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Diss Factsheets

Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Sensitising potential of four textile dyes and some of their metabolites in a modified local nymph node assay
Author:
R. Stahlmann, M. Wegner, K. Riecke, M. Kruse, T. Platzek
Year:
2006
Bibliographic source:
Toxicology 219, 113-123

Materials and methods

Test guidelineopen allclose all
Qualifier:
no guideline followed
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
Authors' experience indicate that the sensitisation-challenge protocol as a 2-phase approach may have a higher sensitivity and specificity and, therefore, may be useful in the detection of weak sensitisers or to clarify ambiguous results.
Principles of method if other than guideline:
Two distinct protocols were followed: Sensitisation protocol and Sensitisation-challenge protocol.

SENSITISATION PROTOCOL:
The test item was applied on the dorsum of the left ear of the animals for 3 consecutive days. The right ears were treated with an equal volume of vehicle (DMSO). Forty-eight hours after the last exposure, mice were euthanised in deep CO2 anaesthesia.

SENSITISATION-CHALLENGE PROTOCOL
Animals were shaved over a surface of approximately 2 cm² on their backs. This surface was treated once daily on days 1–3 with 50µl of the test solution. Mice remained untreated on days 4–14. On days 15–17, animals were challenged with 25 µl of the test solution applied to each dorsum of both ears. Mice were sacrificed on day 19.
Results obtained in mice treated according to this protocol were compared to a control group treated with the vehicle DMSO alone.

In both cases, in addition to the endpoints weight and cell number of the draining ear lymph nodes, lymphocyte subpopulations were analysed by flow cytometry.
GLP compliance:
no
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
4'-aminoacetanilide
EC Number:
204-576-6
EC Name:
4'-aminoacetanilide
Cas Number:
122-80-5
Molecular formula:
C8H10N2O
IUPAC Name:
4'-aminoacetanilide
Test material form:
solid
Details on test material:
Name: 4'-aminoacetanilide
Specific details on test material used for the study:
4'-Aminoacetanilide, kindly provided by the Wollforschungsinstitut Aachen, Germany

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Female NMRI mice
- Source: Winkelmann, Brochen, Germany
- Age at study initiation: 7 weeks
- Diet: Altromin pellet feed (Altromin 1324, Lage, Germany) ad libitum
- Water: tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Housing: Mice were kept in Macrolon® cages in groups of six
- Temperature (°C): 23± 1 °C
- Humidity (%): 45-55%
- Lighting sequence: 12 hours light followed by 12 hours dark

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Remarks:
No separate NC or PC groups were used
Concentration:
10% and 30%
No. of animals per dose:
6
Details on study design:
SENSITISATION PROTOCOL:
Groups of 6 female mice received 25 µl of the test solution on the dorsum of the left ear for 3 consecutive days. The right ears were treated with an equal volume of DMSO. 48h after the last exposure, mice were euthanised in deep CO2 anaesthesia. Ear thickness was measured using a spring-loaded micrometer Oditest (Kroeplin, Schüchtern, Germany). Draining auricular lymph nodes were excised and weighed. Single cell suspensions were prepared by gentle mechanical disagregation through stainless steel gauze to determine the total number of cells counted with the automated cell counter Sysmex F-820 (Sysmex Europe Gmbh, Norderstedt, Germany) and for flow cytometric analysis of cell surface markers.

SENSITISATION-CHALLENGE PROTOCOL:
Animals were shaved over a surface of approximately 2cm2 on their backs. This surface was treated once daily on days 1-3 with 50 µl of the test solution. Mice remained untreated on days 4-14. On days 15-17, animals were challenged with 25 µl of the test solution applied to each dorsum of both ears. Mice were sacrificed on day 19 and draining auricular lymph nodes were processed as described for the sensitisation protocol. Results obtained in mice treated according to this protocol were compared to a control group treated with the vehicle DMSO alone.

FLOW CYTOMETRY:
Cells were stained using commercially available fluorochrome-conjugated antibodies: CD 4, CD 45R CD 69, CD8a, 1A/1E. Whilst CD 4 and CD 8 are T-cell markers characterising T-helper or cytotoxic T-cells, respectively, CD45R, also known as B220, is a mouse specific B-cell marker. The murine MHC-II corresponds to 1A. CD69, also known as very early antigen, is an indicator of activation of lymphocyte activation.

Cell suspensions from individual lymph nodes (sensitisation protocol) or the two lymph nodes of each animal (sensitisation-challenge protocol) were simultaneously incubated with an antibody conjugated with phycoerythrine (PE) and/or an antibody conjugated with fluoresceine isothiocyanate (FITC) protected from light at 4°C over 30 min and then washed twice in phosphate buffered saline (PBS, Biochrom, Berlin, Germany) and sodium azide 0.02% (Sigma, Heidelberg, Germany).

