Registration Dossier

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: Performed to GLP standards

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Evaluating the skin irritation potential of trisodium 6,6',6"-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoate using the EPISKIN reconstituted human epidermis model after a treatment preiod of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay is to measure cytotoxicity in reconstituted human epidermal cultures following topical exposure to trisodium 6,6',6"-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoate by means of the colourmetric MTT reduction assay.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
other: in vitro test
Strain:
other: in vitro test
Details on test animals and environmental conditions:
Test for direct MTT reduction: 37 C, 5% CO2 in air for 3 hours.
Pre-incubation: 37 C, 5% CO2 in air for at least 24 hours.
Main test: 37 C, 5% CO2 in air for 42 hours.

Test system

Type of coverage:
not specified
Preparation of test site:
other: moistened with sterile distilled water
Vehicle:
unchanged (no vehicle)
Controls:
other:
Amount / concentration applied:
10 +/- 2 mg of trisodium 6,6',6"-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoate was applied to the epidermis surface. No vehicle was used. The epidermis surface had previously been moistened with 5 μl of sterile distilled water to improve contact between the solid test material and the epidermis.
Duration of treatment / exposure:
15 minute exposure period
Number of animals:
No animals involved as this was an in vitro test.
Details on study design:
Pre-test (assessment of direct test material): trisodium 6,6',6"-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoate is checked for the ability to directly reduce MTT. 10 mg of trisodium 6,6',6"-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoate is added to 2 ml of a 0.3 mg/ml MTT solution (v/v) prepared in assay medium. The solution was incubated in the dark at 37 C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control. If the MTT solution containing the test substance turns blue/purple, is it considered reduced.

The epidermis tissues were pre-incubated at 37 C, 5% CO2 in air for at least 24 hours before the main test.

Main test: Triplicate tissues were treated with the test material for an exposure period of 15 minutes. 10 +/- 2 mg of trisodium 6,6',6"-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoate was applied to the epidermis surface. No vehicle was used. The epidermis surface had previously been moistened with 5 μl of sterile distilled water to improve contact between the solid test material and the epidermis. Triplicate tissues, treated with PBS are used as negative controls. To ensure satisfactory contact with the positive control material, the SDS solution was spread over the entire surface of the epidermis using a pipette tip. After 7 minutes contact time, the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder fo the contact period. The plates were kept in a biological safety cabinet at room temperature for 15 +/- 0.5 minutes. At the end of the exposure period, each tissue was rinsed with PBS and placed into maintenance medium and incubated at 37 C, 5% CO2 in air for 42 hours. A MTT assay was carried out with the each tissue and the optical density value was measured for each tissue to determine cell viability.

in vitro controls: sodium dodecyl sulphate used as the positive control material; Dulbecco's phosphate buffered saline used as the negative control material.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: Relative mean % viability
Remarks on result:
other:
Remarks:
Remarks: in vitro test: see remarks on results section below for scores. (migrated information)

In vivo

Irritant / corrosive response data:
For each test material, the relative mean tissue viabilities obtained after the 15 minute treatment followed by the 42 hour post-exposure period were compared to the mean of the negative control treated tissue (See table 1 for results).

Any other information on results incl. tables

Pre-test results: The MTT solution containing trisodium 6,6',6"-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoate did not turn blue/purple indicating that the test material did not directly reduce MTT.

Table 1. Mean Optical density values and % viabilities for the negative control material, positive control material and trisodium 6,6',6"-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoate.

Material OD540 of tissues Mean OD540 of triplicate tissues +/- SD of OD540 Relative individual tissue viability Relative mean % viability +/- SD of % viability
Negative control material 0.745 0.824 0.071 90.4 100 8.5
0.847 102.8
0.881 106.9
Positive control material 0.061 0.06 0.002 7.4 7.3 0.23
0.058 7
0.061 7.4
trisodium 6,6',6"-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoate 0.834 0.881 0.048 101.2 106.9 5.85
0.879 106.7
0.93 112.9

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
The relative mean viability of Trisodium 6,6',6"-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoate was 106.9% after a 15-minute exposure period and therefore is considered to be non-irritant.
Executive summary:

In a primary dermal irritation study reconstituted human epidermis model (EPISKIN) was dermally exposed to 10 +/- 2mg of trisodium 6,6',6"-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoate without a vehicle for 15 minutes.  The EPISKIN was incubated for 42 hours, followed by an MTT test.  Irritation was scored by the method of % cell viability.

 

In this study, trisodium 6,6',6"-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoate is not a dermal irritant.