Amines, C12-14-tert-alkyl, bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)

Endpoint summary

Description of key information

The EC3 value for the test substance was estimated to be 0.64% in an OECD 429 study.

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:

18 Oct 2016 to 12 Jan 2017

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:

July 22, 2010

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

6 July 2012

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
- Name of test substance: Orasol Yellow 152
- Batch No.of test material: 001-152602
- Purity test/code: 99.5 area-%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Storage stability: The stability under storage conditions over the study period was guaranteed by the sponsor.
- Solubility and stability of the test substance in the solvent/vehicle: The stability of the test substance in the vehicle was determined indirectly by the concentration control or homogeneity analysis. For this purpose, the samples taken were stored at room temperature over the maximum duration of the application period and were subsequently deep-frozen. Afterwards, these samples were analyzed.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 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.

FORM OF APPLICATION: Suspension

Species:

mouse

Strain:

CBA/Ca

Remarks:

OlaHsd

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: 9 weeks (pretest), 8 weeks (main test)
- Weight at study initiation: 18.7 g – 21.7 g (pretest), 16.7 g – 21.6 g (main test)
- Housing: Polycarbonate cages type MII with mesh wire tops, supplied by BECKER & Co., Castrop-Rauxel, Germany
- 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 before the first test-substance application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24
- Humidity (%): 30 – 70
- Air changes: the animals were housed in fully air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Vehicle:

dimethylformamide

Remarks:

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

Concentration:

0%, 1%, 2.5%, 5%

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 test-substance preparations at different concentrations were formulated in DMF. The highest test-substance concentration which was used in the pre-test was a 50% test substance preparation.
- Irritation: Signs of local irritation were recorded on day 1, 2 and 5 with test substance concentrations of 10% and 50%
- Systemic toxicity: no signs of systemic toxicity
- Ear thickness measurements: After application of the 10% and 50% test substance concentrations the animals showed considerable increases in ear thickness and/or ear weights (compared to current vehicle values).
- Erythema scores: no erythema of the ear skin was observed; at both concentrations, 10% and 50%, the ear skin was slightly yellow discolored.

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION
- Topical application: 3 consecutive epicutaneous applications (day 0 – day 2) of 25 µL were applied to the dorsal part of both ears of each animal.
- Administration of 3H-methyl-thymidine: 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.

DETERMINATION OF EAR WEIGHT, LYMPH NODE WEIGHT AND CELL COUNT
- 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.

DETERMINATION OF INCORPORATED 3HTDR
- 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).
- 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.

EVALUATION OF RESULTS
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.

CRITERIA USED TO CONSIDER A POSITIVE RESPONSE
A test item is regarded as a sensitiser 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).
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.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

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 (for the statistical analyses the statistical test WILCOXON was used).

Key result

Parameter:

EC3

Value:

0.64

Remarks on result:

other: 3H-thymidine incorporation

Cellular proliferation data / Observations:

No signs of systemic toxicity were noticed in all animals during general observation.

When applied as 5%, 2.5% and 1% preparations 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 in the auricular lymph node cell counts was biologically relevant (increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) at 5% and 2.5%. The increases were statistically significant at all concentrations.

In addition, there were statistically significant increases in lymph node weights at all concentrations.

The test-substance preparations did not cause relevant increases in ear weights demonstrating the absence of ear skin irritation. The increases in ear weights of the 5% and 2.5% concentrations were statistically significant.
Slight or moderate yellowish discoloration of the ear skin was noted in all animals treated with the test substance during the observation period. Slight or moderate scaling at the auricular fold was observed in the animals at all concentrations on study day 5. Moderate crust formation on the ear skin or slight scaling at the auricular fold was noted in 1 animal each of the vehicle control group on study day 5.

The threshold concentration for sensitization induction was < 1% for 3H thymidine incorporation. The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H thymidine incorporation was calculated by semi-logarithmical regression from the results of all concentrations to be 0.64%. The EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by linear regression from the results of the 1% and 2.5% concentrations to be 1.6%.

MAIN TEST

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

Test Group	Treatment	^3H-thymidine incorporation [DPM/Lymph Node Pair]
		Mean	S.D.	Stimulation Index^1
1	vehicle DMF	273.0	180.9	1.00
2	1% in DMF	1,182.0	286.7	4.33 ##
3	2.5% in DMF	1.383.2	736.3	5.07 #
4	5% in DMF	2,160.6	519.7	7.92 ##

Test Group	Treatment	Cell Counts [Count/Lymph Node Pair]
		Mean	S.D.	Stimulation Index^1
1	vehicle DMF	11,466,000	2,909,211	1.00
2	1% in DMF	15,376,800	1,904,688	1.34 #
3	2.5% in DMF	20,185,200	3,029,909	1.76 ##
4	5% in DMF	23,076,600	2,139,425	2.01 ##

Test Group	Treatment	Ear Weight [mg/Lymph Node Pair]
		Mean	S.D.	Stimulation Index^1
1	vehicle DMF	5.4	0.8	1.00
2	1% in DMF	6.5	0.8	1.02
3	2.5% in DMF	7.1	1.0	1.11 #
4	5% in DMF	8.7	0.8	1.15 ##

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

Test Group	Treatment	Ear Weight [mg/animal]
		Mean	S.D.	Stimulation Index^1
1	Vehicle DMF	33.2	2.1	1.00
2	1% in DMF	33.8	1.8	1.02
3	2.5% in DMF	36.9	2.9	1.11#
4	5% in DMF	38.2	1.8	1.15 ##

1 test group x / test group 1 (vehicle control)

# = statistically significant for the value p≤0.05

## = statistically significant for the value p≤0.01

Interpretation of results:

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The potency to induce skin sensitisation was assessed in a study according to OECD 429 and in compliance with GLP criteria. In this study five female CBA/CaOlaHsd mice each were treated with 1%, 2.5% and 5% w/w preparations of the test substance in N,N-Dimethylformamide (DMF) or with the vehicle alone. The high concentration was selected based on the presence of ear skin irritation in a pretest using a 10% or 50% preparation in DMF. 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 0.8cm 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 5%, 2.5% and 1% preparations 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 in the auricular lymph node cell counts was biologically relevant (increase to 1.5 fold or above of control value = stimulation index (SI)≥1.5) at 5% and 2.5%. The increases were statistically significant at all concentrations. In addition, there were statistically significant increases in lymph node weights at all concentrations. The test-substance preparations did not cause relevant increases in ear weights demonstrating the absence of ear skin irritation. The increases in ear weights of the 5% and 2.5% concentrations were statistically significant.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data, the test substance has to be classified as skin sensitising Category 1A, H317: May cause an allergic skin reaction according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.