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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Jul - 06 Sep 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
adopted 25 Jun 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Test material

Constituent 1
Reference substance name:
Vinasses, residue of fermentation containing biomass of Corynebacterium glutamicum
EC Number:
932-179-4
Molecular formula:
Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance).
IUPAC Name:
Vinasses, residue of fermentation containing biomass of Corynebacterium glutamicum
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: Dec 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Sterilization at 121 °C for 20 min to avoid contaminations of the cell culture

Test animals / tissue source

Species:
human
Strain:
other: EpiOcularTM, reconstructed three-dimensional human corneal epithelium

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 µL (83.3 µL / cm²)

NEGATIVE CONTROL:
- Amount applied: 50 µL
- Lot No.: RNBG3520

POSITIVE CONTROL:
- Amount applied: 50 µL
- Lot No.: S6943111
Duration of treatment / exposure:
30 ± 2 min at 37 ± 2 °C
Duration of post- treatment incubation (in vitro):
120 ± 15 min at 37 ± 2 °C
Number of animals or in vitro replicates:
2 tissues for each treatment and control group
Details on study design:
DETAILS OF THE TEST PROCEDURE USED
The EpiOcular™ Eye Irritation Test (EIT) consists of a topical exposure of the neat test item to a human reconstructed cornea model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.

RHCE TISSUE CONSTRUCT
- Model used: OCL-200-EIT (MatTek Corporation, Bratislava, Slovakia)
- Tissue Batch number: 27062 (main experiment), 27066 (viable tissue controls), 27027 (killed tissue controls)

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiOcular™ tissues was assessed via MTT cytotoxicity assay. The determined OD (540-570 nm) was in the accepted range of 1.0 - 3.0.
- Barrier function: The barrier function was evaluated by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon treatment with 100 µL of 0.3% Triton X-100. The ET-50 value fits to the acceptance criteria of 12.2 – 37.5 min.
- Contamination: The cells used to produce the EpiOcularTM tissue were screened for the presence of viruses, bacteria, yeast and other fungi. No contamination was detected. Tissue sterility was confirmed by long-term incubation with antibiotic and antimycotic free culture.

DURATION AND TEMPERATURE
- EXPOSURE: 30 ± 2 min at 37 ± 2 °C
- POST-EXPOSURE IMMERSION: 12 ± 2 min at room temperature
- POST-EXPOSURE INCUBATION: 120 ± 15 min at 37 ± 2 °C

REMOVAL OF TEST MATERIAL AND CONTROLS: The tissues were extensively rinsed with Dulbecco's phosphate buffered saline (DPBS).

INDICATION OF CONTROLS USED FOR DIRECT MTT-REDUCERS AND/OR COLOURING TEST CHEMICALS:
The ability of the test substance to directly reduce MTT and to form a blue/purple reaction product was assessed in a pre-experiment. Since the MTT solution colour turned blue/purple, the test substance is presumed to have reduced the MTT. Therefore, an additional test using freeze-killed tissues was performed. A second pre-test revealed that the test item was also colour interfering upon mixing with water. Therefore coloured tissue controls were performed using two additional viable tissues. Moreover, since the test item showed non-specific colouring of living tissues, a third control for non-specific tissue colour in killed tissues was performed to avoid a possible double-correction for colour interference. The true tissue viability was then calculated as the percent living tissue viability obtained with living tissues minus NSMTT minus NSCliving plus NSCkilled.
True Tissue Viability = [%] mean Tissue viability - NSMTT - NSCliving + NSCkilled
NSMTT: non-specific reduction of MTT
NSCliving: non-specific colour of additional viable tissues
NSCkilled: non-specific colour of additional killed tissues
- No. of replicates: 2 tissues (killed/viable) for each test item and controls for determination of NSMTT, NSCliving and NSCkilled.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 15 min at 37 ± 2 °C
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm
- Filter band pass: ± 30nm

EVALUATION CRITERIA
The test substance was considered to be not irritating to eye if the tissue viability after 30 min exposure, 12 min post-exposure immersion and 120 min post-exposure incubation is > 60% relative to the negative control treated tissue.

TEST ACCEPTANCE CRITERIA
- Negative control: The mean absolute OD 570 nm is between 0.8 and 2.5.
- Positive control: The mean relative tissue viability is < 50% of the negative control viability.
- Relative tissue viability difference of replicate tissues is < 20%.

REFERENCE TO HISTORICAL DATA OF THE RHCE TISSUE CONSTRUCT: Please refer to Table 1

Results and discussion

In vitro

Results
Irritation parameter:
other: % tissue viability mean value of 2 tissues
Run / experiment:
30 min exposure
Value:
91
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: tissue viability was corrected for non-specific reduction of MTT and non-specific colour interference
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD of the tissue replicates treated with the negative control was ≥ 0.8 and ≤ 2.8 (1.961).
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control was <50% compared to the negative control (31.1%).
- Relative tissue viability difference of replicate tissues was < 20% (values between 1.5 and 9.7).

Any other information on results incl. tables

Table 2: Results of the test item

Name

Negative Control

Positive Control

Test item

Tissue

1

2

1

2

1

2

OD570 values

1.950

1.957

0.553

0.737

1.781

1.826

1.944

1.995

0.543

0.732

1.803

1.815

OD570 values (blank-corrected)

1.905

1.911

0.508

0.692

1.736

1.781

1.899

1.950

0.498

0.687

1.757

1.770

mean of the duplicates

1.902

1.931

0.503

0.689

1.747

1.775

mean OD

1.916*

0.596

1.761

TODTT- NSMTT

-

-

1.748

TODTTNSMTT and NSCliving

-

-

1.743

tissue viability [%]

99.2

100.8

26.3

36.0

91.1

92.6

relative tissue viability difference [%]

1.5

9.7

1.5

mean tissue viability [%]

100.0

31.1

91.9

mean tissue viability [%]

- NSMTT corrected

-

-

91.2

mean tissue viability [%]

- NSMTT and NSCliving corrected

 

-

 

-

 

90.9

True Tissue Viability

-

-

91.0

*Corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability

OD: optical density

NSMTT: non-specific reduction of MTT

NSC living: non-specific color of additional viable tissues

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008
Conclusions:
CLP: not irritating