N,N'-dibutyl-N,N'-bis(1,2,2,6,6-pentamethylpiperidin-4-yl)-6-(pyrrolidin-1-yl)-1,3,5-triazine-2,4-diamine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-dibutyl-N,N'-bis(1,2,2,6,6-pentamethylpiperidin-4-yl)-6-(pyrrolidin-1-yl)-1,3,5-triazine-2,4-diamine

EC Number:

940-393-4

Cas Number:

1702355-94-9

Molecular formula:

C35 H66 N8

IUPAC Name:

N,N'-dibutyl-N,N'-bis(1,2,2,6,6-pentamethylpiperidin-4-yl)-6-(pyrrolidin-1-yl)-1,3,5-triazine-2,4-diamine

Test material form:

solid: bulk

Details on test material:

- State of aggregation: solid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V. Postbus 6174 5960 AD Horst / The Netherlands
- Age at study initiation: 8-10 weeks
- Housing: in groups
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 23-65
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

other: 1% Pluronic

Concentration:

1. 2. 5%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: highest test item concentration, which could be technically used was a 25% suspension in 1% aqueous Pluronic®
- Irritation: Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 25% once daily each on three consecutive days. On days 2 to 6, the animal treated with 10 and 25% test item concentration, respectively showed an erythema of the ear skin (score 1). Both animals showed visible swelling of the ears on day 6 and test item residues on day 1 only. Furthermore, the animal treated with 10% test item concentration showed an ear weight increase of 35.1%. In the animal treated with 25% test item, an increase in ear thickness of 30.4% was recorded on day 6 of the study. Therefore, a second pre-test was performed using test item concentrations of 2 and 5%. On day 2 and 3, the animal treated with 2 and 5% test item concentration, respectively showed an erythema of the ear skin (score 1). No excessive increase in ear thickness and/or ear weights was recorded.

MAIN STUDY
- Criteria used to consider a positive response: A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
• First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
• Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
- Topical application
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 1, 2 and 5% in 1% aqueous Pluronic®. The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (∅ ∼ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).

- Administration of 3H-methyl-thymidine
Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 19.8 μCi of 3H-methyl thymidine (equivalent to 79.2 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

- Determination of incorporated 3HTdR
Approximately five hours after treatment with 3HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.
The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute.

- Determination of Lymph Node Weight and Cell Count
After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.
Furthermore, the lymph node cell count was determined for each animal. For this, the volume of the cell suspensions was adjusted to an equal final volume and vortexed. Subsequently, individual cell counts were determined using a cell counter (CAS®1, Schärfe System). The values obtained were taken down manually.

-Determination of Ear Weights
After the lymph nodes had been excised, both ears (left and right) of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.

- Observations
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: At least once daily from experimental start to necropsy.
Body weights: In the pre-test: prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with 3HTdR.
Ear thickness: In the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6).
Ear weights: In the pre-test and main experiment after sacrifice; biopsy punches were taken from each ear.
Lymph node weights: After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.
Lymph node cell count: The lymph node cell count was determined in aliquots taken from the prepared single cell suspensions of the lymph nodes using a cell counter.
Clinical signs (local / systemic): Clinical signs (local irritation at the application site or systemic toxicity) were recorded at least once daily. Especially the treatment sites were observed carefully.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node cell count, and for the DPM values (group mean DPM ± standard deviation).
A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as a statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05).
The Dean-Dixon-Test was used for detection of possible outliers (performed with Microsoft Excel 2007). However, both biological and statistical significance were considered together.

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Remarks on result:

other: SI control 1 1% 22.50* 2% 33.75* 5% 41.42*

Results 2

Key result

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM per animal: vehicle 1069.1 1% 24052.5 2% 36081.3 5% 44281.1

Any other information on results incl. tables

The EC3 value could not be calculated, since all S.I.´s are above the threshold value of 3.

No deaths occurred during the study period. No signs of systemic toxicity were observed during the study period. On days 3 to 5, the animals treated with a test item concentration of 5% showed an erythema of the ear skin (score 1). Animals treated with 1 and 2% test item concentration did not show any signs of local skin irritation. The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age. The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant and biologically relevant increase in lymph node weights or –cell counts was observed in all test item treated groups in comparison to the vehicle control group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was reported for a positive response. The indices determined for the lymph node cell count of all test item treated groups exceeded this threshold. The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A biologically relevant or statistically significant increase in ear weights was observed in the groups treated with a test item concentration of 2 and 5%. Furthermore, the cut-off value (1.1) of the ear weight index for a positive response regarding ear skin irritation reported for BALB/c mice was exceeded in all test item treated groups. The threshold value of 25% increase in ear weights for excessive local skin irritation mentioned in OECD guideline 429 was exceeded in the group treated with a test item concentration of 5%, indicating excessive local skin irritation in the high dose group.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Based on the above-mentioned findings regarding ear skin irritation, an influence of irritation on lymphocyte proliferation cannot be excluded in the mid and high dose. Nevertheless, on the basis of the present data and the high S.I. of 22.5 obtained in the low dose (1%) without accompanying excessive increase in ear weights, the test item has to be classified as a sensitiser.
The EC3 value could not be calculated, since all obtained SI´s were considerably above the threshold value of 3.
The test item was found to be a skin sensitiser.