Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10th October 2017 to 9th November 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:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: “The Murine Local Lymph Node Assay: A Test Method for Assessing the Allergic Contact Dermatitis Potential of Chemicals/Compounds”, NIH No. 99-4494, 1999
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
5-[4-Methoxy-2-(propan-2-yl)phenoxy]pyrimidine-2,4-diamine
Cas Number:
865304-71-8
Molecular formula:
C14H18N4O2
IUPAC Name:
5-[4-Methoxy-2-(propan-2-yl)phenoxy]pyrimidine-2,4-diamine
Test material form:
solid: crystalline
Details on test material:

Off-white

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
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:
125 mg of the test article were brought to a total volume of 1000 μl with DMF and sonicated for approximately 30 minutes to yield a 12.5% (w/v) formulation.
No. of animals per dose:
Five
Details on study design:
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.
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.
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 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 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.
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. 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.
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:
SI = 4.5

In vivo (LLNA)

Results
Key result
Parameter:
SI
Value:
0.8
Test group / Remarks:
12.5% (w/v)
Remarks on result:
not determinable
Cellular proliferation data / Observations:
The total number of proliferating lymphocytes was calculated by multiplying the total cell number by the percentage of the cells that have incorporated BrdU.
12.5% (w/v) L-001739764-000M020
Mean Total No. Cell in Node - 3359850
Mean %BrdU+ LNC - 0.95
Lymphocyte Proliferation (No. BrdU+) - 33737

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Topical application of test article L-001739764-000M020 at 12.5% (w/v) in DMF resulted in an SI value less than 3.0. Therefore, this test article is not a dermal sensitizer in the Screening Local Lymph Node Assay.
Executive summary:

Test article L-001739764-000M020 was tested for solubility at 25% (w/v) in acetone:olive oil (4:1, AOO), dimethyl sulfoxide (DMSO), and N,N-Dimethylformamide (DMF), and at 12.5% (w/v) in DMF, and was found to be soluble only at 12.5% (w/v) in DMF. The Study Director, in consultation with the Sponsor, chose DMF as the vehicle for the study. For each test article, one group of five healthy female CBA/J mice was treated by topical application to the dorsum of each ear, once daily for three consecutive days. A vehicle control group of five mice was treated with DMF, and another group of five mice was treated with the positive control, alpha-hexylcinnamaldehyde in DMF (25% HCA), in the exact same manner.
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. Body weights were measured on Day 1 prior to first dose, Day 3, and Day 6 prior to euthanasia.
The mice were given an intraperitoneal injection of the thymidine analog 5-bromo-2’-deoxy-uridine (BrdU) approximately five hours prior to euthanasia. After euthanasia, the auricular lymph nodes were isolated, single-cell suspensions of lymph node cells (LNC) were generated, and the LNC suspensions were analyzed by flow cytometry for BrdU incorporation and the total number of LNC. The number of proliferating LNC (#BrdU+) was calculated and used as a measure of the proliferative response of the local lymph node. The stimulation index (SI) was calculated by dividing the proliferative response (#BrdU+ LNC) of each test article-treated animal by the mean proliferative response of the vehicle control group. The mean SI and standard deviation (S.D.) was calculated for each group from the individual animal data. If any test article or control article treatment group yielded an SI value of 3.0 or more, then the test article or control article was considered to be a sensitizer.


All animals survived the in-life phase of the study and were observed to be normal. There were no difficulties in administration of the test article or with its adherence to the dosed ears. Body weight losses were noted but were not considered significant (less than 2 grams). Ear thickness measurements and individual animal observations indicated that no test article treatment resulted in more than moderate local dermal irritation. The SI of the positive control group, 25% HCA, was 5.8. The SI of 12.5% (w/v) L-001739764-000M020 was 0.8.