Registration Dossier

Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
other: Appendix VI of the French National Register N°302 of December, 1999
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 involves 50% of cells death (IC50) on a cell monolayer, using the Neutral Red Uptake method.
GLP compliance:

Test material

Constituent 1
Reference substance name:
Tara gum
EC Number:
EC Name:
Tara gum
Cas Number:
Oligosaccharides of Caesalpinia Spinosa extract
Test material form:
solid: particulate/powder
Details on test material:
light beige powder

Test animals / tissue source

other: Rabbit cornea fibroblasts
other: SIRC line (ATCC CCL60)

Test system

physiological saline
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
Test item: Dilution at 25% and 50% of the test material are used
Positive control: 0.2%, 0.05%, 0.01% in saline solution
Duration of treatment / exposure:
contact with the test item = 60 secondes
Observation period (in vivo):
Duration of post- treatment incubation (in vitro):
Number of animals or in vitro replicates:
Each sample and positive control are tested on three cultures wells by assessed concentration. Negative control is tested on three culture wells per plate
Details on study design:
The cells are treated with tripsin and counted. 24 wells cell culture plate are seeded with 1 ml of the cell suspension containing 2 x 10^5 cells/ml in complet DMEM medium and then cultured overnight (37°C, 5% CO2).
Neutral red solution is diluted at 0.05 mg/ml in complet culture medium. The culture medium is removed and 1 ml of the solution is placed in each well. The plates are returned to the incubator at 37°C,5% CO2 for 3hours.
After this contact time, the strain solution is removed and replaced with 1 ml of complete culture medium per well.The plates are maintained at room temperature for at least 30 mintutes in order to stabilise the system before contact with the test item and reference item. Each well is firstly rinsed with 2 ml of PBS before being treated with 500 μL of the diluted 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. The plate is shaken manually throughtout the treatment period.
After 55 seconds, the treatment solution is aspirated. At precisely 60 seconds, the well is rinsed 5 times (5x2 mlof PBS). The pipettes used forrinsing must held vertically. The supernatant is aspirated after each rinse. After the final rinse, the well are left without the medium until the development phase.
After the culture plate has been fully treated, 1 ml of the desorption solution is placed in each well.
The plate is shaken approximatively 15 ml until homogenous colouring.
The solutions are placed in 2 wells of a 96 well plate (150μL/well). Absorbancies are measured at 540nm against the blank (desorption solution)

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: Percentage of mortality observed at the 50% dilution
ca. 6
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation
Irritation parameter:
other: Estimated IC 50 (%)
> 50
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The negative control shows an insignificant cytotoxicity.
The IC50 of the positive control is comprised between 0.01% and 0.2%
These results allow to validate the test.

Applicant's summary and conclusion

Interpretation of results:
other: insignificant cytoxicity according to table of French national register N°302, December 1999
Under the retained experimental conditions, the cytotoxicity of the tes item may be classified as in significant 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 (vehicle) controls were used.

The IC50 was up to 50% and the death cells rate at 50% of test item has been asessed to 0%.

Under the retained experimental conditions, the cytotoxicity of the tes item may be classified as insignificant cytotoxicity according to the adopted scale.