Pyrimidine, 2,4-dichloro-5-fluoro-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 July 2016 to 8 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

no

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2,4-dichloro-5-fluoropyrimidine

EC Number:

625-810-7

Cas Number:

2927-71-1

Molecular formula:

C4HCl2FN2

IUPAC Name:

2,4-dichloro-5-fluoropyrimidine

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL- Lot/batch No.of test material: RZ2-H70762-064RADIOLABELLING INFORMATION- Radiochemical purity: not available- Specific activity: 81.8 Ci/mmolSTABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Room temperature, protected from light

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS- Source: Outside vendor- Age at study initiation: 7 to 9 weeks- Weight at study initiation: 15 to 25 g - Housing: Solid bottom cages with bedding and automatic water supply- Diet (e.g. ad libitum): Standard certified commerical laboratory diet- Water (e.g. ad libitum): Municipal tap water- Acclimation period: 7 days- Indication of any skin lesions: All animals with any evidence of disease or physical abnormalities were not be selected for studyENVIRONMENTAL CONDITIONS- Temperature (°C): 20-26- Humidity (%): 30-70- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Phase 1 (Irritation Screen) - Test material was applied at concentrations of 1, 5, 10, 25, and 50% once daily on Days 1, 2, and 3 via dermal application.Phase 2 (main study) - Test material was applied at concentrations of 1, 5, 10 % once daily on Days 1, 2, and 3 via dermal application.

No. of animals per dose:

Phase 1 (Irritation Screen) - 2 animals per concentration of the the test materialPhase 2 (main study) - 5 animals per concentration of the the test material

Details on study design:

PRE-SCREEN TESTS: (Phase 1)- Compound solubility: Yes- Irritation: YesMAIN STUDY Observations for morbidity, mortality, injury, and the availability of food and water were conducted twice daily for all animals. Observations for clinical signs were conducted 2 to 4 hours postdose on days 1 through 3 and daily thereafter for all animals. Body weights were measured and recorded on Days 1 (prior to dosing) and 6 (prior to administration of the cell proliferation marker article for Phase 2 animals). Auricular lymph nodes were harvested approximately 5 hours post [3H]-Methyl Thymidine injection. Cell samples from the harvested lymph nodes were subsequently analyzed for total radioactivity by liquid scintillation counting (LSC) for 5 minutes or 100,000 counts. The raw data were expressed as disintegrations per minute (dpm) per animal and background values were determined.ANIMAL ASSIGNMENT AND TREATMENT- Name of test method: Nurine local lymph node assay- Criteria used to consider a positive response: To cause or elicit skin sensitisation reactions (allergic contact dermatitis)TREATMENT PREPARATION AND ADMINISTRATION: The prepared test material formulation was dispensed on each day of dosing and stored at room temperature. The vehicle, Acetone/Olive Oil (4:1, v:v), was prepared for use on each day of dosing. The test substance was mixed with the appropriate amount of vehicle to achieve concentrations of 1, 5, 10, 25, and 50%. A micropipette was used to apply 25 μL of the test formulation to the outer portion of the ear of the animal. On Day 6, the cell proliferation marker article, [3H]-Methyl Thymidine, was administered to the phase 2 animals via intravenous injection at a dose level of 20 μCi/animal and a dose volume of 0.25 mL/animal.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The mean and standard deviation were calculated for each endpoint by group. For each endpoint, treatment groups were compared to the reference groups using the following analysis: Body weight - Group Pair-wise Comparison (Levene’s/ANOVA-Dunnett’s/Welch’s) Distintegrations per Minute (DPM) - Log Transformation/Group Pair-wise Comparisons

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

ca. 1

Variability:

Standard Deviation: 175.69 dpm

Test group / Remarks:

Vehicle

Results 2

Parameter:

SI

Value:

ca. 13.55

Variability:

Standard Deviation: 1346.02 dpm

Test group / Remarks:

35% HCA

Results 3

Parameter:

SI

Value:

ca. 21.23

Variability:

Standard Deviation: 3490.69 dpm

Test group / Remarks:

Test Substance Concentration 1%

Results 4

Parameter:

SI

Value:

ca. 51.09

Variability:

Standard Deviation: 11852.63 dpm

Test group / Remarks:

Test Substance Concentration 5%

Results 5

Parameter:

SI

Value:

ca. 48.1

Variability:

Standard Deviation: 9867.88 dpm

Test group / Remarks:

Test Substance Concentration 10%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA The cell proliferation marker article, [3H]-Methyl Thymidine, was used as received from the supplier and no adjustments were made for purity. On the day of dosing, the radioactive cell marker was diluted with the appropriate volume of 1X Phosphate Buffered Saline (PBS) to achieve a radioactivity concentration of 80 μCi/mL. The solution was mixed thoroughly and stored at room temperature prior to dosing.DETAILS ON STIMULATION INDEX CALCULATIONIndividual lymph node radioanalysis values, expressed as disintegrations per minute (dpm) per animal were documented. The mean dpm for each group wascalculated, and the Sensitization Index (SI) was calculated as the ratio of the unbiased estimate of the mean of the treated group again the control group. An SI equal to or greater than 3.0 classified the test substance as a potential sensitiser.CLINICAL OBSERVATIONS:There were no significant findings or signs of local irritation and toxicity for animals administered vehicle or 1% test substance during the main study. 3 of 5 animals administered 5% of test mixture were observed with red discoloration of the skin (left and right ear), earliest onset of symptoms occurred on Day 2. All animals administered 10% Compound 2431768 were observed with red discoloration of the skin (left and right ear) in all animals, earliest onset of these symptoms was observed on Day 2 of the study. BODY WEIGHTSThere was no test article-related effect on body weight during the course of this study

Applicant's summary and conclusion

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

Compound 2431768 formulations (1, 5, and, 10%) resulted in an SI ≥3 relative to the vehicle control and therefore, were determined to be sensitisers at all of the tested concentrations in the Murine Local Lymph Node Assay. The positive control resulted in an SI ≥3, confirming the integrity of the assay.

Executive summary:

Skin sensitisation potential of LSN24314768 was investigated using the murine local lymph node assay. Dose response information was used to assign skin sensitisation hazard sub-categories according to UN GHS and EU CLP classification criteria.

The positive control group (35% HCA) resulted in a sensitization index (SI) of 13.55 relative to the vehicle, which is ≥3 confirming the integirty of the assay.

When the 1, 5, and, 10% Compound 2431768 formulations were compared to the vehicle group, the sensitisation indices were 21.23, 51.09, and 48.10, respectively.

Compound 2431768 formulations (1, 5, and, 10%) resulted in an SI ≥3 relative to the vehicle control and therefore, were determined to be sensitisers at all of the tested concentrations in the Murine Local Lymph Node Assay.

Since the estimated concentration of LSN2431768 needed to produce a SI of 3 is <1% i.e. the EC3 is ≤2% this compound will be classified as a skin sensitiser sub-category 1A.