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-)

Endpoint summary

Description of key information

In an in vivo skin sensitising study (LLNA) according to OECD Guideline 429 (BASF Colors&Effects, 2017), the test substance exhibited a skin sensitizing potential.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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

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)

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

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

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

Key result

Parameter:

SI

Value:

> 3

Test group / Remarks:

3H-thymidine incorporation

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.

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

Interpretation of results:

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In a skin sensitising study (radioactive Murine Local Lymph Node Assay) according to OECD Guideline 429 (BASF Colors&Effects, 2017), the skin sensitizing potential of the test substance was assessed. Groups of 5 female CBA/CaOlaHsd mice each were treated with 1 %, 5 %, and 25 % w/w preparations of the test substance in N,N-Dimethylformamid (DMF) or with the vehicle alone. The high concentration was selected based on the presence of excessive ear skin irritation in a pretest using a 50 % preparation in DMF. Each test animal was treated with 25 µL per ear of the appropriate test-substance preparation, applied to the dorsal surfaces 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 application, the mice were injected into the tail vein with 20 µCi of 3H-thymidine in 250 µL of sterile saline. 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 animals pooled lymph nodes were also determined. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation. No signs of systemic toxicity were noticed in all animals during general observation. When applied as 25 % preparation in DMF, the test substance induced a statistically significant increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes, which was above the cut off value for biological relevance (increase above the cut off stimulation index (SI) of 3). The increase of the 5 % test-substance preparation was statistically significant but failed to reach the cut off. The increase in the auricular lymph node cell counts at 25 % was statistically significant, 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)). 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. 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. Thus, 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 > 5 %; < 25 % for 3H thymidine incorporation. 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 15.3 %, while the SI of 1.5 for cell count was > 25 %.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

As a result the substance is classified for skin sensitisation (Category 1B, H317) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.