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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
18 Oct 2016 to 07 Feb 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 guidelineopen allclose all
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)

Test material

Constituent 1
Chemical structure
Reference substance name:
Amines, C10-14-branched and linear alkyl, bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)
EC Number:
285-083-3
EC Name:
Amines, C10-14-branched and linear alkyl, bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)
Cas Number:
85029-58-9
Molecular formula:
C34H24CrN8O6.C10-14H21-29NH2
IUPAC Name:
reaction mass of: branched and linear(C10-C14)ammonium , bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)
Details on test material:
The substance was formerly identified as: 73297-13-9 / 615-953-3 / Chromate(1-), bis[2-[2-[4,5-dihydro-3-methyl-5-(oxo-κO)-1-phenyl-1H-pyrazol-4-yl]diazenyl-κN1]benzoato(2-)-κO]-, hydrogen, compd. with 1-tridecanamine (1:1:1)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of test substance: Orasol Yellow 157
- Test-substance No.: 16/0017-1
- Batch identification: 003-142715
- CAS: 85029-58-9
- Purity: 98.6 area-% (HPLC, 240 nm) 99.0 area-% (HPLC, 430 nm)
- Content: 98.4 g/100 g (titration)
- Identity: Confirmed

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Homogeneity: The test substance was homogeneous by visual inspection.
- Storage stability: The stability under storage conditions over the study period was guaranteed by the sponsor.

ADDITIONAL TEST-SUBSTANCE INFORMATION
- Physical state / color: Solid / brown
- Storage conditions: Room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
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); 18.1 g – 21.9 g
- Housing: singe housed in Polycarbonate cages type MII with mesh wire tops, supplied by BECKER & Co., Castrop-Rauxel, Germany. For enrichment PLEXX mouse tunnel (red, transparent) and nest building material Nestlets NES 3600 (PLEXX b.v.; AB Elst, Netherlands) were provided. Dust-free wooden bedding was used.
- Diet: ad libitum, Kliba mouse/rat maintenance diet “GLP”, supplied by Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum.
- Water: ad libitum
- Acclimation period: at least 5 days before the first test-substance application
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24
- Humidity (%): 30 – 70
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Remarks:
The vehicle was selected because good homogeneity of the preparation was achieved
Concentration:
1%, 2.5% and 5%
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Tested (pre-test) concentrations 10% and 50%
- Compound solubility: tested
- Irritation: Signs of local irritation were recorded on the pre-screen test, at concentrations of 10% or 50% suspensions in the vehicle (DMF).
- Systemic toxicity: No signs of systemic toxicity were observed in the pre screen test.
- Ear thickness measurements: A ≥ 25% increase in ear thickness or ear weights compared to historical control values obtained for the selected vehicle.
- Erythema scores: Severe erythema (Score ≥3)

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: The 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 is 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.

TREATMENT PREPARATION AND ADMINISTRATION:
- Groups of tested animals: 3 treatment groups and 1 vehicle control group, 5 animals per group
- Body weight determination: Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
- Signs and symptoms: Obvious signs of systemic toxicity and/or local inflammation at the application sites were noted for each animal.
- Mortality: A check for moribund and dead animals was made twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.
- Form of administration: Epicutaneous application (simulating dermal contact with the compound which is possible to occur under practical use conditions)
- Application volume: 25 µL per ear
- Site of application: Dorsal part of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
- 3H-thymidine injection: On day 5 of the experiment mice were injected into a tail vain with 20 µCi of 3H-thymidine in 250 µL of sterile saline.

TERMINAL PROCEDURE
- The animals were sacrificed on study day 5 about 5 hours after the 3H-thymidine injection.
- 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 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.
- Measurement of 3H-thymidine incorporation: 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):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
- Parameter: 3H-thymidine incorporation, cell count, lymph node weight and ear weight
- Statistical test: WILCOXON test

Results and discussion

Positive control results:
The application of hexylcinnamaldehyde at concentrations of 1%, 5% and 15% in methyl ethyl ketone resulted in a greater than 3-fold increase in isotope incorporation at the 15 % w/v concentrations. Therefore, hexylcinnamaldehyde was shown to be a skin sensitiser, confirming the validity of the protocol used for the study.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
2.89
Test group / Remarks:
1% in vehicle (DMF vehicle)
Parameter:
SI
Value:
2.54
Test group / Remarks:
2.5 % in vehicle (DMF vehicle)
Parameter:
SI
Value:
4.36
Test group / Remarks:
5% in vehicle (DMF vehicle)
Parameter:
EC3
Value:
3.1
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION AND EC3 VALUE
The threshold concentration for sensitisation induction was > 2.5% and < 5% 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 these concentrations to be 3.1%, while the SI of 1.5 for cell count was > 5%.

EAR WEIGHTS
The test-substance preparations did not cause relevant increases in ear weights demonstrating the absence of ear skin irritation.

BODY WEIGHTS
The expected body weight gain was generally observed in the course of the study.

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

LOCAL FINDINGS
Slight yellowish discoloration of the ear skin was noted in all animals treated with the test substance during the observation period. Slight scaling at the auricular fold was observed in all 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.

Any other information on results incl. tables

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

SD

SI*

1

Vehicle DMF

273.0

178.5

1.00

2

1% in DMF

789.4

369.4

2.89##

3

2.5% in DMF

693.0

298.4

2.54#

4

5% in DMF

1189.5

335.4

4.36##

 

Test group

Treatment

Cell counts (counts/lymph node pair)

 

 

Mean

SD

SI*

1

Vehicle DMF

11466000

2909211

1.00

2

1% in DMF

14161999

4313111

1.24

3

2.5% in DMF

15097200

289785

1.32#

4

5% in DMF

15513000

2445990

1.35#

 

 

Test group

Treatment

Lymph Node Weight (mg/lymph node pair)

 

 

Mean

SD

SI*

1

Vehicle DMF

5.4

0.8

1.00

2

1% in DMF

5.8

0.7

1.07

3

2.5% in DMF

5.6

0.6

1.04

4

5% in DMF

6.5

0.9

1.19

 

 

Test group

Treatment

Ear weight (mg/animal)

 

 

Mean

SD

SI*

1

Vehicle DMF

33.2

2.1

1.00

2

1% in DMF

33.6

1.6

1.01

3

2.5% in DMF

34.2

0.7

1.03

4

5% in DMF

34.8

1.1

1.05

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

# = statistically significant for the value p ≤ 0.05

## = statistically significant for the value p ≤ 0.01

Applicant's summary and conclusion

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