Reaction mass of N-(4-(bis(4-(methylamino)phenyl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate and [4-[[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate and [4-[bis[4-(dimethylamino)phenyl]methylene]-2,5-cyclohexadien-1-ylidene]dimethylammonium acetate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study following official guidelines, GLP compliant, performed on a similar substance

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Tris[4-(diethylamino)phenyl]methylium acetate

EC Number:

263-974-8

EC Name:

Tris[4-(diethylamino)phenyl]methylium acetate

Details on test material:

analogue substance 2

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld- Age at study initiation: 8 - 12 weeks- Weight at study initiation: 18.3 g - 21.1 g- Housing: Single- Type of cage: Makrolon cage, type II- Enrichment: Nest-building material: wood wool (Typ NBF E-011); Abedd ® Lab. and Vet. Service GmbH Vienna, Austria. PLEXX mouse tunnel (red, transparent) EMSICON Jung GmbH, Forstinning, Germany.- Bedding: Lignocel PS14; SSNIFF- Diet: Kliba-Labordiät (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum- Water: Tap water ad libitum- Acclimation period: 7 daysENVIRONMENTAL CONDITIONS- Temperature / Humidity: The animals were housed in fully air-conditioned rooms. Central air-conditioning guaranteed a range of 20 – 24°C for temperature and of 30 – 70% for relative humidity. There were no deviations from these ranges, which influenced the results of the study.- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Remarks:

MEK was used as the vehicle because good homogeneity of the preparation was achieved.

Concentration:

0.1, 0.3 and 1%

No. of animals per dose:

5

Details on study design:

SELECTION OF DOSES/CONCENTRATIONSThe selection of concentrations took into account available information on the chemical/physical properties and the composition of the test substance. In addition the results of pretests with 10% and 1% test-substance preparations in MEK were considered, which showed considerably increased lymph node weights and ear weights as indication of ear irritation in both concentrations. The ears of the mice were discolored by the test substance or showed residues of the test substance at the concentration of 1% or 10% respectively.TREATMENT PREPARATION AND ADMINISTRATION:The test-substance preparations were produced on a weight per weight basis shortly before the application by stirring with a magnetic stirrer. The homogeneity of the test-substance preparation during application was provided by stirring with a magnetic stirrer.CONDUCT OF THE STUDYThe study comprised three treatment groups and a vehicle control group. Each group consisted of 5 mice.- Randomization: Prior to first application, the animals were distributed to the individual groups, received their animal numbers and were allocated to the respective cages according to the randomization instructions of „Nijenhuis, A. and Wilf, H.S.: Combinatorial Algorithms, Academic Press, New York, San Francisco, London, 1978, pp. 62 – 64“.- Body weight determination: Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.- Signs and symptoms: No detailed clinical examination of the individual animals was performed but any obvious signs of systemic toxicity and/or local inflammation at the application sites were noted in the raw data.- Form of application: Epicutaneous application is simulating dermal contact with the compound which is possible to occur under practical use conditions.- Application volume: 25 μL per ear- Site of application: Dorsal part of both ears- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site- Mortality: Twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.³H-THYMIDINE INJECTIONOn study day five (about 66 to 72 hours after the last application of test substance to the ears) the mice were injected intravenously with 20 μCi of 3H-thymidine in 250 μl of sterile saline into a tail vein.TERMINAL PROCEDURESThe animals were sacrificed on study day 5 about 5 hours after 3H-thymidine injection by cervical dislocation.- Determination of ear weight: Immediately after the death of each animal a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each test group. These measurements serve for detecting a potential inflammatory ear swelling.- Removal and weight determination of the lymph nodes: Immediately after removal of the ear punches the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal.- Preparation of cell suspension and determination of cell count: After weight determination, the pooled lymph nodes of each test group were stored in phosphate buffered saline in an ice-water bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing all lymph nodes per test group through an iron mesh (mesh size 200 μm) into 40 mL of phosphate-buffered physiological saline. For determination of cell counts, an aliquot of each suspension was further diluted with Casy®ton in a ratio 1:500. The cell count was determined using a Casy®-Counter.- Measurement of 3H-thymidine incorporation of the lymph node cells: The remaining cell suspensions were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloro-acetic acid (TCA). Each precipitate was transferred to scintillation fluid and incorporation of 3H-thymidine into the cells was measured in a ß-scintillation counter.CALCULATIONSThe stimulation indices of cell count, 3H-thymidine incorporation, lymph node weight and ear weight were calculated as the ratio of the test group values for these parameters divided by those of the vehicle control group.EVALUATION OF RESULTSIn order to reveal a possible induction of sensitization, the response in the draining lymph node after epicutaneous application of several concentrations of the test substance to the skin of the ear backs is determined. The parameters used to characterize the response are lymph node cell count, 3H-thymidine incorporation into the lymph node cells and to a certain extent lymph node weight. Because not only sensitization induction but also irritation of the ear skin by the test substance may induce lymph node responses, the weight of ear punches taken from the area of test-item application is determined as a parameter for inflammatory ear swelling serving as an indicator for the irritant action of the test substance.The increase SI of cell count by a factor of ≥ 1.5 and/or of 3H-thymidine incorporation by a factor of ≥ 3 as compared to the concurrent vehicle control group is generally considered as indicating a sensitizing potential of a test substance.The EC leading to the respective SI values were calculated by semi-logarithmical regression using all data points of the test groups because all concentrations induced effects above the cut-off SIs.In addition the evaluation uses the following considerations:If biologically relevant increases in ear weights are running in parallel to the increase in cell count, 3H-thymidine incorporation and/or lymph node weight, it cannot be ruled out, that the lymph node response was caused by irritation and not by skin sensitization. If a test substance does not elicit a biological relevant increase in cell count, 3H-thymidine incorporation but shows a clear concentration related increase in response, further investigation of the sensitization potential at higher concentrations should be considered.

