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Diss Factsheets

Administrative data

Description of key information

Based on the two available key studies (Orovecz, OECD TG 439, GLP, 2018, Klimisch 1; Orovecz, OECD TG 438, GLP, 2018, Klimisch 1) the test substance Saccharomyces Cerevisiae, lysate was not classified for skin and eye irritation according to CLP criteria.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
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
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)
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.
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
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Test item
Value:
92.3
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Negative Control
Value:
100
Negative controls validity:
valid
Positive controls validity:
valid
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.
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 19 July 2017 to 13 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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 ºC, below 70 Relative Humidity)
- Stability under test conditions: not applicable
- Solubility and stability of the test substance in the solvent/vehicle: The solubility of the test item in physiological saline was tested prior to the experiment (30 mg test material in 1 mL physiological saline). The test item did not dissolve in physiological saline.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was applied in its original form.
Species:
chicken
Strain:
other: ROSS 308 and COBB 500
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Commercial abattoir TARAVIS KFT
- Number of animals: 7 eyes were used in this study
- Characteristics of donor animals (e.g. age, sex, weight): approximately 7 weeks old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to the test facility at ambient temperature at the earliest convenience.
After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box).
- Time interval prior to initiating testing: within 2 hours
- indication of any existing defects or lesions in ocular tissue samples: not lesions
- Indication of any antibiotics used: not specified
Vehicle:
unchanged (no vehicle)
Controls:
yes
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):30 mg
- Concentration (if solution): not applicable

VEHICLE
Not applicable
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
240 minutes
Number of animals or in vitro replicates:
3 eyes wer used for test and positive control conditions and one eye was used for negative control
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES

Eyes selection

After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein- treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

Preparation of eyes

The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

EQUILIBRATION AND BASELINE RECORDINGS

Eyes examination and acclimatization time

The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoid too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus.

The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.

The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

The baseline assessments

At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5% within the -45 min and the zero time. No changes in thickness (0.0%) were observed in the eyes in each experiment. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES
One for negative control condition, three for test and positive control conditions

NEGATIVE CONTROL USED
Physiological saline, (Salsol solution, 0.9% (w/v) NaCl)

SOLVENT CONTROL USED (if applicable)
Not applicable

POSITIVE CONTROL USED
Imidazole

APPLICATION DOSE AND EXPOSURE TIME
30 mg of test item, (or 30µL of controls ) were applied for 10 seconds exposure period

OBSERVATION PERIOD
30, 75, 120, 180 and 240 minutes after the post- treatment rinse

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The time of application was noted, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible.

Additional gentle rinsing with 20 mL saline was performed at each time point when the test item or positive control material remaining on the cornea was observed in each experiment. The test item treated eyes were rinsed additional gentle rinsing with 3x20 mL saline after treatment in each experiment.

- Indicate any deviation from test procedure in the Guideline : no deviation

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal thickness and corneal opacity were measured at all time points.
- Damage to epithelium based on fluorescein retention: Morphological effects including “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test substance to the cornea were observed.
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-with setting: setting of the slit lamp microscope for corneal thickness measurement: depth-measuring device no. I.; slit-width setting: 9.5
- Macroscopic morphological damage to the surface: performed by investigator
- Others (e.g, histopathology): not specified

SCORING SYSTEM: see attached table below
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment

DECISION CRITERIA: see attached table below
Irritation parameter:
cornea opacity score
Run / experiment:
Experiment I Test item
Value:
0
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
Experiment I Test item at 240 minutes
Value:
1.1
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
Experiment I Test item
Value:
0.33
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
Experiment II Test item
Value:
0.17
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
Experiment II Test item at 240 minutes
Value:
1.1
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
Experiment II Test item
Value:
0.17
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no damage

DEMONSTRATION OF TECHNICAL PROFICIENCY: Imidazole, used as positive control, showed positive response to the ICE test as expected.

ACCEPTANCE OF RESULTS:
The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range in each experiment. This study was considered to be valid. (attached table was showed below)

TEST ITEM

 

 

Experiment I

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

0.5 %

I

Mean maximum corneal swelling at up to 240 min

1.1 %

I

Mean maximum corneal opacity

0.00

I

Mean fluorescein retention

0.33

I

Other Observations

Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 30 minutes after

the post-treatment rinse.

Overall ICE Class

3xI

 

 

Experiment II

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

0.0 %

I

Mean maximum corneal swelling at up to 240 min

1.1 %

I

Mean maximum corneal opacity

0.17

I

Mean fluorescein retention

0.17

I

Other Observations

Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 30 minutes after

the post-treatment rinse.

Overall ICE Class

3xI

 

 

POSITIVE CONTROL

 

 

Experiment I

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

10.8%

II

Mean maximum corneal swelling at up to 240 min

28.7%

III

Mean maximum corneal opacity

4.00

IV

Mean fluorescein retention

3.00

IV

Other Observations

Imidazolewas stuck onallcornea surfaces after the post- treatmentrinse.The cornea surfaces (3/3) were not clearedat 240minutesafterthe

post-treatmentrinse.

