1,3,5-Triazine-2,4-diamine, N2-[(1R,2S)-2,3-dihydro-2,6-dimethyl-1H-inden-1-yl]-6-(1-fluoroethyl)-

Endpoint summary

Description of key information

LLNA, OECD 429, mouse: not sensitising up to 100% (w/v) in DMF

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

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: R. Janvier, Le Genest St Isle, France
- Age at study initiation: at least 8 weeks
- Weight at study initiation: 20.3-25.3 g
- Housing: individually in suspended stainless steel, wire-mesh cages in a room within a barrier maintained unit with restricted entry
- Diet: Certified rodent pellet diet: AO4C-10, S.A.F.E. (Scientific Animal Food and Engineering, Route de Saint Bris, Augy, France), ad libitum
- Water: filtered and softened tap water from municipal water supply, ad libitum
- Acclimation period: at least 5 days before beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

dimethylformamide

Concentration:

0, 10, 25, 50, 100% w/v in DMF

No. of animals per dose:

5

Details on study design:

- Compound solubility: The vehicle Dimethylformamide (DMF) was selected to ensure maximum solubility of the test substance. A homogenous solution was obtained for test and control substances

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Measurement of tritiated thymidine incorporation into proliferating lymph node cells
- Criteria used to consider a positive response: A test substance is regarded as a skin sensitiser if one concentration of the test substance results in an increase of ³H-TdR incorporation of three-fold or greater, when compared to control values in the absence of skin irritation and if there is a dose-related response. The concentration of test material causing a three fold increase (i.e., an SI of 3) is known as the EC3.

TREATMENT PREPARATION AND ADMINISTRATION:
Dosing formulations were prepared by dissolving the test substance in DMF to produce the required dosing concentration (w/v). Each mouse was topically dosed once daily with 25 μL of the formulation using an Eppendorf pipette, to the dorsal surface of each ear. Mice were dosed on Days 0, 1 and 2. There was no run off of the formulation during the application.
The site of application was examined for signs of local irritation. The animals were checked for mortality and clinical signs at least once a day during the study. Individual animal body weights were measured at the start of the test and at the scheduled kill of the animals.

On Day 5, animals were moved to a radio-active suite. Each mouse was placed individually in a retention box, intravenously injected via the tail vein with 250 μL of sodium chloride (0.9%) containing 20 μCi of ³H methyl thymidine and placed in a plastic cage for 5 hours. Five hours after intravenous injection, the mice were sacrificed and the two auricular lymph nodes were removed from each mouse. The nodes from each mouse were placed in an individual tube containing physiological saline and were disaggregated by crushing with a plastic piston. A single cell suspension was obtained, free of connective tissue.

Cell suspensions were washed with 5 mL of 0.9% physiological saline, centrifuged for 20 minutes at 1800 rpm and the pellets obtained were resuspended in 2 mL of 5% trichloroacetic acid (TCA) and stored overnight at approximately +4 °C. After a final centrifugation, the pellets were resuspended in 1 mL of saline, mixed and then placed for 25 mins in an Ultrasonic Bath to ensure a thoroughly dispersed suspension. Once prepared suspensions were added to numbered scintillation pots containing 10 mL of scintillation fluid and assayed in a beta-counter. The results were expressed as disintegrations per minute (DPM) per node. Stimulation Indices (SI) were calculated according to the following formula:

SI = DPM of treated group / DPM of control group

In addition, a stimulation index (SI) was calculated using the absolute DPM value for each mouse as the numerator and the mean DPM value for the vehicle control mice as the denominator.

Positive control substance(s):

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

Statistics:

The results indicate that the mean values are an appropriate parameter for evaluating effects of treatment. Statistical analysis was not considered necessary.

Positive control results:

In the positive control group given 25% alpha-Hexylcinnamaldehyde, an SI value >3 was noted (SI = 3.86). The results from the concurrent positive control substance demonstrate the validity of this assay with a positive response to treatment with alpha-Hexylcinnamaldehyde.

Parameter:

SI

Value:

1.05

Test group / Remarks:

10% test substance

Parameter:

SI

Value:

1.17

Test group / Remarks:

25% test substance

Parameter:

SI

Value:

1.16

Test group / Remarks:

50% test substance

Parameter:

SI

Value:

1.44

Test group / Remarks:

100% test substance

Parameter:

SI

Value:

3.86

Test group / Remarks:

25% alpha-hexylcinnamaldehyde (positive control)

Cellular proliferation data / Observations:

Mean DPM per group:
control: 919.66
10%: 961.69
25%: 1076.42
50%: 1066.81
100%: 1321.87
25% HCA: 3547.80

The test substance was found to be a non-sensitising compound in the Local Lymph Node Assay.

According to the criteria of Directive 67/548/EEC and Regulation (EC) No 1272/2008 the test substance does not have to be classified as sensitising to the skin.

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitising characteristics of the test substance were assessed in a Local Lymph Node Assay according to OECD guideline 429 under GLP-conditions. In this study 5 female CBA mice per dose group were exposed to test substance concentrations of 0, 10, 25, 50 and 100% (w/v) in DMF on three consecutive days; the performance of the assay was ensured by the use of a 25% concentration (w/v) of hexyl cinnamic aldehyde in DMF in a positive control group. The sites of application were assessed daily for signs of local irritation. After 5 days the mice received an intravenous injection of ³H methyl thymidine, 5 hours later the auricular lymph nodes were excised, single cell suspensions prepared, and after several preparation steps the disintegrations per minute (DPM) per node were determined and stimulation indices (SI) were calculated.

The stimulation indices for the 10, 25, 50 and 100% test substance concentrations were determined to be 1.05, 1.17, 1.16 and 1.44, respectively, demonstrating a lack of cell proliferation due to sensitisation in the draining lymph nodes after application of the test substance to the skin; the test substance was found to be a non-sensitising compound in the Local Lymph Node Assay. The SI value of 3.86 noted in the positive control group confirmed the validity of the assay with a positive response to hexyl cinnamic aldehyde.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the criteria of Regulation (EC) No 1272/2008 the test substance does not have to be classified as sensitising to the skin.