4'-aminoacetanilide

Endpoint summary

Description of key information

In modified LLNA test, 4'-Aminoacetanilide has been identified as a weak sensitizer in NMRI mice.

When in vitro loose-fit coculture-based sensitization assay (LCSA) of primary human keratinocytes and of allogenic dendritic cell-related cells was used for combined testing of the sensitizing and irritative properties, 4'-Aminoacetanilide showed no sensitizing potential under the experimental conditions.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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)

Specific details on test material used for the study:

4'-Aminoacetanilide, kindly provided by the Wollforschungsinstitut Aachen, Germany

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

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.

Positive control results:

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

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.

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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Assessment of the sensitizing potential is performed by the loose-fit coculture-based sensitization assay (LCSA). This in vitro testing is based on a coculture of primary human keratinocytes and allogenic dendritic cell-related cells (DCrc), which is capable of identifying the sensitizing and irritative potential of substances.

GLP compliance:

not specified

Remarks:

GLP compliance not stated

Type of study:

other: loose-fit coculture-based sensitization assay (LCSA) of primary human keratinocytes and of allogenic dendritic cell-related cells

Justification for non-LLNA method:

Development of new in vitro method for the assessment of the sensitizing potential of chemicals, which could be OECD validated in the future.

Specific details on test material used for the study:

p-aminoacetanilide (CAS No. 122-80-5) was purchased from Sigma-Aldrich (Steinheim, Germany).

Details on the study design:

MEDIA AND REAGENTS
p-aminoactanilide was dissolved in DMSO (DMSO Hybrimax, Sigma-Aldrich). Final concentration of DMSO in cell culture medium did not exceed 0.1%. Cells were cultured in serum-free KGM-2 (Keratinocyte growth medium 2, PromoCell, Heidelberg, Germany) supplemented with complete SupplementPack (PromoCell), 100 U/ml penicillin and 100 µg/ml streptomycin (Biochrom, Berlin, Germany). TNBS (2,4,6-Trinitrobenzenesulfonic acid) was also obtained from Sigma-Aldrich. The cytokines IL-4 and GM-CSF were purchased from ImmunoTools (Friesoythe, Germany) and TGF-β1 from R&D Systems (Minneapolis, USA). For FACS (Fluorescence-activated cell sorting) analysis, cells were stained with PE-conjugated anti-CD86 and PE-conjugated isotype control (clone: MOPC-21, mouse IgG1, BD Biosciences, Heidelberg, Germany) and 7-AAD (Cell Viability Solution, BD Biosciences).

CRYOPRESERVATION OF PRIMARY HUMAN KERATINOCYTES
Human skin from healthy donors was received as anonymous residual material from plastic surgery. After 18 h of incubation in PBS containing 2 U/ml of dispase I (Roche, Mannheim, Germany) at 4°C, epidermal sheets were stripped off the dermal layer and dissociated in PBS containing 0.25% trypsin (Biochrom) and 0.01% DNase (Roche) for 15 min at 37°C. Single-cell suspension was obtained by passaging though a 40-µm cell strainer (BD, Heidelberg, Germany). Cells were washed with PBS, resuspended in KGM-2 and seeded on Costar Cell Culture Flasks (Corning, Shiphol-Rijk, the Netherlands) at a density of 5 x 10^5 cells/cm². Cells were cultured until confuence with medium changes on alternate days. KCs (Keratinocytes) were harvested by trypsinization and cryopreserved in FCS with 10% DMSO (Hybrimax, Sigma) at a density of 2 x 10^6 cells/ml.

CRYOPRESERVATION OF HUMAN PBMCs
PBMCs (Peripheral blood mononuclear cells) were enriched from fresh buVy coat preparations (German Red Cross, Berlin, Germany) by density centrifugation on Ficoll-Paque (Biochrom). PBMCs were cryopreserved as described above for KCs at a density of 4 x 10^7 cells/ml.

