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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 27, 2017 to February 28, 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)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Okra, ext.
EC Number:
294-510-2
EC Name:
Okra, ext.
Cas Number:
91723-07-8
Molecular formula:
not applicable
IUPAC Name:
Hibiscus abelmoschus, ext.
Specific details on test material used for the study:
407/DELIP HIBISCUS SEED;
batch: 0016468911;
Date of manufacture: 09 Dec 2016,
Expiry date: 09 Dec 2019.
state:beige solid

Test animals / tissue source

Species:
human
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
not applicable

The EpiOcularTM model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinozytes used to model the human corneal epithelium. The EpiOcularTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.

To assess the ability of the test material to directly reduce MTT a pretest was performed.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: yes (tissue incubations for positive and negative controls included)
Amount / concentration applied:
0.05 mL
Duration of treatment / exposure:
6 hours
Observation period (in vivo):
18h
Number of animals or in vitro replicates:
Two tissue samples were used per group.
Details on study design:
Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The
quotient of the values indicates the relative tissue viability.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: Viability (%)
Run / experiment:
test run 1
Value:
36.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: Viability (%)
Run / experiment:
test run 2
Value:
20.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: Viability (%)
Run / experiment:
test run 3
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
1st test run:
The final relative mean viability of the tissues treated with the test substance after the 1st test
run was 36.6%. However, the acceptance criteria for the killed controls were not met: High
values for direct MTT reduction (OD values > 0.35 and mean value of the killed control tissues
30.7% with individual values 30.7% and 30.6%) and high variability of the test-substance
treated tissues (difference between the tissues 18.8% with inidividual values 57.9 and 76.6%
close to the classification cut-off) a 2nd test run was performed to verify the result.
2nd test run:
The final relative mean viability of the tissues treated with the test substance after the 2nd test
run was 20.6%. However, the acceptance criteria for the killed controls were not met, becaue
exceptionally high values for direct MTT reduction and high variability between the single killed
control tissues (OD values > 0.35 and mean value of the killed control tissues 30.7% with
individual values 84.3% and 50.4%) were observed. Thus, a 3rd test run was performed.
3rd test run:
The final relative mean viability of the tissues treated with the test substance after the 3rd test
run was 0.0%. However, the acceptance criteria for the killed controls were not met, because
exceptionally high values for direct MTT reduction and high variability between the single killed
control tissues (OD values > 0.35 and mean value of the killed control tissues 115.3% with
single values 153.0% and 77.6%) were observed. In addition high variability of the testsubstance
treated tissues (difference between the tissues 22%) was noted. Further, the final
relative mean viability of the tissues treated with the test substance after the 3rd test run was
set to zero after substracting the mean viabilaity of the killed controls form the man viability of
the test-substance treated tissues.

Any other information on results incl. tables

The positive control methyl acetate decreased the mean viability to 24% of control cultures.

The negative control water did not affect the viability of the control cultures

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification