Diphosphoric acid, zinc salt, compd. with 1,3,5-triazine-2,4,6-triamine (1:1:2)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to OECD guidelines and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

Study pre-dates OECD 439 guideline, but is considered to be equivalent

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 1,3,5-triazine-2,4,6-triamine and zinc bis(dihydrogen phosphate)

EC Number:

690-512-6

Cas Number:

1271172-98-5

Molecular formula:

Zn(C3H7N6)2(P2O7)

IUPAC Name:

Reaction products of 1,3,5-triazine-2,4,6-triamine and zinc bis(dihydrogen phosphate)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals

Species:

other: EpiDerm tissue, human derived

Details on test animals or test system and environmental conditions:

ENVIRONMENTAL CONDITIONS
All incubations, were carried out in a controlled environment.
- Atmosphere containing 5.0 CO2 in air in the dark.
- Temperature: 37.0°C

Test system

Vehicle:

other: DPBS-buffer

Controls:

yes

Amount / concentration applied:

In the main test, 27.7, 27.5 and 24.3 mg were applied. Initially, the pre-incubated tissues were wetted with 25 uL of DPBS buffer before applying the test substance, which was evenly spread to match the tissue size.

Duration of treatment / exposure:

35 minutes

Details on study design:

Test system:
Name: Commercially available Epi-200-SIT-kit.
Source: MatTek corporation, Ashland, USA.
Rationale: One of the validated in vitro skin irritation tests is the EpiDerm test, which is recommended in international guidelines (e.g. OECD, EC and
validated by ECVAM).

Pre-test:
The test item was tested for forming a colour without MTT addition. This pre-test provided a colourless solution. Afterwards the test item was tested for the ability of direct formazan reduction. The MTT solution (with test material) didn't change its colour within one hour, which meant that direct MTT reduction had not occured.

Treatment:
All tissues used were pre-incubated; eight 6-well plates were prepared with 0.9 mL assay medium in 3 of the 6 wells. The tissues were inspected for viability. Viable tissues were transferred in the wells with the medium using sterile forceps in place in an incubator for 1 hour. After this period another three wells of each plate were filled with medium and then further incubated for 18 hours.

The pre-incubated tissues were placed into fresh 6-well plates containing 0.9 mL medium per well. Seperate plates were used for the negative, positive and treatment groups using 30 uL DPBS buffer, 30 uL SDS solution or 25 uL (before applying the test item) respectively. Tissues were dosed in 1 minute intervals. After dosing the last tissue, all plates were transferred into an incubator for 35 minutes. Tissues were then rinsed, dried, fresh medium added and further incubated for 24 hours (excess test material was removed).

The tissue inserts were then transferred into a new 6-well plate and incubated for a further 18 hours (post-incubation).

Cell viability measurement (MTT assay):
After the incubation period (a total of ca. 42 hours) a 24-well plate was prepared with 300 uL freshly prepared MTT-reagent in each well. The tissues were blotted onto the bottom and then transferred. The 24-well plate was placed in the incubator for 3 hours. After this time the MTT reagent was aspirated and replaced by PBS buffer. this was replaced several times. Each insert was throughly dried and set into the empty pre-warmed 24-well plate. Into each well, 2 mL isopropanol were added and shaken for 2 hours at room temperature. After two hours, the insertes in which formazan had been produced were pierced with an injection needle, extracting all the colour. the inserts were then discarded and the contents mixed to ensure homogenisation. Frome ach well, two replicates with 200 uL were added into a 96-well plate which was read in a plate spectral photometer at 570 nm.

Formazan production was calculated as a % photometric absorption compared with the negative control.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

94.4

Vehicle controls validity:

valid

Positive controls validity:

valid

In vivo

Irritant / corrosive response data:

See below for mean tissue viability scores ('Any other information on results incl. tables').

After treatment, the relative absorbance values were decreased to 94.4%, which is above the threshold for irritation (50%). The OD of the negative control was within the required acceptability criteria of 1 < mean OD < 2.5 (2.156) as cited in the report, and also with the range in the most recent OECD guideline ( 0.8 < mean OD < 2.8).

Any other information on results incl. tables

Mean tissue viability score in the in vitro skin irritation test (mean of three replicates):

	Mean tissue viability (percentage of control)
Negative control	100
Test substance	94.4
Positive control	7.3

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substance is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.