2-[(4-aminophenyl)azo]-1,3-dimethyl-1H-imidazolium chloride

Endpoint summary

Description of key information

Evaluation of skin sensitiztion potential in mice using the local lymph node assay (LLNA)

In vitro skin sensitization turnkey testing strategy including protein reactivity (DPRA), activation of keratinocytes (LuSens) and activation of dendritic cells (h-CLAT) tests; classified as H317.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

08 Sep 2014 to 14 Jul 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

There are no official national or international guidelines for In Vitro Sensitization; however,the studies were performed according to the methods described in the following publications:

Bauch C, Kolle SN, Fabian E, Pachel C, Ramirez T, Wiench B, Wruck CJ, van Ravenzwaay B, Landsiedel R. Intralaboratory validation of four in vitro assays for the prediction of the skin sensitizing potential of chemicals. Toxicology in Vitro 25, 1162 – 1168, 2011.

Maxwell G, Aeby P, Ashikaga T, Bessou-Touya S, Diembeck W, Gerberick F, Kern P, Marrec-Fairley M, Ovigne JM, Sakaguchi H, Schroeder K, Tailhardat M, Teissier S, Winkler P. Skin sensitisation: the Colipa strategy for developing and evaluating nonanimal test methods for risk assessment. ALTEX 28(1): 50-5, 2011.

Bauch C, Kolle SN, Ramirez T, Eltze T, Fabian E, Mehling A, Teubner W, van Ravenzwaay B, Landsiedel R, (2012), Putting the parts together: Combining in vitro methods to test for skin sensitizing potentials, Regul Toxicol Pharmacol, 63(3):489-504.

Urbisch D, Mehling A, Guth K, Ramirez T, Honarvar N, Kolle SN, Landsiedel R, Jaworska J, Kern P, Gerberick F, Natsch A, Emter R, Ashikaga T, Miyazawa M, Sakaguchi H. Assessing skin sensitization hazard in mice and men using non-animal test methods. Regulatory Toxicology and Pharmacology 71(2), 135-352. 2015.

For the DPRA test the following publications apply additionally:
The study is conducted based on the following draft test guideline:
OECD: Draft Proposal for a New Guideline on In Vitro Skin Sensitization: Direct Peptide Reactivity Assay (DPRA), accessed on 13 Nov 2013 at http://www.oecd.org In addition the study is performed according to the methods described in the following publications:

Gerberick GF, Vassallo JD, Bailey RE, Chaney JG, Morrall SW, Lepoittevin JP. Development of a Peptide Reactivity Assay for Screening Contact Allergens. Toxicological Sciences 81,332-343, 2004.

Gerberick GF, Vassallo JD, Foertsch LM, Price BB, Chaney JG, Lepoittenvin JP. Quantificationn of Chemical Peptide Reactivity for Screening Contact Allergens: A Classification Tree Model Approach. Toxicological Sciences 97(2), 417-427, 2007.

Based on the results of an ECVAM (European Center for Validation of Alternative Methods) led validation study on the DPRA, it was concluded by the EURL (EU Reference Laboratory for Alternatives to Animal Testing) ECVAM Scientific Advisory Committee (ESAC) that the DPRA is suitable to be used in a weight-of-evidence approach or integrated testing strategy for distinguishing between skin sensitizers and non-skin sensitizers (ECVAM: EURL ECVAM Recommendation on the Direct Peptide Reactivity Assay (DPRA) of 12 Dec 2013).

For the LuSens the following publications apply additionally:
The study is conducted based on the following draft test guideline:

OECD: Draft Proposal for a New Guideline on In Vitro Skin Sensitization: In vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method, accessed on 03 Sep 2014 at http://www.oecd.org In addition the study is performed according to the methods described in the following publications:

Ramirez T, Mehling A, Kolle SN, Wruck CJ, Teubner W, Eltze T, Aumann A, Urbisch D, Ravenzwaay BV, Landsiedel R.., LuSens: A keratinocyte based ARE reporter gene assay for use in integrated testing strategies for skin sensitization hazard identification. Toxicol In Vitro. 2014 Dec;28(8).

