Reaction mass of Amines, C10-14-branched and linear alkyl, [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]chromate(1-) and Amines, C10-14-branched and linear alkyl, bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)]chromate(1-) and Amines, C10-14-branched and linear alkyl, bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]chromate(1-)

Endpoint summary

Description of key information

LLNA according to OECD429: skin sensitiser (category 1B)

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:

31 Jan 2017 to 27 Jul 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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

6 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

- Name as cited in study report: Orasol Black X51
- Test substance No.: 16/0074-1
- Batch: 001-150606
- Purity: ca. 95% can be assumed
- Physical state / color: Solid / black
- Storage conditions: Room temperature

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 and main test,1st experiment) or 8 weeks (main test, 2nd experiment)
- Weight at study initiation: 18.9 g to 20.9 g (pretest) or 15.5 g to 19.8 g (main test, 1st experiment) or 16.7 g to 19.9 g (main test, 2nd experiment)
- Housing: 1 animal per cage in Polycarbonate cages type MII with mesh wire tops, supplied by BECKER & Co., Castrop-Rauxel, Germany. PLEXX mouse tunnel (red, transparent) and nest building material Nestlets NES 3600 (PLEXX b.v.; AB Elst, Netherlands) is used as enrichment. Dust-free wooden bedding was used in this study.
- 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 to 24
- Humidity (%): 30 to 70
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

dimethylformamide

Concentration:

- Experiment 1: 0, 2.5, 10 and 25% (w/w)
- Experiment 2: 0 and 50% (w/w)

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 can be technically used was a 50% test-substance preparation. The test-substance preparations at different concentrations were suspensions in DMF.
- Irritation and systemic toxicity: In order to determine the highest test substance concentration that does not induce local signs of skin irritation and/or systemic toxicity a pretest (experimental conduct in accordance with GLP, but without a GLP status) was performed. Two mice were treated with test substance concentrations of 10% and 50% each on three consecutive days. In the pretest, clinical signs were recorded after each application as well as on day 5. Signs of local irritation were recorded on day 1, 2 and 5. Prior to the first application of the test item (day 0), on day 2 and before sacrifice (day 5) the ear thickness was determined by using a micrometer. Furthermore, the ears were punched after sacrifice at the apical area by using a biopsy punch (Ø 0.8 cm) and were immediately pooled per animal and weighed by using an analytical balance. Additionally, the weight of the pooled lymph nodes from both sides was determined for each animal.
The highest test substance concentration which can be technically used was a 50% test-substance preparation.
No relevant signs of systemic toxicity were observed in the pretest. After application of the 10% and 50% test-substance concentrations the animals did not showed relevant increases in ear weights (compared to current vehicle values) and ear thickness measurements. However, extensive pull out of hair was observed at the head of one animal applied with the 50% concentration. In addition, the ear skin was black discolored in both concentrations and compound residues were observed on the ears of the animals applied with the 50% concentration. Therefore, the following dose levels were selected for the main study: 2.5%, 10%, and 25% in the first experiment.

MAIN STUDY
The study comprised two experiments, the first with three treatment groups and a vehicle control group and the second with one treatment and a vehicle control group. Due to the borderline result obtained for 3H-thymidine incorporation at the 25% concentration, the maximal applicable concentration of 50% was tested in the second experiment.

- 25 µL of the substance preparations was applied on the dorsal part of both ears for 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), 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.
- 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.
- 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.
- 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.
- 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.

OTHER OBSERVATIONS
- Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
- Obvious signs of systemic toxicity and/or local inflammation at the application sites were noted for each animal
- A check for moribund and dead animals was made twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.

EVALUATION CRITERIA
- 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.

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 the ones of the vehicle control group.
- ³H thymidine incorporation, cell count, lymph node weight and ear weigh were statistically analyzed using the WILCOXON Test.
- 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.

Positive control results:

A SI index exceeding 3 was observed at Alpha-Hexylcinnamaldehydeconcentrations of 5 and 15% and shows that the test system is able to detect sensitizing compounds under the test conditions chosen

Parameter:

SI

Value:

1.78

Test group / Remarks:

2.5%

Parameter:

SI

Value:

2.08

Test group / Remarks:

10%

Parameter:

SI

Value:

2.87

Test group / Remarks:

25%

Parameter:

SI

Value:

6.1

Test group / Remarks:

50%

Key result

Parameter:

EC3

Value:

26

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
When applied as 2.5, 10, and 25% suspension in DMF the test substance induced statistically significant and concentration-dependent increases of 3H thymidine incorporation into the cells from the auricular lymph nodes, but the 25% concentration failed to reach the cut off for biologically relevance (increase above the cut off Stimulation Index of 3). When applied as 50% suspension in DMF in a second experiment the test substance induced a statistically significant and biologically relevant increase of 3H thymidine incorporation (increase above the cut off Stimulation Index of 3).
Concomitantly, the 50% test-substance preparation induced 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. In addition, statistically significant increases in lymph node weights were noted at the 25% and 50% concentration.