Overall ICE Class

1xIII 2xIV

 

 

NEGATIVE CONTROL

 

 

Experiment I

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

0.0%

I

Mean maximum corneal swelling at up to 240 min

0.0%

I

Mean maximum corneal opacity

0.00

I

Mean fluorescein retention

0.00

I

Other Observations

None

Overall ICE Class

3xI

 

 

Experiment II

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

0.0%

I

Mean maximum corneal swelling at up to 240 min

0.0%

I

Mean maximum corneal opacity

0.00

I

Mean fluorescein retention

0.00

I

Other Observations

None

Overall ICE Class

3xI

 

VALIDITY OF THETEST

 

Historical Control data (updated on 11 August 2016):

 

Negative Control: Physiological Saline

 

Observation

Min. Value

Max. Value

Maximum corneal swelling at up to 75 min

-3.2 %

3.4 %

Maximum corneal swelling at up to 240 min

-4.8 %

3.4 %

Maximum corneal opacity change

0.00

0.50

Fluorescein retention

0.00

0.50

Number of studies

254

 

Positive Control: Imidazole

 

Observation

Min. Value

Max. Value

Maximum corneal swelling at up to 75 min

-6.6 %

25.0 %

Maximum corneal swelling at up to 240 min

-15.9 %

36.7 %

Maximum corneal opacity change

3.50

4.00

Fluorescein retention

2.00

3.00

Number of studies

117

 

Interpretation of results:
GHS criteria not met
Conclusions:
Based on these in vitro eye irritation assays in isolated chicken eyes with Saccharomyces cerevisiae, lysate, the test item was not classified for eye irritation or serious eye damage as defined by the current GHS and the CLP criteria.
Executive summary:

This GLP compliant study was performed according to OECD 438 guideline for Isolated Chicken Eye Irritation test. The purpose was to assess the potential eye irritation potential of the registered substance Saccharomyces cerevisiae, lysate on chicken eyes.

In each experiment after the zero reference measurements, the eye was held in horizontal position and 30 mg test item was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 µL of physiological saline (0.9% (w/v) NaCl solution). In each experiment, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in each experiment. Thus, the experiment was considered to be valid.

Experiment I: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. No cornea opacity change was observed on three eyes. No significant fluorescein retention change (severity 0.5) was noted on two eyes and no fluorescein retention change was noted on one eye. Test item was stuck on all cornea surfaces after the post-treatment rinse. All cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Experiment II: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on the test item treated eyes. No significant cornea opacity change (severity 0.5) was observed on one eye and no fluorescein retention change was noted on two eyes. No significant fluorescein retention change (severity 0.5) was noted on one eye and no fluorescein retention change was noted on two eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. All cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Based on these in vitro eye irritation assays in isolated chicken eyes with Saccharomyces cerevisiae, lysate, the test item was not classified for eye irritation or serious eye damage as defined by the current GHS and the CLP criteria.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Two key studies were available for skin and eye irritation evaluation:

An in vitro GLP compliant skin irritation test of Saccharomyces cerevisiae, lysate test item was performed in a reconstructed human epidermis model EPISKINTM (SM) (Orovecz, OECD TG 439, GLP, 2017, Klimisch 1). Disks of EPISKIN TM (three units) were treated with the test item and incubated for 15 minutes at room temperature. The epidermis units were then incubated at 37°C for 42 hours in an incubator. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. MTT was quantified thereafter. 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. In conclusion, under the experimental conditions of the study, the results indicate that the test item is non-irritant to skin.

An Isolated Chicken Eye Irritation test was performed was to assess the potential eye irritation potential of the registered substance Saccharomyces cerevisiae, lysate on chicken eyes (Orovecz, OECD TG 438, GLP, 2017, Klimisch 1). The eye was covered by 30 mg of test item was onto the centre of the cornea. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 µL of physiological saline. In each experiment, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined. Two experiments were performed in which no significant corneal swelling was observed during the four-hour observation period on the test item treated eyes. No significant cornea opacity change was observed on one eye and no fluorescein retention change was noted on two eyes. No significant fluorescein retention change was noted on one eye and no fluorescein retention change was noted on two eyes. Under the experimental condition of the study, the test substance Saccharomyces cerevisiae, lysate, did not induced eye irritation or serious eye damage.

Justification for classification or non-classification

Based on the two available key studies (Orovecz, OECD TG 439, GLP, 2018, Klimisch 1; Orovecz, OECD TG 438, GLP, 2018, Klimisch 1), the test substance Saccharomyces Cerevisiae, lysate did not induce skin irritation in a test performed according to OECD TG439 and did not induce eye irritation in an ICE assay performed according to OECD TG 438 guideline. Hence, it can be stated that the test substance Saccharomyces Cerevisiae, lysate does not require classification for skin and eye irritation according to CLP criteria.