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Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Remarks:
Alternative method
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: Appendix VI of the French National Register N°302 of December, 1999
Deviations:
no
Principles of method if other than guideline:
This test is an alternative method which aims to assess a test item eye irritant potential. The principle is based on the test item cytotoxicity assessment by determination of the concentration which leads to 50% of cells death (IC50) on a cell monolayer, using the Neutral Red Uptake Method.
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Saccharides of mannose and galactose from Ceratonia Siliqua seed
IUPAC Name:
Saccharides of mannose and galactose from Ceratonia Siliqua seed
Test material form:
liquid
Details on test material:
Clear yellow

Test animals / tissue source

Species:
other: Rabbit cornea fibroblasts
Strain:
other: SIRC line (Cat N°2-552 CCL60)
Details on test animals or tissues and environmental conditions:
Cells were cultured in the Test facility in DMEM medium supplemented with 10% of foetal calf serum, decomplementized at 56°C for 30 minutes, 1% of a penicillin/streptomycin solution and L-glutamine. Cells were kept and preserved in accordance with the internal procedures of the Test facility for freezing, unfreezing and subculturing.

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Dilution at 5%, 15%, 25%, 35% and 50% of the test material are used
Duration of treatment / exposure:
contact with the test item = 60 secondes
Observation period (in vivo):
N/A
Duration of post- treatment incubation (in vitro):
not specified
Number of animals or in vitro replicates:
Each sample, negative and positive controls are tested on two cultures wells by assessed concentration.
5%, 15%, 25%, 35% in monoplicate and 50% in duplicate
Details on study design:
Cells in culture were trypsined and counted in accordance with the internal procedure od the Test facility. Then cells are seeded in 24wells plate at the rate of 2 x 10^5 cells/well under a volume of 1 ml of complet DMEM medium and then incubated for 24 hours (37°C, 5% CO2).
A stock solution of 0.4% neutral red prepared in sterile conditions with distilled water and diluted at 1/80 in complete culture medium at 37°C.
A 1% solution of 100% acetic acid in 50° ethanol was prepared.

48h after seeding, the culture medium of each well was removed. 1ml of colouring solution of neutral red was deposited per well. The plates were incubated at 37°C,5% CO2 for 3hours.
After this time of contact, the colouring solution was removed and replaced by 1 ml of complete culture medium per well. The plates was maintained at room temperature for at least 30 mintutes before being put in contact with the test item.

Each well is firstly rinsed with 2 ml of PBS before being treated with 500 μL of each dilution of test item. The contact time is 60 seconds. Treatments are applied, preferably, well by well and the stopwatch is started when the treatment is applied.
After 55 seconds, the treatment solution is aspirated. At precisely 60 seconds, the well is rinsed 5 times (5x2 ml of PBS). The pipettes used forrinsing must held vertically. The supernatant is aspirated after each rinse. After the final rinse, the well remained without the medium while waiting the revealing phase.
After the culture plate has been fully treated, 1 ml of the revealing solution (acetic acid/ethanol : 1/100) is placed in each well. The plate is shaken approximatively 15 minutes.
The solutions revealed in each well were put in a 96-well microplate of 200 μL/well in duplicate.
Absorbances are measured at 540 nm against the blank (revealing solution) on an automatic microplate reader.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: Percentage of mortality observed at the 50% dilution (higher dilution tested)
Value:
ca. 22
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
other: Estimated IC 50 (%)
Value:
> 50
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

In vivo

Irritant / corrosive response data:
The cytotoxicity of tested material was judged not very important
IC 50 is greater to 50% and the dealth cells rate at 50% of test item (higher dilution tested) has been assessed to 22%.

Applicant's summary and conclusion

Interpretation of results:
other: not very important cytotoxicity according to the adopted scale.
Conclusions:
Under the retained experimental conditions, the cytotoxicity of the test item may be classified as not
very important cytotoxicity according to the adopted scale.
Executive summary:

An in vitro eye irritation study was performed on rabbit cornea fibroblasts according to French national Method published in December 1999 in National register N° 302. The principle of the method is based on assessing the cytotoxicity of the product tested by identifying the concentration causing 50% mortality (IC50) using the technique of neutral red release.

Positive and Negative controls were used. The IC50 was up to 50% and the death cells rate at 50% of test item (higher dilution tested) has been asessed to 22%.

Under the retained experimental conditions, the cytotoxicity of the tes item may be classified as not very important cytotoxicity according to the adopted scale.