Saccharomyces cerevisiae, lysate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 31 August 2017 to 16 November 2017

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)

Deviations:

yes

Remarks:

Due to technical reason the dead epidermis units were stored lower temperature than indicated in the Study Plan. This fact had no impact on the study on the results or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Saccharomyces cerevisiae, lysate

EC Number:

305-230-8

EC Name:

Saccharomyces cerevisiae, lysate

Cas Number:

94350-12-6

IUPAC Name:

Saccharomyces cerevisiae, lysate

Test material form:

solid: particulate/powder

Remarks:

light beige

Details on test material:

- Source and lot/batch No.of test material:
supplied by the sponsor, batch no. AC17F00560
- Expiration date of the lot/batch: February 2019
- Purity test date: 30 June 2017

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the sponsor; Batch/Lot number: AC17F00560
- Expiration date of the lot/batch: February 2019
- Purity test date: 30 June 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25 °C, ≤ 70 % relative humidity).
- Stability under test conditions: not applicable
- Solubility and stability of the test substance in the solvent/vehicle: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was applied as supplied, no formulation was required.

In vitro test system

Test system:

artificial membrane barrier model

Source species:

human

Cell type:

other: EpiSkin Reconstructed Human Epidermis

Cell source:

other: not applicable

Source strain:

other: not applicable

Justification for test system used:

The irritation potential of a chemical may be predicted by measurement of its cytotoxic effect, as reflected in the MTT assay, on the EPISKIN TM (SM) reconstituted human epidermis. This method is approved by international regulatory agencies as a replacement for the identification of irritants / corrosives in the in vivo Rabbit skin assay (OECD No. 404).
The test is designed to predict and classify the skin irritant potential of chemicals/formulations/products/mixtures according to chemical safety regulations, using the reconstructed human epidermis model EPISKINTM (SM) and parameters related to skin irritation.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM
- Tissue batch number(s): 17-EKIN-036 and 17-EKIN-024 (killed epidermis)
- Production date: not specified
- Expiry date: 11 September 2017 and 19 June 2017 (killed epidermis)
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing:06 September 2017

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: After the 15 minutes incubation time, the EPISKINTM (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
After rinsing the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere
- Observable damage in the tissue due to washing: No damage
- Modifications to validated SOP: No modification

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 3 mg/mL stock solution ; 2 mL of 0.3 mg/mL per wells
- Incubation time: 3 hours ± 5 minutes
- Spectrophotometer: Thermo Fisher Scientific Thermo Fisher Scientific, Catalogue Number: 24072800, Serial Number: 0920-14
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth:not specified
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Historical data PBS : OD of 0.802±0.157 ;
SDS : OD of 0.094±0.048 ;
IC50 of the batch with SDS : 2.2 mg/mL
- Barrier function: Satisfatctory, validated
- Morphology: well differentiated epidermis consisted of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination: All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasman there was no contamination.
- Reproducibility: Yes

NUMBER OF REPLICATE TISSUES: triplicate were used

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues (if applicable): For killed epidermis, living epidermis units (Source: SkinEthic, France, Batch No.:17- EKIN-024, Expiry Date: 19 June 2017) were placed in a 12 well plate with 2 mL of distilled water, then incubated at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere for 48 hours (±1 hour). At the end of the incubation the water was discarded and the dead epidermis units were frozen (at -19.6°C to -29.7°C) on 16 June 2017 (the frozen units can be used up to 6 months). Before use, the killed tissues were thawed at room temperature (at least 30 minutes in 2 mL of Maintenance Medium). Further use of killed tissues was similar to living tissues.
- N. of replicates : two replicates were used
- Method of calculation used:
Test items that interfere with MTT can produce non specific reduction of the MTT. In this case, additional control samples are used to determine the OD value derived from non-specific reduction of the MTT. The measured OD value is corrected by the result of the additional controls before calculation of viability % as detailed below:

Non specific MTT reduction calculation (NSMTT%):
NSMTT (%) = [(ODKT- ODKNC) / ODNC] × 100
with :
ODKNC: negative control killed tissues OD
ODKT: test item treated killed tissues OD
ODNC: negative control OD

If NSMTT% is ≤ 30%, then true MTT metabolic conversion (TODTT) is undertaken as
follows:
TODTT = [ODTT – (ODKT – ODKNC)]
ODTT: test item treated viable tissues

The % relative viability (% RV) for each test item replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / mean ODNC] × 100
% RV 2 = [TODTT2 / mean ODNC] × 100
% RV 3 = [TODTT3 / mean ODNC] × 100
The mean value of the 3 individual relative viability % for test item is calculated:
Mean Relative Viability % = (% RV 1 + % RV 2 + % RV 3) / 3

If NSMTT% is > 30% relative to the negative control, then additional steps must be undertaken if possible, or the test item must be considered as incompatible with the test.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: Three different steps : pre incubation ; incubation with test item ; post exposure period including MTT assay

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 15 minutes exposure and 42 hours post exposure is less than 50%
- The test substance is considered to be non-corrosive to skin if the viability after 15 minutes exposure and 42 hours post exposure is greater than or equal to 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10mg
- Concentration (if solution): not applicable

VEHICLE:
no vehicle used

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): Sodium Dodecyl Sulphate
- Concentration (if solution): 5% (w/v) in solution

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

triplicates were used

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item

Value:

92.3

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Negative Control

Value:

100

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive Control

Value:

5.3

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no damage
- Direct-MTT reduction: the test item was showed being an MTT-interacting substance and the test item had an intrinsic colour
- Colour interference with MTT: As colour change (purple) was observed after three hours of incubation of the test item in MTT working solution, thus the test material might interact with MTT.

DEMONSTRATION OF TECHNICAL PROFICIENCY: the SDS, used as positive control, showed positive reponse to the test sytem, as expected

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD value of the three negative control tissues was in the recommended range (0.674). Standard deviation of the viability results for negative control samples was 1.3%.

- Acceptance criteria met for positive control: The positive control treated tissues showed 5.3% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 0.8%.

- Acceptance criteria met for variability between replicate measurements: The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 1.4%. The mean OD value of the blank samples (acidified isopropanol) was 0.046.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, in this in vitro EPISKINTM (SM) model test with Saccharomyces cerevisiae, lysate (Batch number: AC17F00560), the results indicate that the test item is non-irritant to skin.

Executive summary:

An in vitro GLP compliant skin irritation test of Saccharomyces cerevisiae, lysate test item was performed in a reconstructed human epidermis model EPISKINTM (SM). The irritation potential of the test item was evaluated according to the OECD No. 439 guideline.

Disks of EPISKIN TM (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2, in a >95% humidified atmosphere The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere, protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. The possible MTT interaction potential of the test item was examined using two additional test item treated and two negative control treated killed epidermis units. Furthermore, to avoid a possible double correction for colour interference, a third set of controls for non-specific colour in killed tissues (NSCkilled), two additional disks were used. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 min exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the  test item is considered to be irritant to skin.

Following exposure with Saccharomyces cerevisiae, lysate, the mean cell viability was 92.3% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in  vitro  EPISKINTM  (SM)  model  test  with  Saccharomyces cerevisiae, lysate (Batch number: AC17F00560), the results indicate that the test item is non-irritant to skin.