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

10 Jan 2017 to 10 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

Report date:

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, [2,4-dihydro-4-[(2-hydroxy- 4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-); Amines, C10-14-branched and linearalkyl, 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-branchedand 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:

943-144-8

IUPAC Name:

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

Test material form:

solid

Specific details on test material used for the study:

- Name as cited in study report: Orasol Red 335
- Test substance No.: 16/0125-1
- Batch identification: 002-141706
- Content: 96 g/100 g (100 g/100 g minus water content)
- Physical state / colour: Solid / red
- Storage conditions: Room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Remarks:

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: 9 weeks (pretest) / 8 weeks main test (experiment 1 and experiment 2)
- Weight at study initiation: 17.5 g – 20.4 g (pretest) / 17.2 g – 20.7 g (main test – experiment 1) / 16.7 g – 19.8 g (main test – experiment 2)
- Housing: individual housing 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) were provided as enrichment. Dust-free wooden bedding was used.
- Diet: Kliba mouse 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
- Air: fully air-conditioned
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

50%, 10%, 2.5% and 0 % (experiment 1) and 25%, 10%, 5% and 0% (experiment 2) (w/w)

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The 50% preparation was the maximum technically applicable concentration. The test-substance preparations at different concentrations were suspensions in AOO. Stability and homogenicity of the suspensions were confirmed.
- 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
Two main experiments were conducted. Each main experiment of the study used 3 test groups and 1 control group. The second experiment was conducted due to inconsistent and borderline results observed in the first experiment.
- 25 µL was applied to 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 each animal.
- 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.

ADDITIONAL OBSERVATIONS AND MEASUREMENTS
- 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.

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: 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. lymph node 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 that of the vehicle control group. Subsequent statistical analysis was performed according to the WILCOXON - Test

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:

1.44

Test group / Remarks:

5%

Remarks on result:

other: Second experiment

Results 2

Parameter:

SI

Value:

2.15

Test group / Remarks:

10%

Remarks on result:

other: Second experiment

Results 3

Parameter:

SI

Value:

2.44

Test group / Remarks:

25%

Remarks on result:

other: Second experiment

Results 4

Parameter:

SI

Value:

1.35

Test group / Remarks:

2.5%

Remarks on result:

other: First experiment

Results 5

Parameter:

SI

Value:

3.35

Test group / Remarks:

10%

Remarks on result:

other: First experiment (considered false positive)

Results 6

Key result

Parameter:

SI

Value:

2.4

Test group / Remarks:

50%

Remarks on result:

other: First experiment

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
- When applied as 10% preparation in AOO in the first experiment the test substance induced a biologically relevant (just above the cut off Stimulation Index (SI) of 3) and statistically significant but concentration-independent increase of 3H thymidine incorporation into the cells of the auricular lymph nodes. Concomitantly, the 10% 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. The increases of the 50% preparation in 3H thymidine incorporation and lymph node cell counts were statistical significant but without relevance. In addition, the 10% preparation in AOO induced a relevant increase in lymph node weight. Statistically significant increases in lymph node weights were noted after treatment with all concentrations.
- Due to the inconsistent and borderline results at the 10% concentration and no clear concentration response an additional experiment repeating the 10% preparation and testing additional test substance concentrations was conducted.
- When applied as 25%, 10% and 5% preparation in AOO in the 2nd experiment, 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). However, statistically significant increases were noted in 3H thymidine incorporation into the cells in all test groups (5%, 10% and 25%) and in the auricular lymph node cell counts at the 10% concentration. In addition, statistically significant increases in lymph node weights were noted after treatment with all concentrations.

EAR WEIGHTS
The test substance concentrations did not cause increases (SI > 1.25) in ear weights demonstrating the absence of relevant ear skin irritation in both experiments. However, statistical significant increases in ear weights were noted after treatment with the 10% and 50% test-substance preparations during the 1st experiment.

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

CLINICAL OBSERVATIONS
No relevant signs of systemic toxicity were noticed in all animals during general observation. Except for the 2.5% concentration, all test-substance treated animals showed reddish discolored feces during the observation period.

LOCAL FINDINGS
Slight or moderate discoloration (red) of the ear skin was observed in all test-substance treated groups. In addition, compound residues and crust formation were observed after treatment with the 50% concentration.

Any other information on results incl. tables

Pre-screen test

After application of the 10% and 50% test-substance concentrations the animals did not show relevant signs of local irritation as confirmed by the ear weights (compared to current vehicle values) or ear thickness measurements. After treatment with both concentrations the ear skin was slightly to moderately red discolored and moderate compound residues were observed on the ears of the animals applied with the 50% concentration. No relevant signs of systemic toxicity were observed in the pretest. Feces was discolored red and animals were discolored red in both groups during the observation period.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met