Xanthylium, 9-(2-carboxyphenyl)-3,6-bis(diethylamino)-, hydrogen bis[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]chromate(3-), compd. with 3-[(2-ethylhexyl)oxy]-1-propanamine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan 2017 - Oct 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Jul 2010

Deviations:

no

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:

Xanthylium, 9-(2-carboxyphenyl)-3,6-bis(diethylamino)-, hydrogen bis[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]chromate(3-), compd. with 3-[(2-ethylhexyl)oxy]-1-propanamine

EC Number:

284-723-9

EC Name:

Xanthylium, 9-(2-carboxyphenyl)-3,6-bis(diethylamino)-, hydrogen bis[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]chromate(3-), compd. with 3-[(2-ethylhexyl)oxy]-1-propanamine

Cas Number:

84962-27-6

Molecular formula:

C32H20CrN10O14S2.1.5(C28H31N2O3).1.5(C11H25NO)

IUPAC Name:

Xanthylium, 9-(2-carboxyphenyl)-3,6-bis(diethylamino)-, hydrogen bis[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2- hydroxy-5-nitrobenzenesulfonato(3-)]chromate(3-), compd. with 3-[(2-ethylhexyl)oxy]-1-propanamine

Test material form:

solid

Details on test material:

- Physical state/color: solid/red

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Stable under test conditions
- Homogeneity: Homgogeneous by visual inspection

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance preparations were produced on a weight per weight basis shortly before application by stirring with a magnetic stirrer.

FORM AS APPLIED IN THE TEST
- Suspension

OTHER SPECIFICS:
- Vehicle: AOO (mixture of acetone + olive oil in a ratio 4+1 v%)
- Reason for the vehicle: Good homogeneity of the preparation was achieved
- Homogeneity of the test substance preparation: The homogeneity of the lowest test substance preparation (10%) was investigated once by analysis during the study.
- Concentration control analysis of the test substance preparation: The correctness of the concentration of the test substance preparations (10%, 25%, 50%) was investigated once by analysis during the study.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Remarks:

CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Postbus 6174, 5960 AD Horst, The Netherlands
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 9 weeks (pre-test) and 8 weeks (main test)
- Weight at study initiation: 16.5 g - 21.3 g(pre-test); 16.4 g - 20.8 g (main test)
- Housing: Single housed
- Diet: Kliba mouse/rat maintenance diet, ad libitum
- Water: ad libitum
- Acclimation period: At least five days before the first test substance application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12 (light phase from 6 am to 6 pm)

- IN-LIFE DATES: From: 03 Jan 2017 To: 16 Jan 2017

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Test substance 10%, 25%, and 50% in acetone/olive oil (4:1 v/v)

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429.
- Irritation: Recorded on day 1, 2, and 5
- Systemic toxicity: Recorded after each application as well as on day 5
- Ear thickness measurements: Determined on day 0, 2, and 5
- Erythema scores: not specified

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Murine Local Lymph Node Assay
- Criteria used to consider a positive response: Stimulation index (SI) is equal to or above 3.0

TREATMENT PREPARATION AND ADMINISTRATION:
- Randomization: Animals were randomized to the individual groups
- Body weight determination: Individual body weights were recorded on day 0 prior to application and on day 5 prior to sacrifice
- Signs and Symptoms: Obvious signs of systemic toxicity and/or local inflammation at the application sites were noted
- Mortality: A check for moribund and dead animals was made twice on working days and once on Saturdays, Sundays, and on public holidays
- Form of application: Epicutaneous
- Application volume: 25 µl per ear
- Site of application: Dorsal part of both ears
- Frequency of application: Three consecutive applications (day 0 - day 2) to the same application site
- 3H-thymidine injection: On day 5, 20 µCi 3H-thymidine in 250 µl sterile saline were injected into the tail vein of the mice

Statistics:

WILCOXON-test for the parameter: 3H-thymidine incorporation, cell count, lymph node weight and ear weight

Results and discussion

Positive control results:

A concurrent positive control was not included in the study. The results of the last periodic positive control studies as well as positive control data of further study-related experiments are shown in table 1 in "any other information on results".

