Diphosphoric acid, compound with 1,3,5-triazine-2,4,6-triamine (1:2)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-04-05 to 2017-12-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

July 28, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Behörde für Gesundheit und Verbraucherschutz

Test material

Test material information

Constituent 1

Reference substance name:

Diphosphoric acid, compound with 1,3,5-triazine-2,4,6-triamine (1:2)

EC Number:

236-860-0

EC Name:

Diphosphoric acid, compound with 1,3,5-triazine-2,4,6-triamine (1:2)

Cas Number:

13518-93-9

Molecular formula:

C3H6N6.1/2H4O7P2

IUPAC Name:

diphosphoric acid, compound with 1,3,5-triazine-2,4,6-triamine (1:2)

Test material form:

solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human donors

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: reconstituted three-dimensional human skin model EpiDerm (MatTek)
- Tissue batch number(s): 25829

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: yes
- Wavelength: 540 nm
- Filter: no

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.857 ± 0.107
- Barrier function: 5.4 h
- Contamination: Screened for HIV-1, hepatits B and C, bacteria, yeast, and other fungi. No contaminates were detected.

NUMBER OF REPLICATE TISSUES: triplicate

PREDICTION MODEL / DECISION CRITERIA
The irritant potential was predicted from the relative mean tissue viabilities compared to the negative control. If the mean relative tissue viability is > 50% the test material is not considered an irritant. If the mean relative tissue viability is ≤ 50%, the test material is considered an irritant.

The test meets acceptance criteria if:
- mean absolute OD570 of the three negative control tissues is ≥ 1.0 and ≤ 2.5
- mean relative tissue viability of the three positive control tissues is ≤ 20%
- standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 mg
- Concentration: 25 mg test item were mixed with 300 μL sterile deionised water

NEGATIVE CONTROL
- Amount(s) applied: 30 μL

POSITIVE CONTROL
- Amount(s) applied: 30 μL

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 111

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY: The standard deviation determined for all triplicates was below the limit of acceptance of 18 %. Hence, all acceptance criteria required were fulfilled.

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

Table 1. Summary of Results

	Optical density [OD540] mean tissue (n = 3)	Mean Viability (n = 3) [%]	CV [%]
Dimelamine pyrophosphate	1.550	111.0	1.2
D-PBS (negative control)	1.396	100.0	7.6
5% SDS (positive control)	0.092	6.5	10.9

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

In an in vitro skin irritation human skin model test (EpiSkin) according to OECD guideline 439 and in compliance with GLP, a cell viability of 111% was measured when compared to the untreated control. Therefore, the test item should not be classified.