Reaction mass of sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)

Endpoint summary

Description of key information

In a local lymph node assay (LLNA) according to OECD Guideline 429, the test substance did not show skin sensitising properties (BASF Colors&Effects, 2017).

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:

2016-11-02 to 2017-07-19

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

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2012-07-06

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: N01-131001

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

OTHER SPECIFICS: Solid / dark brown

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 weeks
- Weight at study initiation: 17.6 g – 21.8 g
- Housing: single housed in polycarbonate cages type MII with mesh wire tops
- Diet: Kliba mouse/rat maintenance diet “GLP” (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
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 hours light from 06:00 h to 18:00 h; 12 hours darkness from 18:00 h to 06:00 h

Vehicle:

methyl ethyl ketone

Remarks:

MEK was used as the vehicle because good homogeneity of the preparation was achieved.

Concentration:

5, 10, 25 %

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: 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.
- Systemic toxicity: No signs of systemic toxicity were observed in the pretest.
- Irritation: Excessive local skin irritation was observed after application of the 50 % concentration. At the tested 10 % concentration no relevant signs of local irritation were observed.
- Ear weights measurements: ≥ 25 % increase (50 % concentration)

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- 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.
If applicable, 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.


TREATMENT PREPARATION AND ADMINISTRATION:
Epicutaneous application of 25 µL at the dorsal part of the ear is simulating dermal contact with the compound which is possible to occur under practical use conditions. 3 consecutive applications (day 0 – day 2) to the same application site were performed. 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.

Positive control substance(s):

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

Statistics:

Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. 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.
3H-thymidine incorporation, cell count, lymph node weight and ear weight were analysed via WILCOXON - Test.

Key result

Parameter:

SI

Value:

< 3

Test group / Remarks:

25 %

Key result

Parameter:

SI

Value:

< 3

Test group / Remarks:

10 %

Key result

Parameter:

SI

Value:

< 3

Test group / Remarks:

5 %

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
A biologically relevant and statistically significant response (increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts was observed at the 25 % and 10 % concentrations. The increase at 5 % was statistically significant, but failed to reach the cut off.
The test-substance concentrations did not cause increases (SI > 1.25) in ear weights, demonstrating the absence of relevant ear skin irritation.

DETAILS ON STIMULATION INDEX CALCULATION

EC3 CALCULATION

CLINICAL OBSERVATIONS
Slight or moderate dark brown discoloration of the ear skin was noted during the observation period at all concentrations. No signs of systemic toxicity were noticed in all animals during general observation, but one animal of the 25 % concentration was found dead 3 days after the first application. Dark brown discoloration of feces was noted at all concentrations during the observation period. No macroscopic pathologic abnormalities (Organs without gross lesions) were noted in the animal which was found dead 3 days after the 1st treatment with the 25% concentration.


BODY WEIGHTS
No relevant influence on the body weights was observed in the course of the study.

Table 1: Summary of the stimulation indices

Treatment	3H-thymidine Incorporation Stimulation Index	Cell Count Stimulation Index	Lymph Node Weight Stimulation Index	Ear Weight Stimulation Index
Vehicle Control	1.00	1.00	1.00	1.00
5 % in MEK	0.88	1.29*	1.19*	1.05
10 % in MEK	1.80	1.74**	1.31	1.03
25 % in MEK#	1.44	1.67**	1.33	1.07
* p ≤ 0.05; ** p ≤ 0.01 # Calculation on basis of 4 animals because 1 animal was found dead 3 days after the first application

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitizing potential of the test substance was assessed using the radioactive Murine Local Lymph Node Assay according to OECD Guideline 429.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 each were treated with 5 %, 10 % and 25 % 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 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 animal’s 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.

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.

 

Treatment	3H-thymidine Incorporation Stimulation Index	Cell Count Stimulation Index	Lymph Node Weight Stimulation Index	Ear Weight Stimulation Index
Vehicle Control	1.00	1.00	1.00	1.00
5 % in MEK	0.88	1.29*	1.19*	1.05
10 % in MEK	1.80	1.74**	1.31	1.03
25 % in MEK#	1.44	167**	1.33	1.07
* p ≤ 0.05; ** p ≤ 0.01 # Calculation on basis of 4 animals because 1 animal was found dead 3 days after the first application

 

A statistically significant response in the auricular lymph node cell counts was observed at all concentrations. However, this increase is not considered as biologically relevant, since the increase in lymph cell counts (SI ≥ 1.5) was not reflected in the lymph node weights as well as in the 3H-thymidine incorporation. In addition statistically significant increase in lymph node weights was noted at the 5% concentration. The test-substance concentrations did not cause increases (SI > 1.25) in ear weights, demonstrating the absence of relevant ear skin irritation. 

Thus 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.

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 should not be classified for skin sensitisation under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.