Hydrogen [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]hydroxychromate(1-), compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2017 - Jan 2018

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:

Hydrogen [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]hydroxychromate(1-), compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

EC Number:

287-257-4

EC Name:

Hydrogen [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]hydroxychromate(1-), compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

Cas Number:

85443-67-0

Molecular formula:

C16 H11 Cr N5 O8 S .C11 H25 N O .H

IUPAC Name:

1-Propanamine, 3-[(2-ethylhexyl)oxy]-,(T-4)-[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulfonato(3-)]hydroxychromate(1-)

Test material form:

solid

Details on test material:

- Physical state/color: solid/orange

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: MEK (methyl ethyl ketone)
- 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: 8 weeks (pre-test and main test)
- Weight at study initiation: 17.9 g - 20.9 g(pre-test); 17.6 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: 13 Jun 2017 To: 26 Jun 2017

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

Test substance 10%, 25%, and 50% in methyl ethyl ketone

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: Were determined

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

Results 1

Parameter:

SI

Value:

3.44

Test group / Remarks:

High dose group (50%)

Results 2

Parameter:

EC3

Value:

36.3

Test group / Remarks:

Given in percent (%)

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: At a concentration of 50%, the test substance induced a dose-dependent, statistically significant and biologically relevant increase of 3H-thymidine incorporation into the cells from the auricular lymph node above the cut-off SI of 3.0 (SI of 3.44).

EC3 CALCULATION: The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation was calculated by linear regression to be 36.3%.

CLINICAL OBSERVATIONS: No test substance related signs of systemic toxicity were noticed in all animals during general observation.

BODY WEIGHTS: The expected body weight gain was generally 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. However, discolored feces (orange) was observed in the high dose group (50%) on day 2 and day 5. In both, the low (10%) and high (50%) dose groups, slightly discolored ear skin (orange) was observed from day 1 to day 5. In low and high dose group, animals showed slight swelling of the ears on day 2 and day 1, respectively, which intensified for the high dose group to moderate swelling on day 2. Furthermore, for the high dose group, moderate test substance residues and slight to moderate hair loss from day 2 to day 5 were noted.

Due to the discoloration caused by both test substance preparations, potential erythema could not be assessed in the pretest. Regarding ear thickness, an increase of thickness by 28 and 24% on day 2, as well as an increase by 17 and 30% on day 5 was noted in animal 7 and 8 of the high dose group, respectively. But, as no accompanying increase of ear weight > 25% could be determined, the observed increase of ear thickness is considered to be caused by remaining test-substance residues. In conclusion, the test substance did not elicit excessive local skin irritation/inflammation up to the tested concentration of 50%.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

It is concluded that the test substance exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen. The threshold concentration for sensitization induction was > 25% < 50%. The threshold concentration for sensitization induction was >25% <50%. The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation was calculated by linear regression from the results of these concentrations to be 36.3%.

Executive summary:

The 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 methyl ethyl ketone (MEK) or with the vehicle alone. 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 MEK	1.00	1.00	1.00	1.00
2	10% in MEK	2.23**	1.08	1.25*	1.00
3	25% in MEK	2.64**	1.14	1.40**	1.01
4	50% in MEK	3.44**	1.34	1.37*	1.16

SI = Stimulation index; ² = test group x / test group 1 (vehicle control); * for p ≤ 0.05, ** for p ≤ 0.01

No signs of systemic toxicity were noticed in all animals during general observation. The topical application of the test substance at a concentration of 50% (w/w) in MEK, induced a dose-dependent, statistically significant and biologically relevant increase of 3H-thymidine incorporation into the cells from the auricular lymph node above the cut-off Stimulation Index (S.I.) of 3 (S.I. of 3.44). In addition, the observed effect was reflected by the statistically significant increase of lymph node weight. In the low and mid dose groups, applied with 10 and 25% (w/w) in MEK, a statistically significant increase of 3H-thymidine incorporation into the lymph node cells was observed, but was considered to be not biologically relevant as the corresponding S.I.s did not reach or exceed the cut-off value of 3. The lymph node weights for

both test groups were also statistically significant. However, regarding the auricular lymph node cell counts the 50% preparation and also both lower preparations induced a statistically non-significant but dose-dependent increase below the cut-off Stimulation Index of 1.5. All applied test-substance concentrations did not cause a statistically significant and biologically relevant increase of ear weight above the cut-off S.I. of 1.25. This demonstrates the absence of relevant ear skin irritation. Slight discoloration (orange) of the ear skin was observed in all test-substance treated groups during the observation period. In addition, slight test-substance residues were noted after application of the 50% concentration on day 2 and 5. However, the observed test substance residues could have contributed to the slightly increased ear weights in the high dose group (50%).