Flow cytometry was performed with a FACScan flow cytometer (Becton Dickinson, Heidelberg, Germany) equipped with a 488nm argon-laser. Calibration was done by using CaliBRITE Beads (Becton Dickinson). Analysis was restricted to lymphocytes by appropriately gating by cell size (forward scatter) and cell granularity (side scatter). This approach also excluded debris and cell clumps from analysis. 104 lymphocytes were counted per sample. Data were saved as listmode files and analysed using Winlist 2.0 software (Verity Software House, Topsham, USA). Analysis of lymphocyte subpopulations were done as proposed by Loken et al. (1990).
Positive control substance(s):
other: none
Statistics:
In assays according to the sensitisation protocol, the treated left ears and lymph nodes were compared to the respective vehicle treated right ears and lymph nodes with regard to ear thickness, lymph node weight, lymph node cellularity and flow cytometric data using a paired non-parametric test, the Wilcoxon test. In assays performed according to the sensitisation-challenge protocol, where both ears were treated, mean values were calculated for the ear thickness, lymph node weight and lymph node cellularity of each animal. Results were compared to those obtained from DMSO-treated control animals using the non-parametric Mann–Whitney test. Statistical analysis was done with the software SPSS 10.0 (SPSS Inc., Chicago, USA). Statistical significance was assumed when two-sided P < 0.05.

Results and discussion

Positive control results:
No (historical) positive control is used in this test.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
other: Lymph node weight variation compared with vehicle control
Value:
0.03
Variability:
+ 3% compared with DMSO
Test group / Remarks:
Sensitisation protocol; 10% concentration in test substance
Remarks on result:
other: No significant effect
Parameter:
other: Lymph node cellularity variation compared with vehicle control
Value:
0.04
Variability:
- 4% compared with DMSO
Test group / Remarks:
Sensitisation protocol; 10% concentration in test substance
Remarks on result:
other: No significant effect
Parameter:
other: Lymph node weight variation compared with vehicle control
Value:
0.41
Variability:
+ 41% compared with DMSO
Test group / Remarks:
Sensitisation-challenge protocol; 30% concentration in test substance
Parameter:
other: Lymph node cellularity variation compared with vehicle control
Value:
0.61
Variability:
+ 61% compared with DMSO
Test group / Remarks:
Sensitisation-challenge protocol; 30% concentration in test substance
Parameter:
other: % Median (proportion of CD4+ cells VS DMSO treated ears)
Value:
68.7
Variability:
-0.43% in comparison to DMSO
Test group / Remarks:
Concentration of 10% in test item / CD4 cell marker in Sensitisation protocol
Remarks on result:
other: No significant effect
Parameter:
other: % Median (proportion of CD8+ cells VS DMSO treated ears)
Value:
19.48
Variability:
-0.64% in comparison to DMSO
Test group / Remarks:
Concentration of 10% in test item / CD8 cell marker in Sensitisation protocol
Remarks on result:
other: No significant effect
Parameter:
other: % Median (proportion of CD45R+ cells VS DMSO treated ears)
Value:
8.6
Variability:
+ 6.70% in comparison to DMSO
Test group / Remarks:
Concentration of 10% in test item / CD45R cell marker in Sensitisation protocol
Remarks on result:
other: No significant effect
Parameter:
other: % Median (proportion of CD69 cells VS DMSO treated ears)
Value:
7.05
Variability:
+ 12.08% in comparison to DMSO
Test group / Remarks:
Concentration of 10% in test item / CD69 cell marker in Sensitisation protocol
Remarks on result:
other: No significant effect
Parameter:
other: % Median (proportion of CD4+ cells VS DMSO treated ears)
Value:
13.39
Variability:
-3.18% in comparison to DMSO
Test group / Remarks:
Concentration of 10% in test item / 1A cell marker in Sensitisation protocol
Remarks on result:
other: No significant effect
Parameter:
other: % Median (proportion of CD4+ cells VS DMSO treated ears)
Value:
66.7
Variability:
+ 5.5% in comparison to DMSO
Test group / Remarks:
Concentration of 10% in test item / CD4 cell marker in Sensitisation-challenge protocol
Remarks on result:
other: No significant effect
Parameter:
other: % Median (proportion of CD4+ cells VS DMSO treated ears)
Value:
65.2
Variability:
+ 3.0% in comparison to DMSO
Test group / Remarks:
Concentration of 30% in test item / CD4 cell marker in Sensitisation-challenge protocol
Remarks on result:
other: No significant effect
Parameter:
other: % Median (proportion of CD8+ cells VS DMSO treated ears)
Value:
15.9
Variability:
-3.7% in comparison to DMSO
Test group / Remarks:
Concentration of 10% in test item / CD8 cell marker in Sensitisation-challenge protocol
Remarks on result:
other: No significant effect
Parameter:
other: % Median (proportion of CD8+ cells VS DMSO treated ears)
Value:
16.1
Variability:
-2.8% in comparison to DMSO
Test group / Remarks:
Concentration of 30% in test item / CD8 cell marker in Sensitisation-challenge protocol
Remarks on result:
other: No significant effect
Parameter:
other: % Median (proportion of CD45R+ cells VS DMSO treated ears)
Value:
9.1
Variability:
-13.8% in comparison to DMSO
Test group / Remarks:
Concentration of 10% in test item / CD45R cell marker in Sensitisation-challenge protocol
Remarks on result:
other: No significant effect
Parameter:
other: % Median (proportion of CD4+ cells VS DMSO treated ears)
Value:
8.9
Variability:
-15.1% in comparison to DMSO
Test group / Remarks:
Concentration of 30% in test item / CD45R cell marker in Sensitisation-challenge protocol
Remarks on result:
other: No significant effect
Parameter:
other: % Median (proportion of CD69+ cells VS DMSO treated ears)
Value:
6.3
Variability:
+ 39.1% in comparison to DMSO
Test group / Remarks:
Concentration of 10% in test item / CD69 cell marker in Sensitisation-challenge protocol
Remarks on result:
other: Significant effect
Parameter:
other: % Median (proportion of CD69+ cells VS DMSO treated ears)
Value:
4.8
Variability:
+ 6.3% in comparison to DMSO
Test group / Remarks:
Concentration of 30% in test item / CD69 cell marker in Sensitisation-challenge protocol
Remarks on result:
other: No significant effect
Parameter:
other: % Median (proportion of 1A+ cells VS DMSO treated ears)
Value:
13
Variability:
-20.1% in comparison to DMSO
Test group / Remarks:
Concentration of 10% in test item / 1A cell marker in Sensitisation-challenge protocol
Remarks on result:
other: No significant effect
Parameter:
other: % Median (proportion of 1A cells VS DMSO treated ears)
Value:
12.5
Variability:
-23.3% in comparison to DMSO
Test group / Remarks:
Concentration of 30% in test item / 1A cell marker in Sensitisation-challenge protocol
Remarks on result:
other: No significant effect
Cellular proliferation data / Observations:
None of the animals studied showed any abnormal clinical sign. In animals where one ear was treated with textile dyes according to the sensitisation protocol, the contralateral ears showed no relevant crosscontamination with the test substance.

