Reaction mass of Amines, C12-14-tert-alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-08-10 to 2016-11-23

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:

2010-07-22

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:

Reaction mass of Amines, C12-14-tert-alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-)

EC Number:

916-868-7

Molecular formula:

C32H22CoN10O8.C10-14H21-29NH2 and C32H22CoN10O8.Na

IUPAC Name:

Reaction mass of Amines, C12-14-tert-alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-)

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No. of test material: 002-151203

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

OTHER SPECIFICS: Solid / orange

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., 5960 AD Horst, The Netherlands
- Age at study initiation: 8 – 10 weeks
- Weight at study initiation: 16.4 g – 21.1 g
- Housing: single housed in Polycarbonate cages type MII with mesh wire tops in fully air-conditioned rooms.
- Diet. Kliba mouse/rat maintenance diet “GLP”, supplied by Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum.
- Water: Drinking water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24
- Humidity (%): 30 – 70
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

1 %, 5 %, and 25 %

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. The highest test-substance concentration which was used in the pretest was a 50 % test-substance preparation. The test-substance preparations at different concentrations were formulated in DMF.
- Irritation: After application of the 50 % concentration the animals showed considerable (> 25 %) increases in ear weights (compared to current vehicle values) as indication of excessive ear skin irritation. At the 10 % concentration no relevant signs of local irritation were observed.
- Systemic toxicity: No signs of systemic toxicity were observed in the pretest.
- Ear thickness measurements: ≥ 25 % increase in ear thickness or ear weights compared to historical control values obtained for the selected vehicle.
- Erythema scores: ≥ 3

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-thymidine incorporation, cell count and lymph node weight
- Criteria used to consider a positive response: In 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 dorsal surface of both ears is determined by measuring 3H-thymidine incorporation into the lymph node cells. Additional parameters used to characterize the response are, lymph node cell count and, to a certain extent, lymph node weight. Because irritation by the test substance may also induce lymph node responses, the weights of ear punches taken from the area of test-substance application are determined as an indicator for inflammatory ear swelling due to any irritant action of the test substance. A test item is regarded as a sensitizer in the LLNA if exposure to at least one concentration of the test item results in an incorporation of 3H thymidine at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index (SI ≥ 3.0). However, the biological relevance of the obtained stimulation indices is considered in conjunction with the other assessed end points (i.e. cell counts, lymph node weights, ear weights). Hereby, the thresholds used for assessment of cell counts and ear weights are represented by stimulation indices (SI) of 1.5 and 1.25, respectively.

TREATMENT PREPARATION AND ADMINISTRATION:
The test-substance preparation was 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. The test substance suspension was applicated epicutaneously at the dorsal part of both ears (25 µL each ear) with 3 consecutive applications (day 0 – day 2) to the same application site. On study day five (about 66 to 72 hours after the last application of test substance to the ears) the mice were injected into a tail vein with 20 µCi of 3H-thymidine in 250 µL of sterile saline.
The animals were sacrificed on study day 5 about 5 hours after 3H-thymidine injection by cervical dislocation under Isoflurane anesthesia.
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 animal. 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 animal were stored in phosphate buffered saline (PBS) in an ice-water bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing the lymph nodes through an iron mesh (mesh size 200 µm) into 6 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 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 β-scintillation counter.

Positive control substance(s):

other: A concurrent positive control (reliability check) with a known sensitizer was not included in this study.

Statistics:

3H-thymidine incorporation, cell count, lymph node weight and ear weight: WILCOXON - Test

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

> 3

Test group / Remarks:

3H-thymidine incorporation

Results 2

Key result

Parameter:

EC3

Value:

15.3

Test group / Remarks:

3H thymidine incorporation

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
The increase in the auricular lymph node cell counts at 25 % was statistically significant (SI = 1.39), but failed to reach the cut off for biological relevance (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5.
In addition, there were statistically significant increases in lymph node weights at 25 % (SI = 1.32) and 5 % (SI = 1.44).
The ear weights of all test-substance treated animals were not relevantly elevated as compared to the vehicle control group demonstrating the absence of ear skin irritation.

DETAILS ON STIMULATION INDEX CALCULATION
The stimulation indices of 3H-thymidine incorporation, cell count, lymph node weight and ear weight measurements were calculated as the ratio of the test group mean values for these parameters divided by those of the vehicle control group.

EC3 CALCULATION
the EC (estimated concentration) leading to the respective SI values were calculated by linear or semi-logarithmical regression between the data points directly below and above the SI if possible or using the two nearest points below or above the SI.

CLINICAL OBSERVATIONS:
No signs of systemic toxicity were noticed in all animals during general observation. Red discoloration of the ear skin of the animals treated with all concentrations was noted during the study period. In addition, encrusted compound residues (slight to severe) around the application site were observed in all test-substance treated animals on study day 5. The animals of the vehicle control group showed slight scaling on study day 5.


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

Any other information on results incl. tables

Table 1: Mean stimulation indices (SI) (expressed as multiples of the vehicle control) for 3H-thymidine incorporation, cell count, lymph node weight and ear weight

Test Group	Treatment	3H-thymidine incorporation SI	Cell count SI	Lymph node weight SI	ear weight SI
1	Vehicle (DMF)	1.00	1.00	1.0	1.00
2	1 %	1.037	1.12	1.11	1.00
3	5 %	2.18*	1.22	1.32**	1.08*
4	25 %	3.77**	1.39*	1.44**	1.03

* p ≤ 0.05; ** p ≤ 0.01

Applicant's summary and conclusion

Interpretation of results:

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