Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 June 2018 - 29 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. certificate)

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: 99.29%

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was crushed to a fine powder, using a mortar and a pestle.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
other: DPBS
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The test was carried out with the reconstituted three-dimensional human skin model EpiDerm(MatTek). The certificate of analysis from the EpiDerm™ model is presented in Annex 1.
- Tissue batch number(s): Lot No.: 28623
- Production date: 13 June 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for 35 mins and 25 mins at room temperature
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C for 42 ± 2 h

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 3
- Observable damage in the tissue due to washing: no


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (final concentration)
- Incubation time: 3 h ± 5 min
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm
- Linear OD range of spectrophotometer: 1.0-3.0

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 2.176 ± 0.022, within the acceptable ranges.
- Barrier function: 4.95 hrs, within the acceptable ranges.
- Morphology: appropriate formation of the epidermal barrier, a presence of a function stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination: sterile

NUMBER OF REPLICATE TISSUES: In each time interval three tissues were used per test item, three tissues for the positive control and three tissues for negative control.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- N. of replicates : two killed tissues were treated with 25 mg of the test item (KT) and two killed tissues were left untreated as a control (KU)
- Method of calculation used: Non-specific reduction of MTT (NSMTT) was calculated relative to the negative control of living tissues (NK) according to the following formula: NSMTT [%] = [(ODKT - ODKU)/ODNK] * 100
If the test item is classified as non-irritant and if non-specific MTT reduction is ≤ 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) of the test item treated living tissues TM was corrected according to the following formula: TODTT = ODTM – (ODKT – ODKU)
If non-specific MTT reduction is > 30% relative to the negative control of living epidermis, the test item is considered as incompatible with the test method.

To check the colouring potential of the test item 25 mg of the test item were mixed per 300 μL aqua dest. and per 300 μL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for 60 min. If the test item is classified as non-irritant and colouring is detected by unaided eye-assessment, and the chemical in water and/or isopropanol absorbs light in the range of 570 ± 30 nm, the test item was checked for its tissue-colouring potential.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if the viability after 60 minutes exposure is less than 50%.
- The test substance is considered to be non-irrating to skin if the viability after 60 minutes exposure is greater than or equal to 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS; Gibco, Cat. No. 14040-091, Lot No.: 1838067

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% SDS solution; MatTek, CAS No.: 151-21-3, Lot No.: 040418SVA).
Duration of treatment / exposure:
60 ± 1 min (37 ± 1 °C for 35 mins and 25 mins at room temperature)
Duration of post-treatment incubation (if applicable):
42 ± 2 h
Number of replicates:
3

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
test item
Value:
9.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
positive control
Value:
3.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.
- Colour interference with MTT: The mixture of 25 mg of the test item per 300 μl aqua dest. and per 300 μL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (1.806). (Table 3)
- Acceptance criteria met for positive control: The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.7%).
- Acceptance criteria met for variability between replicate measurements: Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.2% – 1.7%).
- Range of historical values if different from the ones specified in the test guideline: Historical control data (n=40) from 2015-2018 was presented and was within guideline standards (Table 4)

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
In the in vitro skin irritation test using the reconstructed human epidermal model EpiDerm, 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine was irritating.
Executive summary:

In an in vitro skin irritation assay in a human epidermal model EpiDerm (183804), reconstructed human epidermis tissue was exposed to 25 mg of 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (99.29%) for 60±1 minutes (35 minutes at 37±1°C and 25 minutes at room temperature). PBS was used for the negative control and 5% SDS was used for the positive control. After removal of the test substance, tissues were post-incubated for 42±2 hours. After removal of the test substance, tissues were incubated with MTT for 3 hours incubation. The OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

The controls confirmed the validity of the study. The mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (1.806). The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.7%). Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.2% – 1.7%).

The colour of the test substance did not interfere with the endpoint. The test substance is not directly MTT reducing. The average viability of tissues treated by the positive control, 5% SDS, was 3.7 % of the negative control average value. The average viability of tissues treated by the test item, 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine, was 9.4 % of the negative control average value i.e. viability was > 50 %. According to these results, the test substance is irritating.