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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
07 December 2018 - 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Colour: light yellow
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: SUQIAN UNITECH CO., LTD; 2018041002
- Purity test date: 99.29%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light
- Stability under test conditions: stable at room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:

FORM AS APPLIED IN THE TEST
All test item solutions were freshly prepared immediately prior to use.
The test item was dissolved in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity ≥99%; AppliChem; Lot No.: 0001179895; 0001336139). A stock solution of 200 mM was prepared by preweighing the test material into a glass vial. Vortex mixing was used to aid solubilisation. Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent. These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo, 5800 AN Venray, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: The animals were derived from a controlled full-barrier maintained breeding system (SPF).
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 17.6-20.4 g
- Housing: Full barrier in an air-conditioned room; the animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding.
- Diet: Free access to Altromin 1324 maintenance diet for rats and mice
- Water: Free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: at last 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): at least 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
Pre-screen test: 25% and 50% (w/v)
Main study: 0, 6.25%, 12.5% and 25% (w/v)
No. of animals per dose:
Pre-screen test: 2 mice per dose
Main study: 5 mice per dose
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The maximum technically applicable concentration of the test item in the vehicle was found to be 50% (w/v) in DMSO. Two animals were treated with a test item concentration of 50% (diluted with DMSO). Two animals were treated with a test item concentration of 25% (diluted with DMSO).

- Irritation: Sticky fur was observed at both application sites of each animal treated with the test item from day 3 until day 5. Furthermore, it was noted that the test item at the concentrations of 25% and 50% in DMSO had a very intensive smell.

- Systemic toxicity: Cageside observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge). The animals treated with the test item at a concentration of 50% in DMSO showed moderately reduced spontaneous activity on day 2 and slightly reduced spontaneous activity on day 3. The animals treated with the test item at a concentration of 25% in DMSO showed slightly reduced spontaneous activity on day 2 and 3. All animals showed the expected weight development, which allows for a weight loss of up to 2 g throughout the duration of the prescreen test.

- Ear thickness measurements: Ear thickness increased in all dose groups at Day 6

The test item concentration 25% in DMSO was selected as the maximum dose level to use in the main test. According to OECD guideline 429, marked change in activity level might indicate systemic toxicity. Therefore, the test item concentration 50% in DMSO was excluded as both treated animals showed moderately reduced spontaneous activity during the prescreen test.

Refer to Tables 2-4.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).

TREATMENT PREPARATION AND ADMINISTRATION:
Topical Application
Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear. Topical applications were performed once daily over three consecutive days. The first treatment day is defined as study day 1.

Administration of 3H-Methyl Thymidine
Five days after the first topical application all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250 µL of 3H-methyl thymidine, diluted with PBS to a working concentration of 80µCi/mL.

Preparation of Cell Suspension
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4° C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.

Determination of Incorporated 3H -Methyl Thymidine
The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.
Positive control substance(s):
other: 1% phenylenediamine; a shared positive control was performed concomitantly, using 5 animals.

Results and discussion

Positive control results:
The positive-control substance exceeded the stimulation index of 3 confirming the reliability of the test system (10.4 ± 2; Table 7).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
14
Test group / Remarks:
6.25%
Remarks on result:
not determinable
Parameter:
SI
Value:
18.8
Test group / Remarks:
12.5%
Remarks on result:
not determinable
Parameter:
SI
Value:
12.6
Test group / Remarks:
25%
Remarks on result:
not determinable
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA/DETAILS ON STIMULATION INDEX CALCULATION/EC3 CALCULATION

All of the three tested concentrations of the test item exceeded the stimulation index of 3. The stimulation index at a concentration of 6.25% was 14.0 The stimulation index at a concentration of 12.5% was 18.8 The stimulation index at a concentration of 25% was 12.6 (Table 5). As an excessive local skin irritation by the test item cannot be excluded (see clinical observations below), the data were considered as not conclusive.

CLINICAL OBSERVATIONS: All animals survived throughout the test period without showing any systemic effects. However, significant local effects were recorded on day 6 in all animals treated with the test item. At both application sites signs of alopecia, hardening of the ears, oedema and crust were observed. During excision of the draining auricular lymph nodes it was noted that the ear tissue of all animals treated with the test item at a concentration of 12.5% and 25% was necrotic, detached from surrounding skin tissue and undermined with pus-like discharge (Table 8)

BODY WEIGHTS: All animals showed the expected weight development, which allows for a weight loss of up to 2 g throughout the study (Table 6).

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
Under the conditions of the present study the sensitising properties of the test item 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine could not be fully determined. With the significant local effects observed, a relevant impact on the test results cannot be excluded. Therefore, the results obtained are considered as inconclusive and further testing is required.
Executive summary:

In a dermal sensitization study (188590) with 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (99.29%), young adult CBA/Ca/Ola/Hsd mice (5 females) were tested in the Local Lymph Node Assay. The doses tested were 0, 6.25, 12.5 and 25% (w/v) in DMSO, based on the results of the pre-screen test (25 and 50% (w/v)).The reliability of the test system was confirmed by a positive control test with 1% phenylenediamine in DMSO that was performed concomitantly, using 5 animals.

The positive control, 1% phenylenediamine, gave the appropriate response (stimulation index 10.4 ± 2). All animals survived throughout the test period without showing any systemic effects. However, significant local effects were recorded on day 6 in all animals treated with the test item. At both application sites signs of alopecia, hardening of the ears, oedema and crust were observed. During excision of the draining auricular lymph nodes it was noted that the ear tissue of all animals treated with the test item at a concentration of 12.5% and 25% was necrotic, detached from surrounding skin tissue and undermined with pus-like discharge. All animals showed the expected weight development, which allows for a weight loss of up to 2 g throughout the study. Treatment with 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine at 6.25, 12.5 and 25% (w/v) in DMSO resulted in stimulation indices of 14, 18.8 and 12.6, respectively. As an excessive local skin irritation by the test item cannot be excluded, an EC3 value could not be derived and the data were considered as not conclusive; further testing is required.