LOOSE-FIT COCULTURE-BASED SENSITIZATION ASSAY
KCs were defreezed at 37°C, washed with PBS and resuspended in KGM-2. KCs were seeded in 12-well plates (Costar Cell Culture Cluster, Corning) at a density of 6 x 10^4 cells/cm². When cells were adherent, wells were rinsed with PBS to remove dead cells. PBMCs were first defreezed and resuspended in KGM-2, then seeded onto KCs at a density of 3 x 10^6 cells/cm². IL-4, GM-CSF and TGF-β were added to the coculture at final concentrations of 100, 100 and 10 ng/ml, respectively. After 48 h of incubation, test substances were added. Increasing concentrations of test substances were tested until significant cytotoxicity occurred as measured by 7-AAD (7-Aminoactinomycine D) staining. Every substance was tested with three different DC-rc/KC-donor pairs to account for interindividual variability.
TNBS at a concentration of 100 µmol/l served as positive control. After another 48 h, floating cells were pipetted off the coculture and prepared for FACS analysis.

FACS ANALYSIS
FACS analysis was performed on a FACS Calibur flow cytometer (BD Biosciences). Three cell populations CD14+, CD4+CD8+ and CD86+ were gated according to their scatter properties. The latter is considered to be the DC-rc (Dendritic cell-related cells) population and is therefore gated (details in Schreiner et al. 2007, 2008 (A loose-fit coculture of activated keratinocytes and dendritic cell-related cells for prediction of sensitizing potential. Allergy 62:1419–1428; A new dendritic cell type suitable as sentinel of contact allergens. Toxicology 249:146–152)). The experiments were assumed to be reliable, when the addition of TNBS led to a 1.5- to 3-fold increase in CD86 cell population expression compared with zero controls. Relative upregulation of CD86 expression was calculated by the following equation: MFI (CD86 of treated cells)/MFI (CD86 of vehicle control). 7-AAD staining was used to determine and exclude dead cells from the calculation. Dose-eVect relationships for relative CD86 expression and viability were determined using GraphPad Prism® software (GraphPad Software, San Diego, USA).

CATEGORISATION
For categorisation of sensitising potential, the substance concentration that led to half-maximal increase of CD86 expression (EC50) was assessed. Substances were categorised as follows: half-maximal increase in CD86 expression <12.5 µM, extreme; <50 µM, strong; <100 µM, moderate; and >100 µM, weak. Substances failing to elicit significant rise in CD86 expression up to the maximum test concentration were deemed non-sensitisers. Likewise, categorisation of irritative potential is based on the concentration that resulted in cell death of 50% (EC50%) of DC-rc as compared to vehicle controls. Substances eliciting 50% cell death at concentrations below 50 µM were considered to be irritative. Those with an EC50% value from 50 to 1,000 µM were considered to be weakly irritative. Substances that did not reach the 50% threshold or with EC50% values above 1,000 µM were rated as non-irritative according to Wanner et al. 2010 (Classification of sensitizing and irritative potential in a combined in vitro assay. Toxicol. Appl. Pharmacol. 245:211–218).

Positive control results:

A post hoc analysis of CD86 expression after application of 100 µM TNBS (which served as positive control in all test series) revealed a relatively low variability: 2.3 ± 0.48-fold increase in CD86 (§SEM) with 12 different donor pairs of keratinocytes and dendritic cell-related cells.

Run / experiment:

other: Sensitisation EC 50 (µmol/l)

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Irritation EC50% (µmol/l)

Remarks on result:

other: No indication of skin irritation

Interpretation of results:

study cannot be used for classification

Conclusions:

p-aminoacetanilide showed no sensitizing properties up to concentration of 300µM. The irritative potential of cleavage product p-aminoacetanilide was not assessable.

Executive summary:

Loose-fit coculture-based sensitization assay (LCSA) of primary human keratinocytes and of allogenic dendritic cell-related cells was used for combined testing of the sensitizing and irritative properties of p-Aminoacetanilide. The test substance showed no sensitizing potential under the experimental conditions.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The endpoints followed in the 2 published studies are not the same criteria defined in CLP Regulation. It is therefore not possible to conclude the Hazard category of 4'-Aminoacetanilide. However, based on the weight of evidence approach, 4'-Aminoacetanilide is not considered as skin sensitizer.