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EC number: 305-230-8 | CAS number: 94350-12-6
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
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
- 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.
Reference
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Eye irritation
Link to relevant study records
- 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) - 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.
Reference
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 |
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.
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