EC3 CALCULATION
The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H thymidine incorporation and 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 these concentrations to be 26% and 31%, respectively.

CLINICAL OBSERVATIONS:
No relevant signs of systemic toxicity were noticed in all animals during general observation. Black discolored feces were noted in all animals treated with the test substance during the observation period.

IRRITATION AND LOCAL FINDINGS
- Ear weights: The test substance concentrations did not cause increases (SI > 1.25) in ear weights demonstrating the absence of relevant ear skin irritation. However, a statistical significant increase in ear weights was noted after treatment with the 2.5%, 25% and 50% test-substance preparations.
- Local findings: Slight or moderate gray discoloration of the ear skin of the animals treated with the 2.5%, 10% and 25% test-substance preparation were noted during the observation period. Moderate compound residues (black discolored at 50%) were noted on the ear skin of the animals treated with the 25% and 50% test-substance preparation during the observation period. In addition, slight pull out of hair at the head was noted in all animals at the 50% concentration on study day 5. Slight crust formation on the ear skin was noted in 1 animal of the vehicle control group on study day 5.

BODY WEIGHTS
The expected body weight gain was generally observed during the study.

Interpretation of results:

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

LLNA test (OECD429)

The skin sensitisation potential of the test substance has been tested using a Local Lymph Node Assay according to OECD 429 and GLP principles. Test concentrations were determined based on a solubility test and a pre-screen tests. From the solubility test it appeared that the 50% preparation was the maximum technically applicable concentration. In the pre-screen test two mice were treated with test substance concentrations of 10% and 50% each on three consecutive days as a suspension in a vehicle of dimethylformamide (DMF). No relevant signs of systemic toxicity were observed in the pretest. After application of the 10% and 50% test-substance concentrations the animals did not showed relevant increases in ear weights (compared to current vehicle values) and ear thickness measurements. However, extensive pull out of hair was observed at the head of one animal applied with the 50% concentration. In addition, the ear skin was black discoloured in both concentrations and compound residues were observed on the ears of the animals applied with the 50% concentration. Therefore, in the first main study 5 animals were exposed to 0, 2.5 10 and 25% (w/w) test substance. Due to the borderline result obtained for 3H-thymidine incorporation at the 25% concentration, the maximal applicable concentration of 50% was tested in the second experiment (which included a vehicle control as well). When applied as 2.5, 10, and 25% suspension in DMF the test substance induced statistically significant and concentration-dependent increases of 3H thymidine incorporation into the cells from the auricular lymph nodes (SI of 1.78, 2.08 and 2.87, respectively), but the 25% concentration failed to reach the cut off for biologically relevance. When applied as 50% suspension in DMF in a second experiment the test substance induced a statistically significant and biologically relevant increase of 3H thymidine incorporation (SI of 6.1). Concomitantly, the 50% test-substance preparation induced a biologically relevant and statistically significant response in the auricular lymph node cell counts. In addition, statistically significant increases in lymph node weights were noted at the 25% and 50% concentration. No relevant signs of systemic toxicity were noticed in all animals during general observation. Black discoloured faeces were noted in all animals treated with the test substance during the observation period and the test substance did not cause increases in ear weights demonstrating the absence of relevant ear skin irritation. However, a statistical significant increase in ear weights was noted after treatment with the 2.5%, 25% and 50% test-substance preparations. Slight or moderate grey discoloration of the ear skin of the animals treated with the 2.5%, 10% and 25% test-substance preparation were noted during the observation period. Moderate compound residues (black discoloured at 50%) were noted on the ear skin of the animals treated with the 25% and 50% test-substance preparation during the observation period. In addition, slight pull out of hair at the head was noted in all animals at the 50% concentration on study day 5. Slight crust formation on the ear skin was noted in 1 animal of the vehicle control group on study day 5. The expected body weight gain was generally observed during the study. The EC 3 for 3H thymidine incorporation was calculated by linear regression from the results of these concentrations to be 26%. Based on the results, the substance was considered to be a skin sensitizer (BASF 2017).

Justification for classification or non-classification

Based on the available data, the substance should be classified as Skin Sens. 1B (H317: May cause an allergic skin reaction) according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (Regulation (EC) No. 1272/2008 and its updates).