3,7-dihydropurin-6-one;sodium

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-10-05 to 2018-01-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 28 July 2015

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3,7-dihydropurin-6-one;sodium

Cas Number:

45738-97-4

Molecular formula:

C5H3N4NaO

IUPAC Name:

3,7-dihydropurin-6-one;sodium

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: White powder
- Purity / Composition: 98%
- Test item storage: Room temperature

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

EPISKIN Small ModelTM is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum. In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM (EPISKIN-SMTM), 0.38 cm²
- Batch No: 17 EKIN 044
- Source: SkinEthic Laboratories, Lyon, France

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- rinsed with phosphate buffered saline (PBS)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (3 mg/mL in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/mL)
- Incubation time: 3 hours
- Spectrophotometer: the amount of extracted formazan was determined at 570 nm in duplicate with the TECAN Infinite M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin in accordance with regulation EC 1272/2008 (UN GHS "Category 2") if the viability after exposure and post-incubation is less or equal to 50%.
- The test substance is considered to be non-irritant to skin in accordance with regulation EC 1272/2008 (UN GHS "No Category") if the viability after exposure and post-incubation is more than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 11.2 to 12.0 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 μL PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 μL
- Concentration (if solution): 5% SDS

Duration of treatment / exposure:

15 +/- 0.5 minutes at room temperature

Duration of post-treatment incubation (if applicable):

42 hours at 37 °C

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of triplicates

Value:

112

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Sodium hypoxanthine was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no color changes were observed it was concluded that Sodium hypoxanthine did not interact with the MTT endpoint. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Sodium hypoxanthine compared to the negative control tissues was 112%. Since the mean relative tissue viability for Sodium hypoxanthine was above 50% Sodium hypoxanthine is considered to be non-irritant. The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 10%. The absolute mean OD570 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 < 6%, indicating that the test system functioned properly.

Any other information on results incl. tables

Table 1: Mean Tissue Viability

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	5.7
Test item	112	5.7
Positive control	10	2.3

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no irritant effects in an validated in vitro system (EPISKIN). The test item is classified as ‘non-irritant’ in accordance with UN GHS ‘No Category’.

Executive summary:

The potential for the test item to induce skin irritation was tested by using the three-dimensional human skin model EpiSkin-SM (SkinEthic) comprising a reconstructed human epidermis with functional stratum corneum. The test item was applied directly atop the EpiSkin-SM tissue for 15 min, followed by 42 h post incubation period and immediate determination of cytotoxic effects via MTT reduction assay. Irritant potential from the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with PBS. The test item showed no irritant effects. The mean relative tissue viability (% negative control) was ≥ 50% (112%) after 15 min treatment and 42 h post incubation. The controls confirmed the validity of the study. The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 10%.  The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. In this study under the given conditions the test item showed no irritant effects. The test item is classified as ‘non-irritant’ in accordance with UN GHS ‘No Category’.