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Description of key information

Based on the solubility of the test item, the available in vitro tests are not applicable for the test item and thus an in vivo study according to OECD Guideline 429 was conducted. The test item showed no potential for skin sensitization in the Local Lymph Node Assay.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Nov 2018 - Mar 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The initial in vitro testing showed that due to limited solubility, in vitro assessment is technically impossible. Therefore, the in vivo test according to OECD 429 was performed to support a conclusion for skin sensitisation.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo (formed partially from Harlan in September 2015), 5800 AN Venray, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: spf
- Age at study initiation: 8–9 weeks
- Weight at study initiation: 19 +/- 1 g
- Housing: Full barrier in an air-conditioned room
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least five days
- Indication of any skin lesions: no indication

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): 12/12 h
- IN-LIFE DATES: From: To: day 1 - day 6
Vehicle:
dimethyl sulphoxide
Concentration:
25%, 50% and 100% (w/v), each suspended in DMSO
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The maximum technically applicable concentration of the test item in the vehicle was found to be 100% (w/v) in DMSO
- Irritation: no findings
- Systemic toxicity: no findings
- Ear thickness measurements: no findings
- Erythema scores: no findings

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD 429
- Criteria used to consider a positive response: The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.
EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation {EC3=c+[(3-d)/(b-d)]x(a c)}, between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated.


TREATMENT PREPARATION AND ADMINISTRATION:

Topical Application
Immediately before the first application the thickness of both ears of all animals was measured. Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear. A second measurement of the ear thickness of all animals was carried out approximately 48 hours after the first application.
Topical applications were performed once daily over three consecutive days. The first treatment day is defined as study day 1.
Administration of 3H-Methyl Thymidine
Five days after the first topical application all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250µL of 3H-methyl thymidine, diluted with PBS to a working concentration of 80µCi/mL.
Preparation of Cell Suspension
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. Shortly before sacrificing the thickness of the ears of all animals was measured for a third time. The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4 °C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.
Determination of Incorporated 3H-Methyl Thymidine
The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.
Positive control substance(s):
other: 1% phenylenediamine (Sigma-Aldrich, lot no.: WXBC5926V, expiry date 05/12/2020) in DMSO
Positive control results:
SI = 10.4 +/- 2.0
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
Test Group 25 % in DMSO
Key result
Parameter:
SI
Value:
1.1
Test group / Remarks:
Test Group 50% in DMSO
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
Test Group 100 % in DMSO
Interpretation of results:
GHS criteria not met
Conclusions:
The informtion for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 429. No indication for skin sensisitasion was found under the condictions of this assay.
Endpoint:
skin sensitisation: in chemico
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because the available in vitro test methods are not applicable for the substance and therefore an in vivo skin sensitisation study was conducted
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
The test material is not soluble in the standard solvents recommended by the guideline.
Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because the available in vitro test methods are not applicable for the substance and therefore an in vivo skin sensitisation study was conducted
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
The solubility assessment showed that there is no solvent that is compatible with the assay as requested in the OECD 442D assay. Therefore, the in vitro assay is technically impossible.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the data provided, the test item is not classified and labelled for skin sensitization according to Regulation (EC) No 1272/2008.