Sulfates of potassium, sodium and calcium, byproduct from fermentation

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 5 April 2017 To 24 May 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:

calcium dipotassium disodium trisulfate

Molecular formula:

SO42-, K+, Na+, Ca2+

IUPAC Name:

calcium dipotassium disodium trisulfate

Test material form:

solid

Remarks:

light to beige solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: EXP LIberica Lote: AI16503047
- Expiration date of the lot/batch: November 2019
- Purity test date: 1 February 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25 ºC, below 70 RH%),
protected from light and humidity.
- Solubility and stability of the test substance in the solvent/vehicle: Solubility of the test item in physiological saline was tested prior to the experiment (30 mg test item in 1 mL physiological saline). The test item was dissolved in physiological saline.

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

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT (slaughterhouse)
- Number of animals: 7 eyes were used for ICE assay, 3 were used for test item and positive control and one for physiological saline condition
- Characteristics of donor animals (e.g. age, sex, weight): Approximately 7 weeks old and mean weight 2.40kg in each experiment
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Heads were collected by a slaughter house technician and heads transported to testing facility at ambient temperature at the earliest convenience. They were processed within two hours of collection.
- Time interval prior to initiating testing: Eyes were checked, and acclimatization period was conducted for approximately 45-60 minutes
- indication of any existing defects or lesions in ocular tissue samples: No indication of lesions in ocular tissue samples. Samples were discarded in the case of 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

CONTROL
- Amount(s) applied (volume or weight with unit): 30µL of physiological saline or 30mg powdered imidazole

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

240 minutes

Number of animals or in vitro replicates:

3 replicates for positive and test item condition, one replicate for physiological saline solution

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES

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

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.

Eyes were examined for slit lamp again when placed in the steel clamp for corneal opacity and fluorescein staining.

EQUILIBRATION AND BASELINE RECORDINGS

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

Triplicates were used.

NEGATIVE CONTROL USED

Physiological Saline Solution

POSITIVE CONTROL USED

Powdered Imidazole

APPLICATION DOSE AND EXPOSURE TIME

30 mg of test item or 30 mg of powdered imidazole

OBSERVATION PERIOD

The control eyes and test eyes were evaluated pre-treatment and at approximately 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 observed, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed throughly with 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual of the test item 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. The test item treated eyes were rinsed additional gentle rinsing with 3x20 mL saline after treatment in each experiment.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: slit lamp microscope [Haag-Streit BP 900 slit lamp microscope, setting measurements with depth-measuring device no.I; slit-width setting: 9.5)
- Damage to epithelium based on fluorescein retention: 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
- Swelling: measured on a slit-lamp microscope
- Macroscopic morphological damage to the surface: Morphological effects include “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test substance to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the investigator.
- Others (e.g, histopathology): at the termination of the observation period, eyes were kept in neutral buffered formalin.

SCORING SYSTEM:
See attached tables 1 ,2 and 3 below

DECISION CRITERIA: The descision criteria used was the same as used in the OECD Guideline 438 method for ICE Assay

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment I : at 75 minutes

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment I : at 240 minutes

Value:

0.5

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment I

Value:

0.17

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment I

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Results 5

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment II : at 75 minutes

Value:

0.5

Negative controls validity:

valid

Positive controls validity:

valid

Results 6

Irritation parameter:

percent corneal swelling

Run / experiment:

Experiment II : at 240 minutes

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Results 7

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment II

Value:

0.17

Negative controls validity:

valid

Positive controls validity:

valid

Results 8

Irritation parameter:

fluorescein retention score

Run / experiment:

Experiment II

Value:

0.17

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: 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.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The positive control (Imidazole) was used and classified as inducing serious eye damage (Category 1)

ACCEPTANCE OF RESULTS:
The results from all eyes used met the quality control standards. The negative control and positive control results were in line with historical control data. This experiment was considered to be valid.

Any other information on results incl. tables

Table4 :Summary of Experiment I

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	0.5%	I
Mean maximum corneal opacity	0.17	I
Mean fluorescein retention	0.00	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

 

Table5 :Summary of Experiment II

 

Experiment II
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	0.0%	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

 

Table 6 SUMMARY TABLE FOR UN GHS CLASSIFICATION EXPERIMENT I AND EXPERIMENT II

 

 

Criteria for “No category” (all true)
3 endpoints classed as I or 2 endpoints classed as I and 1 endpoint classed as II:	True
No severe corneal morphological changes:	True
Test item was not stuck to the cornea at 240 minutes after the post- treatment rinse:	True

 

Criteria for “Category 1” (one or more true)
2 or more endpoints classed as IV:	False
Corneal opacity ≥ 3 at 30 min (in at least 2 eyes):	False
Corneal opacity = 4 at any time point (in at least 2 eyes):	False
Severe loosening of epithelium (in at least 1 eye):	False

 

Criteria for “No prediction can be made” (one or two true)
Based on the endpoints not classifiable for No Category, or for Category 1:	False
Particles of test item were stuck to the cornea and could not be washed off during the study:	False

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on this in vitro eye irritation assay in the isolated chicken eyes with Sulfates of potassium, sodium and calcium, by-product from fermentation, the test item is not classified for eye irritation or serious eye damage according to GHS and CLP criteria.

Executive summary:

The purpose of this GLP compliant study was to evaluate the potential eye irritation effect of the test substance when tested in an Isolated Chicken's Eye Assay (performed according to OECD 438 guideline method).

After the zero reference measurements,  the eye  was held  in  horizontal position and 30 mg of powered test item was applied onto the centre of the cornea such 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 the study, 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 in good correlation with the  historical control data. 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 significant cornea opacity change (severity 0.5) was observed on one eye. No fluorescein retention change was noted on three eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. The 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 test item treated eyes. No significant cornea opacity change (severity 0.5) was observed on one eye. No significant fluorescein retention change (severity 0.5) was noted on one eye. Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Based on this in vitro eye irritation assay in the isolated chicken eyes  with Sulfates of potassium, sodium and calcium, by-product from fermentation, the test item is not classified for eye irritation or serious eye damage according to GHS and CLP criteria.