Saccharomyces cerevisiae, lysate

Eye irritation

Administrative data

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

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 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)

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 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.

Test animals / tissue source

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

Test system

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

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment I Test item

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

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

Results 3

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment I Test item

Value:

0.33

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment II Test item

Value:

0.17

Negative controls validity:

valid

Positive controls validity:

valid

Results 5

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

Results 6

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)

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.