Treatment of mice according to the sensitisation protocol with 10% 4-aminoacetanilide produced no consistent effects on lymph node weight (+3%) and cellularity (−4%). No significant effects were seen by phenotyping of lymphocytes.

In the sensitisation-challenge protocol no significant changes were detected at 10% 4-aminoacetanilide, but at a concentration of 30%, where both the lymph node weight (+41%) and the lymph node cellularity (+61%) increased significantly. Regarding the epitope markers, the number of CD69+ cells were significantly higher (+39%) at the 10% concentration compared to the control group, but not at the 30% dye concentration.

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
Sensitising potential of 4-aminoacetanilide has been investigated in a modified LLNA.
Test item was applied either to the dorsum of the mice ears (sensitisation protocol) or they were first applied to the skin of their backs and 2 weeks later to their ears (sensitisation-challenge protocol).
In the sensitisation protocol, 4-aminoacetanilide did not induce significant effects, whereas in the sensitisation-challenge protocol cell number and lymph node weight increased significantly indicating a sensitising potential in NMRI mice. Hence, two-phase treatment (skin of the back, ear) increased the sensitivity of this assay.
The authors have defined 4-aminoacetanilide as a weak sensitizer in NMRI mice.
Executive summary:

The sensitising and allergenic potential of 4'-Aminoacetanilide has been assessed using modified protocols of the murine "local nymph node assay" (LLNA). The test substance was applied either to the dorsum of the mice ears (sensitisation protocol) or was first applied to the skin of their backs and 2 weeks later to their ears (sensitisation-challenge protocol). In addition to the endpoints weight and cell number of the draining ear lymph nodes, lymphocyte subpopulations were analysed by flow cytometry. In the sensitisation protocol, 4'-aminoacetanilide did not induce significant effects, whereas in the sensitisation-challenge protocol cell number and lymph node weight increased significantly indicating a sensitising potential in NMRI mice. The authors qualified 4'-Aminoacetanilide as a weak sensitiser.