For the h-CLAT test the following publications apply additionally:
The study is conducted based on the following draft test guideline:
OECD: Draft Proposal for a New Guideline on In Vitro Skin Sensitization: human Cell Line Activation Test (h-CLAT), accessed on 01 Sep 2014 at http://www.oecd.org In addition the study is performed according to the methods described in the following publications:

Ashikaga T, Sakaguchi H, Sono S, Kosaka N, Ishikawa M, Nukada Y, Miyazawa , Ito Y, Nishiyama N, Itagaki H. (2010) A comparative evaluation of in vitro skin sensitisation tests: the human cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA). Altern Lab Anim. 38(4):275-84.

Sakaguchi H, Ryan C, Ovigne JM, Schroeder KR, Ashikaga T. (2010) Predicting skin sensitization potential and inter-laboratory reproducibility of a human Cell Line Activation Test (h-CLAT) in the European Cosmetics Association (COLIPA) ring trials. Toxicol In Vitro. 24(6):1810-20.

GLP compliance:

not specified

Remarks:

Adverse oucome pathway investigated using generally accepted scientific principles, this study type is not suitable for classification and the results can only be used as an indication of the potential for sensitisation.

Type of study:

other: protein reactivity (DPRA), activation of keratinocytes (LuSens), and activation of dendritic cells (h-CLAT).

Specific details on test material used for the study:

Lot 0008205061

Details on the study design:

The objective was to assess the skin sensitizing potential of Vibracolor flame orange. A combination of several in vitro methods addressing key events of the adverse outcome pathway (AOP) for skin sensitization1 as defined by the OECD, were part of this in vitro Skin Sensitization Turnkey Testing Strategy:

1)protein reactivity (DPRA):
The reactivity of Vibracolor flame orange towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose the test substance was incubated with synthetic peptides for ca. 24 hours at room temperature and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm.
The test substance was prepared at 100 mM, 10 mM, 5 mM and 1 mM concentrations in deionized water. Two test runs were performed: in the 1st test run 1 mM, 10 mM and 100 mM were tested. In the 2nd test run 1 mM, 5 mM and 10 mM were tested. Per test run, two samples per concentration were incubated with each peptide in ratios of 1:10 (for C-peptide) or 1:50 (for K-peptide). Additionally duplicates of the concurrent vehicle control (= VC) were incubated with the peptides.
Further, a co-elution control was assessed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover the samples were analyzed by measuring UV absorbance

2)activation of keratinocytes (LuSens),:
The keratinocyte activating potential of test substance Vibracolor flame orange was evaluated in the LuSens assay. For this purpose the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer.
In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 9 concentrations of the test substance and cytotoxicity was determined by MTT assay. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve.
Due to the intense color of the test substance a color control plate was assayed in parallel with the pre-test in order to differentiate between formazan production and test-substance residues in the colorimetric test. The cells were treated with the test substance concentrations in the same way as the test plate but received medium without MTT during MTT incubation.
The color control plate demonstrated that the color of the test substance did not have a relevant influence on the colorimetric test (individual results are available with the raw data and are not included in the report).
In the main test luciferase activity was measured after 48 hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance.

3)activation of dendritic cells (h-CLAT):
The potential of test substance Vibracolor flame orange to induce the cell membrane marker CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h-CLAT). For this purpose the test substance was incubated with human promonocytic cell line THP-1 for ca. 24 hours at 37°C and membrane markers expression measured by flow cytometry.
In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 10 concentrations of the test substance and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve.
In the main test after 24 hour exposure THP-1 cells were stained with FITC labeled antihuman- CD86/ anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analyzed using flow cytometry. A total of 5 valid experiments were performed.

Run / experiment:

other: protein reactivity (DPRA)

Parameter:

other: EC 6.38%

Value:

2.55

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Run / experiment:

other: activation of keratinocytes (LuSens)

Parameter:

other: EC1.50 in (ug/mL)

Value:

1.69

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Run / experiment:

other: activation of dendritic cells (h-CLAT)

Parameter:

other: EC150 for CD86 (ug/mL)

Value:

112

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Run / experiment:

other: activation of dendritic cells (h-CLAT)

Parameter:

other: EC200 for CD54 (ug/mL)

Value:

18

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Interpretation of results:

study cannot be used for classification

Remarks:

Support classification as a skin sensitizer.

