Adenosine 5'-(trihydrogen diphosphate), 2'-(dihydrogen phosphate), 5'→5'-ester with 3-(aminocarbonyl)-1-β-d-ribofuranosylpyridinium hydroxide, inner salt, disodium salt

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: test conducted under GLP conditions and acc. to OECD guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 439

Principles of method if other than guideline:

In vitro skin irritation: reconstructed human epidermis test method.
The test is based on the experience that irritant chemicals show cytotoxic effects following short term exposure to the stratum corneum of the
epidermis. The test is designed to predict and classify the skin irritation potential of a test substance by assessment of its effect on a three
dimensional human epidermis model (1-10).

The test consists of topical application of b-NADP, Disodium salt on the skin tissue for 15 minutes. After exposure the skin tissue is thoroughly
rinsed to remove the test substance and transferred to fresh medium. After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect is performed.

Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Adenosine 5'-(trihydrogen diphosphate), 2'-(dihydrogen phosphate), 5'→5'-ester with 3-(aminocarbonyl)-1-β-d-ribofuranosylpyridinium hydroxide, inner salt, disodium salt

EC Number:

246-129-8

EC Name:

Adenosine 5'-(trihydrogen diphosphate), 2'-(dihydrogen phosphate), 5'→5'-ester with 3-(aminocarbonyl)-1-β-d-ribofuranosylpyridinium hydroxide, inner salt, disodium salt

Cas Number:

24292-60-2

Molecular formula:

C21H28N7O17P3.2Na

IUPAC Name:

Adenosine 5'-(trihydrogen diphosphate), 2'-(dihydrogen phosphate), 5'→5'-ester with 3-(aminocarbonyl)-1-β-d-ribofuranosylpyridinium hydroxide, inner salt, disodium salt

Details on test material:

- Physical state: white powder
- Storage condition of test material: in refrigerator (2-8°C) in the dark

Test system

Type of coverage:

other: substance added into 12-well plates on top of the skin tissues

Vehicle:

water

Amount / concentration applied:

TEST MATERIAL
- Amount:10.2 to 10.5 mg
VEHICLE
- Amount: 5 µl

Duration of treatment / exposure:

15 minutes

Details on study design:

Application/Treatment of the test substance
The test was performed on a total of 3 tissues per test substance together with negative and positive controls. The solid test substance (10.2 to 10.5 mg; with a small glass weight boat) with 5 µl Milli-Q water was added into 12-well plates on top of the skin tissues. Three tissues were treated with 10 µl PBS (negative control) and 3 tissues with 10 µl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all
tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.
Cell viability measurement
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-medium (0.3 mg/ml). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 µl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for approximately 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

104

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The relative mean tissue viability of three individual tissues after 15 minutes of exposure to NADP,diNa and 42 hours post incubations was > 50% of the mean viability of the negative controls.

Any other information on results incl. tables

Mean tissue viability in thein vitroskin irritation test with b-NADP, Disodium salt

	Mean tissue viability (percentage of control)
Negative control	100
b-NADP, Disodium salt	104
Positive control	5

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

it is concluded that this test is valid and that b-NADP, Disodium salt is non-irritant in the in vitro skin irritation test under the experimental
conditions described in this report.

Executive summary:

In vitroskin irritation test with b-NADP, Disodium salt using a human skin model (EPISKIN Standard model (EPISKIN-SM^TM)). The possible skin irritation potential of b-NADP, Disodium salt was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

 Skin tissue was moistened with 5 µl of Milli-Q water and 10.2 to 10.5 mg of b-NADP, Disodium salt was applied directly on top of the skin tissue for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with b-NADP, Disodium salt compared to the negative control tissues was 104%. Since the mean relative tissue viability for

b-NADP, Disodium salt was above 50% after 15 minutes treatment b-NADP, Disodium salt is considered to be non-irritant.

 

The positive control had a mean cell viability of 5% after 15 minutes exposure. The absolute mean OD₅₇₀(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 5%, indicating that the test system functioned properly.

 

Finally, it is concluded that this test is valid and that b-NADP, Disodium salt is non-irritant in thein vitroskin irritation test under the experimental conditions described in this report.