In vivo (LLNA)

Results

Parameter:

SI

Value:

1.11

Test group / Remarks:

High dose group (50%)

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: The test substance did not induce a biologically relevant nor statistically significant increase of 3H-thymidine incorporation at any concentration tested. In addition, no biologically relevant increase in lymph node cell counts and no statistically significant or relevant increases of lymph node weights were observed

EAR WEIGHTS: None of the concentrations of the test substance did cause relevant increase in ear weights demonstrating the absence of excessive ear skin irritation.

CLINICAL OBSERVATIONS: No signs of systemic toxicity wre noticed in all animals during general observation. Discoloration (reddish) of the feces and urine was observed in all test substance treated animals.

LOCAL FINDINGS: No local findigs were noted in all animals during the observation period.

BODY WEIGHTS: No relevant influence on the mean body weights was observed during the study.

Any other information on results incl. tables

Pre-test/Irritation Screening:

No relevant signs of systemic toxicity were observed in the pre-test. At the 10% and 50% concentrations, the animals did not show relevant signs of local irritation as confirmed by determination of the ear weights (compared to current vehicle values) and ear thickness measurements. After application of both test substance concentrations discoloration of feces and urine (red) was observed. After application of the 50% concentration slightly discolored ear skin (red) was observed during the study period.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

It is concluded that the test substance does not exhibit a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.

Executive summary:

The skin sensitizing potential of the test substance was assessed by using the radioactive Murine Local Lymph Node Assay. The assay simulates the induction phase for skin sensitization in mice. It determines the response of cells in the auricular lymph nodes to repeated application of the test substance to the dorsal skin of the ears. Groups of 5 female CBA/CaOlaHsd mice per dose were treated with 10%, 25% and 50% (w/w) preparations of the test substance in acetone/olive oil (4:1 v/v%; AOO) or with the vehicle alone. The 50% preparation was the maximum technically applicable concentration. The study used 3 test groups and 1 control group. Each test animal was treated with 25 μL per ear of the appropriate test-substance preparation applied to the dorsal surfaces of both ears on three consecutive days. The control group was applied with 25 μL per ear of the vehicle alone. Three days after the last application, 20 μCi 3H-thymidine in 250 μL sterile saline were injected into the tail vein of the mice. About 5 hours after the 3H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring 3H-thymidine incorporation (indicator of cell proliferation). Cell counts and weights of each animal’s pooled lymph nodes were also determined. In addition, a sample with a diameter of 0.8 cm was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined to obtain an indication of possible skin irritation.

The mean stimulation indices (expressed as multiples of the vehicle control) for 3H-thymidine incorporation, cell count, lymph node weight and ear weight are summarized for each test group in the table below.

Table 1: Stimulation indices

Test group	Treatment	3H-thymidine incorporation SI²	Cell count SI²	Lymph Node Weight SI²	Ear Weight SI²
1	vehicle AOO	1.00	1.00	1.00	1.00
2	10% in AOO	1.00	1.03	1.08	1.03
3	25% in AOO	0.82	1.09	1.10	1.02
4	50% in AOO	1.11	1.17	1.15	0.99

SI = Stimulation index; ² = test group x / test group 1 (vehicle control)

No relevant signs of systemic toxicity were noticed in all animals during general observation. The epicutaneous application of the test substance at a concentration of 10%, 25% and 50% (w/w) in AOO, did not induce a biologically relevant nor statistically significant increase of 3Hthymidine incorporation into the cells from the auricular lymph node above the cut-off Stimulation Index of 3. In addition, no statistically significant or biologically relevant increases of lymph node cell counts or lymph node weights were noted. None of the test-substance concentrations did cause relevant increases (SI ≥ 1.25) in ear weights demonstrating the absence of excessive ear skin irritation.