Conclusions:

Based on the results and applying the evaluation criteria, the test item is peptide reactive, activates keratinocytes and activates dendritic cells. The test item is predicted to be a skin sensitizer.

Executive summary:

The objective was to assess the skin sensitizing potential of the test item. A combination of several in vitro methods addressing key events of the adverse outcome

pathway (AOP) for skin sensitization as defined by the OECD, were part of this in vitro Skin Sensitization Turnkey Testing Strategy:

protein reactivity (DPRA),

activation of keratinocytes (LuSens), and

activation of dendritic cells (h-CLAT).

Based on the results and applying the evaluation criteria, Vibracolor flame orange is peptide reactive, activates keratinocytes and activates dendritic cells. The test item is predicted to be a skin sensitizer.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16Nov2001 to 09Apr2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

supplier: Ciba Specialty Chemicals
. batch number:013673A1
. description: violet powder
. purity: 94.1%

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Age/weight: on the first day of treatment, the animals were approximately 9 weeks old and had a mean body weight ± standard deviation of 20.8±1.6g.
Acclimation: at least 5 days before the beginning of the study.
Allocation: on day 1, animals were assigned to the treatment groups by hand procedure.
Identification : individually by a number on the tail.

The conditions in the animal room were set as follows:
. temperature: 22 ± 2°C
. relative humidity: 30 to 70%
. light/dark cycle: 12 h/12 h
. ventilation: approximately 12 cycles/hour of filtered, non-recycled air.
The temperature and relative humidity were under continuous control and recording. The records were checked daily and filed. In addition to these daily checks, the housing conditions and corresponding instrumentation and equipment are verified and calibrated at regular intervals. The animals were housed individually in disposable crystal polystyrene cages (22.00 cm x 8.50 cm x 8.00 cm). Each cage contained autoclaved sawdust (SICSA, Alfortville, France). Sawdust is analysed by the supplier for composition and contaminant levels.

Vehicle:

other: Ethanol/water (50:50)

Concentration:

0.25-5%

No. of animals per dose:

4

Details on study design:

Compound solubility:
Due to the unsatisfactory solubility of the test item in the first recommended vehicle (acetone/olive oil (4/1, v/v)) and in dimethylformamide (DMF), a mixture ethanol/water (50/50 (v/v)) was chosen among the other proposed vehicles; a homogeneous preparation was obtained at the maximal concentration of 5%.

MAIN STUDY
On days 1, 2 and 3, a dose-volume of 25 µL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip. In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration. No massage was performed but the tip was used to spread the preparation over the application site. No rinsing was performed between each application.

Clinical signs, morbidity and mortality
The animals were observed at least once a day during the study for clinical signs, signs of morbidity or mortality.

Bodyweight
The animals were weighed individually on the first day of the study (day 1) and on the day of sacrifice (day 6).

Ear thickness measurements and recording of local reactions
On days 1, 2 and 3 (before application) and on day 6 (after sacrifice), the thickness of the left ear of each animal of the vehicle control and treated groups was measured using a micrometer. No measurement of ear thickness was carried out for animals of the reference treated group. Any irritation reaction (erythema and oedema) was recorded in parallel. Any other observation (coloration, presence of residual test item,...) was noted.

Intravenous injection of 3H-TdR and sampling of auricular lymph nodes
Lymph node cell proliferative responses were measured as described by Kimber, I. and Dearman R.J. (1991). On day 6, all animals of all groups received a single intravenous injection of 250 µL of 0.9% NaCl containing 20 µCi of 3H-TdR, via the tail vein. Approximately 5 hours later, the animals were killed by cervical dislocation and the auricular lymph nodes were excised. The lymph nodes were pooled for each experimental group. A single cell suspension of auricular lymph node cells (ALNC) was prepared by mechanical disaggregation in Petri dishes with the plunger of a syringe.

