Hydrogen [3-[[1-[anilinocarbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzene-1-sulphonato(3-)]hydroxychromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

Endpoint summary

Description of key information

Skin sensitisation

In an LLNA according to OECD Guideline 429 in mice, the test substance did not show a skin sensitising potential (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:

2017-03-10 to 2017-08-10

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: 002-152503

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

OTHER SPECIFICS: Solid / orange to brown

Species:

mouse

Strain:

CBA

Remarks:

CBA/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
- Age at study initiation: 8 – 9 weeks
- Weight at study initiation: 17.1 g – 20.6 g
- Housing: single housed 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:

methyl ethyl ketone

Concentration:

0 (vehicle control), 10, 15, 50 % in methyl ethyl ketone

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: MEK was used as the vehicle because good homogeneity of the preparation was achieved.
- Irritation: After application of the 10 % and 50 % test substance 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.
- Systemic toxicity: No signs of systemic toxicity were observed in the pretest.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H thymidine injection
- 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 by using the two nearest points below or above the SI.

TREATMENT PREPARATION AND ADMINISTRATION:
The test-substance preparation was produced on a weight per weight basis shortly before application by stirring with a magnetic stirrer. The homogeneity of the test-substance preparation during application was provided by stirring with a magnetic stirrer. Epicutaneous application is simulating dermal contact with the compound which possibly occurs under practical use conditions. 25 µL per ear of the test substance was applicated onto the dorsal part of both ears. 3 consecutive applications (day 0 – day 2) were performed to the same application site.
On study day five (about 66 to 72 hours after the last application of test substance to the ears), 20 µCi 3H thymidine in 250 µL sterile saline were injected into the tail vein of the mice.
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 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 by 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: not included

Statistics:

Table(s) and/or figure(s) of measured parameters presented in the report were produced by using a PC-based tabular calculation software. The mean and individual data were not always rounded but the significant digits were produced by changing the display format. Consequently, calculation of mean values by using the individual data presented in the report will in some instances yield minor variations in value.
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 the ones of the vehicle control group.
For 3H thymidine incorporation, cell count, lymph node weight and ear weight, a WILCOXON - Test was performed.

Key result

Parameter:

SI

Value:

< 3

Test group / Remarks:

3H thymidien incorporation

Key result

Parameter:

SI

Value:

< 1.5

Test group / Remarks:

lymph node cell count

Cellular proliferation data / Observations:

CLINICAL OBSERVATIONS: No 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.

Table 1: 3H thymidine incorporation, cell count and lymph node weight: test group mean values, standard deviations and stimulation indices 

	3H-thymidine incorporation [DPM/Lymph Node Pair]
Treatment	Mean	SD	Stimulation index*
Vehicle in MEK*	342.9	107.5	1.00
10 %	536.8	285.3	1.57
25 %	695.5	262.5	2.03 #
50 %	648.8	297.1	1.89

 

	Cell Counts [Counts/Lymph Node Pair]
Treatment	Mean	SD	Stimulation index*
Vehicle in MEK*	11192000	500572	1.00
10 %	13621200	2618316	1.22
25 %	14427600	1815243	1.29 #
50 %	13017600	1890193	1.16

 

	Lymph Node Weight [mg/Lymph Node Pair]
Treatment	Mean	SD	Stimulation index*
Vehicle in MEK*	5.8	0.3	1.00
10 %	6.4	0.8	1.09
25 %	6.6	0.2	1.13 #
50 %	6.4	0.6	1.09

 

Table 2: Ear weight: test group mean values, standard deviations and stimulation indices

	Ear Weight [mg/animal]
Treatment	Mean	SD	Stimulation index*
Vehicle in MEK*	32.2	0.6	1.00
10 %	32.1	2.0	1.00
25 %	34.1	2.1	1.06
50 %	36.4	3.7	1.13 #

 

* test group x/test group (vehicle control

# statistically significant for the value p ≤ 0.05

* Calculation on basis of 3 animals due to irregularities during sample processing

 

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In a dermal sensitization study according to OECD guideline 429 with the test substance in methyl ethyl ketone (10, 25, and 50 %), female CBA/CaOlaHsd mice were tested (BASF Colors&Effects, 2017). A concurrent positive control (reliability check) with a known sensitizer was not included in this study. Studies using the positive control substance Alpha-Hexylcinnamaldehyde, techn. 85 % are performed twice a year in the performing laboratory in order to show that the test system is able to detect sensitizing compounds under the test conditions chosen. The response in the draining lymph node after epicutaneous application of the test substance to the skin of the dorsal surface of both ears was determined by measuring 3H-thymidine incorporation into the lymph node cells. Skin sensitisation was initiated by 3 consecutive applications 8day 0 - day 2) to the same application site. No mortality was observed. No relevant signs of systemic toxicity were noticed in all animals during general observation. The expected mean body weight gain was generally observed during the study. 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.

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.

No sensitising properties were observed. As a result the substance is not considered to be classified for skin sensitisation under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.