Enzymatic hydrolysis products of Ophiopogon japonicus, Liliaceae, root

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

Study performed according to French offical method published in December 1999 in National register N° 302. No deviation has been reported. No Eye irritation classification can be made, only based on this cytotoxic experiment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Appendix VI of the French National Register N°302 of December, 1999

Principles of method if other than guideline:

The method is an alternative to animal experimentation, to determine the ocular irritant potential of cosmetic products. The principle 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.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Enzymatic hydrolysis products of Ophiopogon japonicus, Liliaceae, root

EC Number:

812-241-6

Cas Number:

952500-62-8

IUPAC Name:

Enzymatic hydrolysis products of Ophiopogon japonicus, Liliaceae, root

Details on test material:

Cohesium batch number 07 304 1
slightly yellow liquid
pH 4.5
solution of the test material with phenoxyethanol 0.5% and Ethylhexylglycerin 0.2%

Test animals / tissue source

Species:

rabbit

Strain:

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

Details on test animals or tissues and environmental conditions:

no animal test
Rabbit cornea fibroblasts: SIRC line from ATCC Cat N°2-552 - CCL60

Test system

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

Dilution at 5%, 15%, 25%, 35% and 50% of the test material are used.

Duration of treatment / exposure:

Cells are exposed 1 minute to the dilution of the test material

Observation period (in vivo):

not specified

Number of animals or in vitro replicates:

Dilution at 5%, 15%, 25%, 35% were tested in Monoplicate, Dilution 50% was tested in duplicate

Details on study design:

Positive and negative control are used.
A suspension at 200,000 cells/ ml complete DMEM medium is prepared 24 hours before the assay is performed.
The day of the experiment, 1 ml of the solution of neutral red at 0.05mg/ml, is deposited on the cells, and placed for 3 hours in the incubator.
Then cells are exposed to a dilution of the test substance for 1 minute. Every dilution is tested one time except the dilution 50% which is tested in duplicate.
After exposure, cells are washed with 1 ml of PBS, and are treated with a revelatory solution (acetic acid/ Ethanol :1/100) .
The optical density of the resulting solution is measured at 540 nm.
The optical density (OD) values obtained for each test chemical are then used to calculate cell viability. The relative cell viability is expressed as a percentage and obtained by dividing the OD of test chemical by the OD of the solvent control.
The following formula is used: % mortality = 100- (OD cells exposed to test mateiral)/ (OD of negative control) X 100.
The curve of per cent cell mortality vs. Concentration of the product is plotted. the IC50 of the test product is obtained by extrapolation from the dose response curve.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: Percentage of mortalité observed at the 50% dilution

Value:

31

Vehicle controls validity:

not applicable

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

31% of mortality observed at the 50% dilution

Results 2

Irritation parameter:

other: Estimated IC 50 (%)

Value:

> 50

Vehicle controls validity:

not applicable

Negative controls validity:

not specified

Positive controls validity:

valid

In vivo

Irritant / corrosive response data:

The cytotoxicity of tested material is slightly important.
IC 50 is greater to 50% and the percentage of mortality observed at the dilution 50% is 31%

Any other information on results incl. tables

Table 1: Optical density means for the test material and percentage of cells mortality

Optical density (OD) and cells mortality (%)
Test material concentration % (p/p)	0	5	10	25	35	50
OD Well 1	1.319	1.035	0.937	0.928	0.902	0.843
OD Well 1	1.380	1.028	0.934	0.930	0.903	0.856
OD means Well 1	1.350	1.032	0.936	0.929	0.903	0.850
OD Well 2	1.059					0.829
OD Well 2	1.064					0.822
OD means Well 2	1.062					0.826
OD means	1.206	1.032	0.936	0.929	0.903	0.838
% mortality	0	14	22	23	25	31

Applicant's summary and conclusion

Interpretation of results:

other:

Remarks:

table of French national register N°302, December 1999

Conclusions:

The test material is considered as slightly irritant considering the fact that the cytotoxicity is not very important according to guideline followed

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.

The IC50 was up to 50% and the percentage of mortality at the dilution 50% is 31% .

According to the method the cytotoxicity is considered not very important.

The cytotoxicity is slightly important according to guideline followed.