Positive control substance(s):

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

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

ca. 3.03

Test group / Remarks:

0.1%

Results 2

Parameter:

SI

Value:

ca. 1

Test group / Remarks:

0%

Results 3

Parameter:

SI

Value:

ca. 13.62

Test group / Remarks:

0.3%

Results 4

Parameter:

SI

Value:

ca. 25.52

Variability:

1%

Cellular proliferation data / Observations:

disintegration per minute: 772.3, 2342.6, 10515.6 and 19707.8 for control, 0.1%, 0.3% and 1%, respectively.

Any other information on results incl. tables

CELL COUNT,³H-THYMIDINE INCORPORATION AND LYMPH NODE WEIGHT

When applied as 0.1%, 0.3% and 1% preparations in MEK, the test substance induced a biologically relevant response (increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts.

There was an increase in lymph node weights, as well.

Concomitantly, the increase of 3H-thymidine incorporation into the cells was biologically relevant (increase above the cut off stimulation index of 3) at all concentrations.

 

EAR WEIGHT

The 1% test-substance preparation caused some increase in ear weights. On d1, d2 and on the day of lymph node removal the ears of all test-group mice were lilac discolored.

 

BODY WEIGHTS

The expected body weight gain was generally observed in the course of the study.

 

OTHER FINDINGS

No abnormalities were observed during general observation.

The increases in lymph node weight, cell counts and 3H-thymidine incorporation were concentration dependent at all concentrations.

Thus it is concluded that Ethylviolet Acetate shows a skin sensitizing effect in the Murine Local Lymph Node Assayunder the test conditions chosen.

The threshold concentration for sensitization induction was ca. 0.1%. The estimated concentration that leads to the SI of 1.5 for cell count (EC 1.5) and the estimated concentration that leads to the SI of 3.0 for 3H-thymidine incorporation (EC 3) were calculated by semi-logarithmical regression from the results of all concentrations to be 0.09% and 0.1%, respectively.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

Analogue substance 2 was tested for skin sensitization following OECD 429 (LLNA). Under the experitmental conditions the substance is a skin sensitizer

Executive summary:

The sensitising potential of analogue substance 2 was investigated in a Local Lymph Node Assay in mice according to OECD Guideline 429 under GLP conditions (BASF SE, 2010a). Based on the results of a pre-test, three groups of five animals were treated with test substance concentrations of 0.1%, 0.3% and 1.0% on three consecutive days on the dorsal part of both ears. Five control animals were similarly treated, but with the vehicle alone (methyl ethyl ketone, MEK). Each test animal was applied with 25 μL per ear of the respective test-substance preparation to the dorsum of both ears for three consecutive days. The control group was treated with 25 μL per ear of the vehicle alone.

Three days after the last exposure of the substance the mice were injected intraveneously with 20 µCi of³H-thymidine in 250 μL of sterile saline into a tail vein and after 5 hours the auricular lymph nodes were removed. The weights of each animal’s pooled lymph nodes were determined. Thereafter lymph nodes were pooled group wise and further evaluated by measuring their cellular content and³H-thymidine incorporation into the lymph node cells (indicators of cell proliferation).

No signs of systemic toxicity were noticed.

When applied as 0.1%, 0.3% and 1% preparations in MEK, the test substance induced a biologically relevant response (increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. There was an increase in lymph node weights, as well.

Concomitantly, the increase of³H-thymidine incorporation into the cells was biologically relevant (increase above the cut off stimulation index of 3) at all concentrations.

The 1% test-substance preparation caused some increase in ear weights. On day 1, day 2 and on the day of lymph node removal the ears of all test-group mice were lilac discolored. The increases in lymph node weight, cell counts and³H-thymidine incorporation were concentration dependent at all concentrations. Thus it is concluded that ethylviolet acetate shows a skin sensitising effect in the Local Lymph Node Assay under the test conditions chosen.

The threshold concentration for sensitisation induction was ca. 0.1%. The estimated concentration that leads to the SI of 1.5 for cell count (EC 1.5) and the estimated concentration that leads to the SI of 3.0 for³H-thymidine incorporation (EC 3) were calculated by semi-logarithmical regression from the results of all concentrations to be 0.09% and 0.1%, respectively.