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Diss Factsheets

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Experimental Toxicology and Ecology, BASF SE, 67056 Ludwigshafen, Germany

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3,5-triazine-2,4,6-triamine phosphate
EC Number:
255-449-7
EC Name:
1,3,5-triazine-2,4,6-triamine phosphate
Cas Number:
41583-09-9
Molecular formula:
C3H6N6.xH3O4P
IUPAC Name:
1,3,5-triazine-2,4,6-triamine phosphate
Details on test material:
- Physical state: solid
- Stability under test conditions: stable
- Storage condition of test material: room temperature
- Other: The substance was homogenous by visual inspection

Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 00228CN9
- Expiration date of the lot/batch: January 2, 2014

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

OTHER SPECIFICS: The substance was homogenous by visual inspection

In vitro test system

Test system:
human skin model
Remarks:
EpiDermTM model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPIDERM(TM) 200 kit from MaTek Corp, Ashland MA, USA

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: tissues were incubated in the incubator at 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: washed with sterile PBS once
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Wavelength: 570 nm
- spectrometer: SunriseTM Absorbance Reader

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: freeze-killed tissue
- N. of replicates : 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability is greater than 50 %
- The test substance is considered to be non-irritant to skin if the viability is lower or equal than 50 %


25 μL sterile PBS was applied first. Thereafter, a bulk volume of 25 μL of the test material was applied with a sharp spoon and homogeneously distributed together with the fluid. Control tissues are concurrently applied with 30 μL sterile PBS or 5% SDS (positive control).

The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator. The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. After all tissues had been rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were incubated in the incubator at 37°C for 24 ± 2 hours. After that the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period.

After the postincubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in 2 mL isopropanol for at least 2 hours at room temperature on a plate shaker (ca. 120 rpm). After shaking the isopropanol extract and piercing the tissues, 2 aliquots of each extract per tissue were transferred to a 96-well microtiter plate. The optical density of the extracted formazan complex was determined spectrophotometrically using a filter with a wavelength of 570 nm.




Control samples:
yes, concurrent negative control
Amount/concentration applied:
25 μL applied to each tissue, spread to match tissue size.
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
ca. 46 h
Number of replicates:
3

Test system

Type of coverage:
open

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1st
Value:
90
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
7%
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2nd
Value:
99
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
5%
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

first experiment   Tissue 1 Tissue 2 Tissue 3 mean SD
Negative control mean OD570 nm 2.2577 2.1342 2.1507 2.1808  
viability [% of negative control] 103.5 97.9 98.6 100 3.07
             
melamine phosphate mean OD570 nm 1.9412 * 2.0047 1.9729  
viability [% of negative control] 89 * 91.9 90 2.06
             
5% (w/v) sodium dodecyl sulfate mean OD570 nm 0.1302 0.1507 0.1487 0.1432  
viability [% of negative control] 6 6.9 6.8 7 0.52
"invalid tissue
second experiment   Tissue 1 Tissue 2 Tissue 3 mean SD
Negative control mean OD570 nm 1.8535 2.3 * 2.0768  
viability [% of negative control] 89.3 110.7 * 100 15.2
             
melamine phosphate mean OD570 nm 2.152 1.924 2.1105 2.0622  
viability [% of negative control] 103.6 92.6 101.6 99 5.85
             
5% (w/v) sodium dodecyl sulfate mean OD570 nm 0.1005 0.114 0.1 0.1048  
viability [% of negative control] 4.8 5.5 4.8 5 0.38

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met