Enzymatic hydrolysis products of Ophiopogon japonicus, Liliaceae, root

Endpoint summary

Description of key information

Cohesium was tested for eye irritation by an alternative method (application to fibroblast of rabbit cornea by the neutral red release method). The substance has not important cytotoxicity.

OF14 AT was tested for cytotoxicity in a GLP study, and the IC50 resulted to be 102.75 mg/mL.

OF14 AT was found to be non-irritant for skin in a GLP study conducted according to OECD 439.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Test system:

human skin model

Remarks:

human reconstructed epidermis (tissues)

Cell type:

other: human reconstructed epidermis (tissues)

Cell source:

other: SkinEthic Laboratories

Vehicle:

unchanged (no vehicle)

Details on test system:

SkinEthicTM RHE
Description: SKINETHIC RhE: RECONSTRUCTED HUMAN EPIDERMIS, 0.5cm² reconstructed epidermal human keratinocutes. Cells are grown on inert polycarbonate filter on chemically defined medium, airlifted for 17 days, till a highly differentiated and stratified epidermis model is obtained comprising the main basal, supra basa, spinous and granular layers and a functional stratum corneum. The RHE model presents a histological morphology comparable to the in vivo human tissue.

Type of coverage:

other: not applicable

Preparation of test site:

other: not applicable

Vehicle:

other: not applicable

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL of sterile water and 16 +/-2 mg of the test item was applied to the epidermis surface.

CONTROLS
- Negative control:16 +/- 0.5 μL of Dulbecco’s Phosphate-Buffered Saline (D-PBS)
- Positive control: 16 +/- 0.5 μL of 5 % (w/v) aqueous solution of Sodium Dodecyl Sulfate (SDS)

Duration of treatment / exposure:

Triplicate tissues were treated with the test item for an exposure period of 42 +/- 1 minutes at room temperature.
At the end of the exposure period, tissues were rinsed and incubated at 37 °C, 5 % CO2 in a humidified incubator for 42 h.

Number of animals:

Triplicate tissues for test item, negative and positive controls

Details on study design:

PRE-INCUBATION STEP
After arrival, 9 RHE will be transferred into new 6-well plates with 1 mL/well of roome temperature SkinEthic Growth Medium (SGM). The absence of air bubbles will be checked by observing underneath the well of the plate. The plate will be then incubated from 1 hour and 30 minutes to 24 hours at 37°C +/- 1°C, 5+/-1% CO2.

PREPARATION OF TEST SOLUTIONS:
32 mg/cm² of test substance will be used
16 +/- 2 mg of test substance, will be applied to each insert, in triplicate.
Positve control: 5% SDS in sterile water
Negative control: DPBS

TREATMENT OF TISSUES
Before starting the test, RHE inserts will be transferred into a new 24- well plate, supplemented with 300µL of fresh SkinEthic Maintenance Medium (SMM) in each well.
10µL of sterile water and 16+/-2mg of solid test substance will be applied in the center of the insert in triplicate.
For negative and positive controls, 16 +/- 0.5 µL will be applied in triplicate.
The exposure of the tissues to the test and control items was performed at room temperature for 42 minutes (± 1 minute).

RINSING OF TISSUES AND INCUBATION FOR 42 HOURS
At the end of the treatment period, each tissue was removed from the well of the treatment plate, and rinsed with D-PBS to remove any residual test or control items. The rinsed tissues were incubated at 37 °C, 5 % CO2 in a humidified incubator for 42 h.

MTT VIABILITY ASSAY
Following the 42 hour post-exposure incubation period: MTT test (MTT Loading/Formazan Extraction) was performed and tissues were incubated for 3 h (± 5 minutes) at 37 °C, 5 % CO2 in a humidified incubator.

OPTICAL DENSITY MEASUREMENTS
At the end of the formazan extraction period: The optical density was measured at 570 nm using a plate reader.

Irritation / corrosion parameter:

% tissue viability

Value:

79.55

Positive controls validity:

valid

Remarks:

Viability: 1.36%

Irritant / corrosive response data:

Following a 42 minutes exposure and 42 h of recovery period, the relative mean viability of the tissues treated with the test item was 79.55 % with a Variation coefficient of 3.49 as assessed by the MTT assay.

Other effects:

None

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, "OF14 AT" was concluded to be NON IRRITANT.

Executive summary:

The test substance "OF14 AT" was subjected to skin irritation assay, conducted according to OECD 439 (2015).

The test was carried out using reconstructed human epidermis (RHE), in triplicate. The exposure of the insert to the test susbtance was carried out for 42 minutes at room temperature.

After treatment the inserts were rinsed with D-PBS and post-incubated with growth medium for additional 42 hours at 37 +/- 1°C and 5 +/-1% CO2. Finally, inserts were incubated with MTT solution in order to evaluate cell viability which is a direct measure of the irritant potential of the test substance.

Under these conditions, the test substance "OF 14 AT" was concluded to be non-irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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.

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

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

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.

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

Irritation parameter:

other: Estimated IC 50 (%)

Value:

> 50

Vehicle controls validity:

not applicable

Negative controls validity:

not specified

Positive controls validity:

valid

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%

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

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.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

October- November 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Internal laboratory guidelines

Principles of method if other than guideline:

The method is designed to evaluate the biological response of mammalian cells in vitro, by assessment of cell viability after exposure to the test substance.

GLP compliance:

yes

Irritation parameter:

other:

Value:

> 102.75

Interpretation of results:

other: the IC50 of the test item resulted to be 102.75 mg/mL

Conclusions:

Under the test condition applied, the IC50 of the test item "OF14 AT", resulted to be 102.75 mg/mL: this means that at concentrations of the test substance higher than 102.75 mg/mL the cytotoxicity will increase (>50% of cell mortality), while at lower concentrations cytotoxicity will decrease (< 50% of cell mortality).

Executive summary:

The cytotoxicty test was carried out by applying the direct contact method on 8 concentrations of the test substance. The dilutions of the substance, were applied in 6 replicas on BALB-3T3 cells for 24 hours at 37°C. Cytotoxicity was qualitatively evaluated by microscopy and quantitatively by neutral red uptake (NRU) assay. The NRU assay allows the quantitative determination of cytotoxicity expressed as IC 50 value (inhibitory concentration estimated to affect the endpoint by 50%).

Under the test condition applied, the IC50 of the test item "OF14 AT", resulted to be 102.75 mg/ml: this means that at concentrations of the test substance higher than 102.75 mg/mL the cytotoxicity will increase (>50% of cell mortality), while at lower concentrations cytotoxicity will decrease (< 50% of cell mortality). In details, 4 concentrations of the test substance (C5: 64.247 mg/mL, C6: 43.705 mg/mL, C7: 29.732 mg/ml and C8: 20.226 mg/mL) resulted to be non-toxic, as their relative cell viability was superior or equal to 70% of the control group (see ISO 10993 -5:2009, par A.2.4).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In an in vitro eye irritation study Cohesium was found to be only slightly cytotoxic. In an OECD 439 study, OF 14 AT was found to be non-irritant. OF14 AT was tested for cytotoxicity in a GLP study, and the IC50 resulted to be 102.75 mg/mL.

Also, considering the nature of the main constituents of the registered substance (oligo/polysaccharides and the corresponding monomers), the test item does not need to be classified as irritating for eyes and skin under CLP Regulation (1272/2008 CE).