Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
06 Oct 2017 to 16 Mar 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
revised July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
Final Guideline (March 2003)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
reduced LLNA

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
White powder

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
Test Animals
Female CBA/J mice were received from Jackson Laboratories (Bar Harbor, ME) on 27 Sep 2017.
Following an acclimation period of at least five days, twenty nulliparous, non-pregnant female mice were assigned to the test groups without conscious bias.
The animals were born on 01 Aug 2017 (±3 days). The Day 1 body weight range for the study animals was 20.3 - 25.3 grams. The weight variation of the animals at study start did not exceed ±20% of the mean body weight. The animals were observed prior to the study start to ensure that no skin lesions were present on the ears.
The animals were identified by cage notation and indelible tail marks. During the acclimation period the animals were housed two per cage in suspended wire-bottom cages; during the study the animals were housed one per cage. Bedding was placed beneath the cages and changed at least three times per week. Fresh PMI Rodent Chow (Diet No. 5001) and water were available ad libitum. The animal room, reserved exclusively for mice on acute tests, was temperature-controlled, had a 12-hour light/dark cycle, and was kept clean and vermin free. Temperature and humidity were continuously monitored using automatic recording devices.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
25% (w/v) in DMF
No. of animals per dose:
5 animals per dose
Details on study design:
Dosing
Groups of five CBA/J mice were treated by a topical application of a single test article concentration, vehicle control (DMF) or positive control (25% HCA) to the dorsum of each ear once daily for three consecutive days. The test substance was spread over the entire dorsal surface of the ear using a micropipette to deliver 25 μl/ear. The test article dose was 12.5% (w/v) for test article L-001739764-000M020 and 25% (w/v) for test article L-006266209-000K003, based on solubility. The moderate sensitizer (and irritant) 25% HCA was tested as a positive control.

Dose Groups
Treatment Animal I.D.
DMF (Vehicle Control) 1-5
25% HCA (Positive Control) 6-10
12.5% (w/v) L-001739764-000M020 11-15
25% (w/v) L-006266209-000K003 16-20

Type and Frequency of Observations
All animals in the study were observed once daily throughout the study for clinical signs, either of local irritation at the application site or systemic toxicity, and for mortality.

Measurements
Body weights were recorded immediately prior to dosing on Day 1 and prior to euthanasia on Day 6. Ear thickness measurements were performed on Day 1 prior to dosing, on Day 3 before the third test article application (approximately 48 hours after the first test article application), and on Day 6 before euthanasia (approximately 120 hours after the first dose). Changes in ear thickness on Day 3 and Day 6 relative to Day 1 were expressed as a percent of the Day 1 pre-dose values. Ear thickness increases of 25% or more were considered biologically significant (based on the scientific literature and historical laboratory data) and deemed indicative of a greater than moderate local dermal irritation response.

Lymph Node Isolation and Processing
On Day 6, approximately 120 hours following the initial dose, and approximately five hours prior to euthanasia, the mice were injected with BrdU in Dulbecco's phosphate-buffered saline (DPBS) at a dose of 200 μl per mouse (15 mg/ml). The BrdU solution was administered by intraperitoneal injection. This thymidine analog becomes incorporated into the DNA of proliferating cells, including proliferating nodal lymphocytes. Each mouse was euthanized using CO2 asphyxiation, and the jugular vein was opened for complete exsanguination. Gross observations of the auricular lymph nodes were made, and the lymph nodes were collected. The auricular lymph nodes were combined for each animal and single-cell suspensions were generated in RPMI-10 medium. An aliquot of each cell suspension was reserved for immunophenotyping analysis; the remaining cell suspensions were fixed with 85% ethanol. The live, unfixed cell suspensions aliquots were used to determine BrdU incorporation into the lymphocyte DNA (percentage of proliferating BrdU+ Lymph Node Cells [%BrdU+ LNC]) and the total number of cells in the nodes, for each individual animal.

