Sodium bis[2,4-dihydro-4-[[2-hydroxy-5-(methylsulphonyl)-4-nitrophenyl]azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Mar 2017 - Aug 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:

Sodium bis[2,4-dihydro-4-[[2-hydroxy-5-(methylsulphonyl)-4-nitrophenyl]azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)

EC Number:

276-075-0

EC Name:

Sodium bis[2,4-dihydro-4-[[2-hydroxy-5-(methylsulphonyl)-4-nitrophenyl]azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)

Cas Number:

71839-91-3

Molecular formula:

C34 H26 Cr N10 O12 S2 . Na

IUPAC Name:

sodium bis[2,4-dihydro-4-[[2-hydroxy-5-(methylsulphonyl)-4-nitrophenyl]azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)

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: 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: 9 weeks (pre-test) and 8 weeks (main test)
- Weight at study initiation: 19.5 g - 23.0 g(pre-test); 17.1 g - 19.9 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: 14 Mar 2017 To: 27 Mar 2017

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

Test substance 10%, 25%, and 50% in MEK

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:

2.77

Test group / Remarks:

High dose group (50%)

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: The test substance did not induce a biologically relevant increase of 3H-thymidine incorporation at any concentration tested. In addition, no biologically relevant increase in lymph node cell counts and no relevant increases of lymph node weights were observed. The increases of the 10%, 25% and 50% preparation in 3H-thymidine incorporation and of the 25% preparation in lymph node cell counts were statistical significant but without relevance.

EAR WEIGHTS: The test substance concentrations did not cause increases (SI > 1.25) in ear weights demonstrating the absence of relevant ear skin irritation. However, a statistical significant increase in ear weights was noted after treatment with the 50% test substance preparation.

CLINICAL OBSERVATIONS: No relevant signs of systemic toxicity were noticed in all animals during general observation. Red discolored feces were observed in all animals treated with the 50% concentration from study day 1 up to study day 5.

LOCAL FINDINGS: Slight red discoloration of the ear skin was noted in all test-substance treated animals during the whole observation period.

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. 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.

Table 1: Historical control data on the periodic positive control

Project No.	Date of performance	Name of positive control substance	Concentrations	Vehicle	Stimulation index 3H-thymidine incorporation	Stimulation index cell counts
58V0288/98A008	Jun 2016	Alpha-hexylcinnamaldehyde, techn. 85%	1%, 5%, 15%	MEK (methyl ethyl ketone)	2.60, 7.08, 14.77	1.50, 2.21, 3.01
58V0288/98A009	Jan 2017	Alpha-hexylcinnamaldehyde, techn. 85%	1%, 5%, 15%	MEK	1.56, 3.20, 9.16	1.22, 2.0, 3.42

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 methyl ethyl ketone (MEK) or with the vehicle alone. However, due to irregularities during sample processing the data of only three animals of the concurrent vehicle control group could be used for calcualtion of the mean values and for determination of the stimulation indices (SI) of the test substance. Thus, calculation of the SI using the historical data for the vehicle MEK are also shown in the summary table below.

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 actual vehicle control or historical 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 SI²	3H-thymidine SI (HC³)	Cell Count SI	Cell Count SI (HC)	Lymph Node weight SI	Lymph Node Weight SI (HC)	Ear Weight SI	Ear Weight SI (HC)
1#	Vehicle MEK	1.00		1.00		1.00		1.00
2	10% in MEK	2.29*	1.46	1.17	1.28	1.05	1.21	0.98	1.00
3	25% in MEK	1.98*	1.26	1.18*	1.28	1.06	1.22	1.03	1.05
4	50% in MEK	2.77*	1.77	1.25	1.36	1.14	1.31	1.17*	1.19

SI = Stimulation index; ² = SI calculated using test group x / test group 1 (vehicle control); ³ = SI calculated using test group 2 -4 / historical control (HC) data for the vehicle MEK; * = p ≤ 0.05; # = calculation on basis of three animals due to irregularities during sample processing

No relevant signs of systemic toxicity were noticed in all animals during general observation. When applied as 50%, 25% and 10% preparation in MEK, the test substance did not induce biologically relevant increases of 3H-thymidine incorporation into the cells from the auricular lymph nodes (no increase above the cut off SI of 3) as well as no biologically relevant increases in lymph node cell counts (no increase to 1.5-fold or above of control value = SI ≥ 1.5).

In addition, there were no relevant increases in lymph node weights at all test-substance preparations. The increases of the 10%, 25% and 50% preparation in 3H-thymidine incorporation and of the 25% preparation in lymph node cell counts were statistical significant but without relevance. The test-substance concentrations did not cause increases (SI > 1.25) in ear weights demonstrating the absence of relevant ear skin irritation. However, a statistical significant increase in ear weights was noted after treatment with the 50% test-substance preparation. Slight red discoloration of the ear skin was noted in all test-substance treated animals during the whole observation period. The data of only 3 animals could be used for calculation of the mean values for the concurrent control group and for determination of the stimulation indices (SI) of the test item. Nevertheless, the calculation of the stimulation indices using the historical data for the vehicle MEK confirmed the evaluation of the results using the concurrent control group.