent control of 6 animals treated with Alpha Hexyl Cinnamic Aldehyde was included.

In the LLNA no antigen specific activation of the cells of the immune system via dermal route was determined after application of up to and including 30% C.I. Direct Blue 264. Therefore, the concentration of 30% turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitization.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guiudeline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Principles of method if other than guideline:
The modified Local Lymph Node Assay (IMDS) was performed on 24 female NMRI mice of the strain Crl:NMRI BR (6 animals/test item group and 6 control animals) to determine if there is any specific (sensitizing) or non-specific (irritant) stimulating potential of the test item C.I. Direct Blue 264.
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
NMRI
Sex:
female
Vehicle:
dimethylformamide
Concentration:
Group 1 Vehicle (DMF)
Group 2 3% C.I. Direct Blue 264
Group 3 10% C.I. Direct Blue 264
Group 4 30% C.I. Direct Blue 264
Group 5 30% Alpha Hexyl Cinnamic Aldehyde (in DMF)
No. of animals per dose:
6 animals/test item group and 6 control animals
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
A modified Local Lymph Node Assay (IMDS) was carried out in mice. The modifications refer to the measurement of cell proliferation by cell counting instead of radioactive labeling. In addition, the acute inflammatory skin reaction is determined to discriminate specific from non-specific activation of immune competent cells in the draining lymph nodes, as also recommend in the update of OECD TG 429. 1. Direct LLNA (NMRI mice, female, 6 animals/group) Groups Weight index Cell count index (index of mean +/- SD in %) Gr. 1 1.00 +/- 9.70 1.00 +/- 17.40 Gr. 2 1.17 +/- 16.65 1.16 +/- 23.10 Gr. 3 1.22 +/- 7.87 1.37 +/- 15.12 Gr. 4 1.16 +/- 14.77 1.16 +/- 25.40 Gr. 5 1.66 +/- 14.40 1.63 +/- 18.66 2. Ear swelling (NMRI mice, female, 6 animals/group, in 0.01 mm) Groups day 1 day 4 Index day 4 (mean +/- SD in %) Gr. 1 17.42 +/- 2.96 20.00 +/- 7.69 1.00 Gr. 2 17.67 +/- 4.41 19.25 +/- 7.39 0.96 Gr. 3 17.75 +/- 3.50 20.92 +/- 6.59 1.05 Gr. 4 17.58 +/- 2.93 20.92 +/- 13.28 1.05 Gr. 5 17.50 +/- 2.98 24.25 +/- 8.64 1.21 * = statistically significant increase (p< 0.05) 3. Ear weight (NMRI mice, female, 6 animals/group, in mg per 8 mm diameter punch) Groups day 4 Index day 4 (mean +/- SD in %) Gr. 1 13.90 +/- 5.76 1.00 Gr. 2 13.22 +/- 6.87 0.95 Gr. 3 13.58 +/- 6.55 0.98 Gr. 4 14.58 +/- 6.86 1.05 Gr. 5 16.33 +/- 7.45 1.18 * = statistically significant increase (p< 0.05)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: not applicable
Parameter:
SI
Remarks:
Cell count index
Value:
1
Test group / Remarks:
6 animals/group
Remarks on result:
other: Gr. 1
Parameter:
SI
Remarks:
Cell count index
Value:
1.16
Test group / Remarks:
6 animals/group
Remarks on result:
other: Gr. 2
Parameter:
SI
Remarks:
Cell coung index
Value:
1.37
Test group / Remarks:
6 animals/group
Remarks on result:
other: Gr. 3
Parameter:
SI
Remarks:
Cell count index
Value:
1.16
Test group / Remarks:
6 animals/group
Remarks on result:
other: Gr. 4
Parameter:
SI
Remarks:
Cell count index
Value:
1.63
Test group / Remarks:
6 animals/group
Remarks on result:
other: Gr. 5
Interpretation of results:
GHS criteria not met
Executive summary:

A LLNA/IMDS was carried out in female NMRI mice after epicutaneous application of a formulation containing 0%, 3%, 10% or 30% of the test item C.I. Direct Blue 264 for 3 consecutive days onto both ears of the animals.

This study does neither point to a non-specific (irritant) nor to a specific immunostimulating (sensitizing) potential of the test item.

This applies to NMRI mice, for weight and cell count indices of the draining lymph nodes as well as ear swelling and ear weight indices evaluated after application of C.I. Direct Blue 264.

These findings were verified by the results obtained with the positive control compound Alpha Hexyl Cinnamic Aldehyde.

Taken together, no antigen specific activation of the cells of the immune system via dermal route was determined after application of up to and including 30% C.I. Direct Blue 264 by the method used.

Therefore, the concentration of 30% turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitization.

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A LLNA/IMDS was carried out in female NMRI mice after epicutaneous application of a formulation containing 0%, 3%, 10% or 30% of the test item C.I. Direct Blue 264 for 3 consecutive days onto both ears of the animals.

This study does neither point to a non-specific (irritant) nor to a specific immunostimulating (sensitizing) potential of the test item. No antigen specific activation of the cells of the immune system via dermal route was determined after application of up to and including 30% C.I. Direct Blue 264.

Therefore, the concentration of 30% turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitization.

The sensitivity of the test system was verified by results obtained with the positive control compound Alpha Hexyl Cinnamic Aldehyde.


Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the negative data in the LLNA assay, according to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.