Reaction mass of Amines, C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-) (1:1) and Amines,C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Jan 2017 to 17 Aug 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

July 22, 2010

Deviations:

no

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Amines, C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-) (1:1) and Amines,C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-)

EC Number:

943-145-3

IUPAC Name:

Reaction mass of Amines, C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-) (1:1) and Amines,C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)] chromate(1-)

Specific details on test material used for the study:

Name as cited in study report: Orasol Red 395
Test substance No.: 16/0124-1
Batch identification: 001-152202
EC No.: 943-145-3
Purity: 97.1% (HPLC, 242 nm) or 98.3% (HPLC, 272 nm) main component
Content: 96.5 g/100 g
Homogeneity: The test substance was homogeneous by visual inspection.
Physical state: Red to brown solid
Storage conditions: Room temperature

In vivo test system

Test animals

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)
- Weight at study initiation: 18.7 g to 20.2 g (pretest) and 16.1 g to 20.4 g (main test)
- Housing: Single housing in polycarbonate cages type MII with mesh wire tops supplied by BECKER & Co., Castrop-Rauxel, Germany with PLEXX mouse tunnel (red, transparent) and nest building material Nestlets NES 3600 (PLEXX b.v.; AB Elst, Netherlands) as enrichment.
- 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

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0, 10, 25, 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.
- 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.

MAIN STUDY
- 25 µL 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.

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.

Results and discussion

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

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.52

Test group / Remarks:

10%

Results 2

Parameter:

SI

Value:

3.26

Test group / Remarks:

25%

Results 3

Parameter:

SI

Value:

3.83

Test group / Remarks:

50%

Results 4

Key result

Parameter:

EC3

Value:

22.8

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
- When applied as 25% and 50% preparations in DMF, the test substance induced a biologically relevant (increase above the cut off Stimulation Index (SI) of 3) and statistically significant increase of ³H thymidine incorporation into the cells of the auricular lymph nodes. The increase at the 10% concentration was statistically significant.
- However, none of the tested concentrations induced a biologically relevant response (no increase to 1.5-fold or above of control value = SI ≥ 1.5) in the auricular lymph node cell counts. The increase at the 25% concentration was statistically significant.
- In addition, statistically significant increases in lymph node weights were noted at the 25% and 50% concentration.

EC3 CALCULATION
The threshold concentration for sensitization induction was ca. 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 the 10% and 25% concentrations to be 22.8%.

CLINICAL OBSERVATIONS:
No relevant signs of systemic toxicity were noticed in all animals during general observation. Red discoloration of feces was noted in all animals of all concentrations and red discoloration of urine was observed at the 25% and 50% concentration during the observation period.

IRRITATION AND LOCAL FINDINGS
- The test-substance concentrations did not cause relevant increases (SI > 1.25) in ear weights demonstrating the absence of excessive ear skin irritation. However, statistically significant increases in ear weights were noted in all concentrations.
- Slight or moderate red discoloration of the ear skin was noted at all concentrations during the whole observation period. At the 25% and 50% concentrations, slight compound residues were observed in all animals and slight pull out of hair at the head was noted at the 50% concentration on study day 2 and 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.

Any other information on results incl. tables

Pre-screen

- No obvious signs of systemic toxicity were observed in the pre-test. Considerable reduction in body weight was noted in one animal treated with the 50% concentration. However, it remained unclear, if the body weight loss could be attributed to the test-substance application.

- After application of the 10% and 50% test-substance concentrations the animals did not show relevant increases in ear weights (compared to current vehicle values) or ear thickness measurements. At both concentrations, the ear skin was red discoloured and compound residues were observed on the ears of the animals applied with the 50% concentration.

Applicant's summary and conclusion

Interpretation of results:

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