Preparation of auricular lymph node cell suspensions and determination of proliferation
Cell suspensions were washed with 15 mL of 0.9% NaCl and pellets obtained were re-suspended in 0.9% NaCl for numeration of lymphocytes (cellularity) and determination of their viability by exclusion of Trypan blue. Each cell suspension was then centrifuged and pellets were precipitated with 3 mL of 5% (w/v) trichloroacetic acid (TCA) in purified water at +4°C overnight. After a last centrifugation, the pellets were precipitated with 1 mL of 5% TCA.
Three mL of Ultima Gold'* scintillation fluid (Packard) were added in order to measure incorporation of 3H-TdR using P-scintillation counting. The results were expressed as disintegrations/mn (dpm) per group. Stimulation Indices (SI) were calculated.

Interpretation of results
The test item was considered as a skin sensitizer when the SI for a dose group is > 3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness were also taken into account for the interpretation of results.

Determination of the EC3 value
Calculation of the EC3 value (theoretical concentration of the test item resulting in a SI value of 3) was performed on the basis of a dose-effect response.

Statistics:

Stimulation Indices (SI) were calculated.

Interpretation of results
The test item was considered as a skin sensitizer when the SI for a dose group is > 3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness were also taken into account for the interpretation of results.

Determination of the EC3 value
Calculation of the EC3 value (theoretical concentration of the test item resulting in a SI value of 3) was performed on the basis of a dose-effect response.

Positive control results:

In the positive control group given HCA at the concentration of 25%, an increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI = 13.56) were noted. The study was therefore considered valid.

Key result

Parameter:

SI

Value:

1.55

Test group / Remarks:

0.25% dose group

Key result

Parameter:

SI

Value:

2.26

Test group / Remarks:

0.50% dose group

Key result

Parameter:

SI

Value:

3.26

Test group / Remarks:

1 % dose group

Key result

Parameter:

SI

Value:

2.52

Test group / Remarks:

2.5% dose group

Key result

Parameter:

SI

Value:

3.88

Test group / Remarks:

5% dose group

Key result

Parameter:

EC3

Value:

3.12

Test group / Remarks:

Curve calcualted from 0.25, 0.5, 2.5 and 5% results

Cellular proliferation data / Observations:

The quantity of cells obtained in each group was satisfactory and the cell viability was higher than 80% in each group.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Under our experimental conditions, the test item induces delayed contact hypersensitivity in the murine Local Lymph Node Assay.

Executive summary:

The aim of this study was to evaluate the potential of the test item to induce delayed contact hypersensitivity using the murine Local Lymph Node Assay (LLNA). This study was conducted in compliance with the principles of Good Laboratory Practice Regulations.

Methods

Twenty-eight female CBA/J mice were allocated to seven groups of four animals each: five treated groups receiving the test item MIP 3100 at the concentrations of 0.25, 0.5, 1, 2.5 and 5%, one negative control group receiving the vehicle (a mixture of ethanol/water (50/50, v/v)), one positive control group receiving the reference item, a-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25%.

The test item, vehicle or reference item were applied over the ears (25µL per ear) for three consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of the lymph node cells in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate stimulation indices (SI). The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1,2,3 and 6.

Results

Due to the unsatisfactory solubility of the test item in the first recommended vehicle (acetone/olive oil (4/1, v/v)), a mixture ethanol/water (50/50 (v/v)) was chosen among the other proposed vehicles; a homogeneous preparation was obtained at the maximal concentration of 5%.

Systemic clinical signs and mortality

No mortality and no clinical signs were observed during the study.

 

Local irritation

No increase in ear thickness was observed in the animals of the treated groups. A red coloration of the skin which could have masked a possible discrete to moderate erythema was noted on the ears of all treated animals from day 2 up to the end of the study (day 6).

 

Proliferation assay

A dose-related increase in the stimulation index was noted and the threshold positive value of 3 was exceeded at the concentrations of 1 and 5%

 

In the absence of local irritation, the positive lymphoproliferative responses observed at the concentrations of 1 and 5% were attributed to delayed contact hypersensitivity. The extrapolated EC3 value for the test item was 3.12%

Conclusion

Under our experimental conditions, the test item induces delayed contact hypersensitivity in the murine Local Lymph Node Assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A 1996 maximisation test in guinea pigs is also available for this test substance with a negative outcome. However it was considered that the 2002 LLNA (this is the preferred in vivo study method) and 2015 in vitro test battery results were key studies for this test substance. On the basis of the key studies it was considered that the test substance was a skin sensitizer.