Flow Cytometry
Flow cytometric analyses were conducted using a FACScan flow cytometer (Becton Dickinson, San Jose, CA) equipped with an Omnichrome argon laser emitting at 488 nm with 15 mW of power. For DNA and/or BrdU determinations, debris and clumps of nuclei were excluded from analysis using gates set on integrated red fluorescence signals. The histograms generated in these experiments were analyzed using Cell Quest Software (Becton Dickinson Immunocytometry Systems, San Jose, CA).

Determination of Stimulation Index
Proliferation of Lymphocytes (#BrdU+ cells): Measured aliquots of fixed cells were washed and resuspended in 1 N HCl containing 0.5% Triton® X-100. This acid denaturation step allows the BrdU antibody to quantitatively interact with BrdU that has been incorporated into the cellular DNA. Following a one-hour denaturation period, the samples were neutralized by washing with sodium tetraborate. The cell nuclei were then washed with a staining buffer [1.0% bovine serum albumin, 0.03% sodium azide and 0.5% Tween®20 in phosphate-buffered saline (PBS)] and incubated with the BrdU-specific (fluoresceinconjugated) antibody (Becton Dickinson Immunocytometry Systems, San Jose, CA 95131, USA). The nuclei were again washed with the staining buffer, resuspended in DPBS containing the DNA-specific dye propidium iodide and the percentage of nuclei staining positive for BrdU (i.e., proliferating cells) was determined using flow cytometry.
Cell Number Determination: To determine total number of cells in the nodes for each animal (#LNC), a measured aliquot of fixed cells was transferred to a tube containing propidium iodide, papain and Tween®20. The number of cells in each aliquot (representing a 1/500 dilution of the LNC suspension) was determined by flow cytometry.
The total number of cells in the lymph nodes was determined by sampling an aliquot that represented
1/500 (dilution factor = 500) of the total number of cells in the nodes as follows:
(Mean No. of Cells in Aliquot) X (Dilution Factor) = Total No. of Cells in Lymph Nodes (#LNC)
To determine the number of BrdU+ cells in the lymph nodes, the total number of LNCs counted in an aliquot of the cell suspension was multiplied by the percentage of LNCs that incorporated BrdU as follows:
(Total #LNC in Node) X [(%BrdU+ Cells)/100%] = No. Proliferating Lymphocytes (#BrdU+ LNC)
The mean values and standard deviations (SD) were calculated from individual animal data for each group.

Analysis of Stimulation Index Data
Calculation of Stimulation Index (SI): For each animal, lymph node cell proliferation as measured by the number of proliferating lymphocytes was determined by flow cytometry and the mean ± S.D. was then calculated for each group. The number of proliferating lymphocytes in each animal was then divided by the mean number of proliferating lymphocytes in the vehicle control group. This “Test / Control Ratio” is the “Stimulation Index” (SI) and was calculated for each animal as follows:
Total #BrdU+ LNC; Proliferating Lymphocytes per animal = Stimulation Index (SI)
Mean #BrdU+ LNC, Proliferating Lymphocytes of Vehicle Control group per animal
The mean SI and S.D. were calculated for each group.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
For each test article-treated group, the individual animal SI values along with the mean group SI and standard deviation were calculated, and ANOVA followed by the Students’ t-Test was performed to statistically compare each test article dose group to the vehicle control group. Although specified in the test guidelines, these calculations and results were not incorporated into the interpretation of the data. An SI value of 3.0 or more is the sole determinant for a positive sensitization response.

Results and discussion

Positive control results:
Valid

In vivo (LLNA)

Results
Parameter:
SI
Value:
4.1
Variability:
0.9
Test group / Remarks:
SI ≥ 3 indicates a sensitizing response
Remarks on result:
positive indication of skin sensitisation based on QSAR/QSPR prediction
Cellular proliferation data / Observations:
Body weight losses were noted but were not considered significant (less than 2 grams).

Mortality and Systemic Observations
All animals survived the in-life phase of the study and were observed to be normal. Prior to the study start, all animals were observed to ensure that no skin lesions were present on the ears.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Topical application of test article L-006266209-000K003 at 25% (w/v) in DMF resulted in an SI value greater than 3.0. Therefore, this test article is a dermal sensitizer in the Screening Local Lymph Node Assay.