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Description of key information

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:
19/04/2016-22/04/2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Version 02A
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Aspect : liquid
Color : yellow
Storage conditions : room temperature
Test item nature : cosmetic ingredient
Expiry date : 09/02/2017
Physical state at 20°C : Yes
Purity : NA (mixture)
Homogeneity : yes
pH : 3.8
Test system:
human skin model
Remarks:
SkinEthic(TM) RHE model, 0.5 cm²
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Source strain:
other: PK2 PKP.ABC-4
Details on animal used as source of test system:
Batch n° 16-RHE-040
Storage : Prepared/packaged using aseptic techniques. Stored in an incubator at 37°C, 5% CO2 with saturated humidity

Vehicle:
not specified
Details on test system:
Biological safety (blood of the donor) :
- absence of H1V1 and 2 antibodies
- absence of hepatitis C antibodies
- absence of hepatitis B antigen HBs
Biological safety (epidermal of the donor)
- absence of mycoplasma

Quality controls :
- Histological observation : 6.5 cell layers / absence of significant histological abnormalities
- Cell viability : O.D. = 1.1 (CV = 4.2%)
- Barrier function integrity test : 8.1h

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps:
- Observable damage in the tissue due to washing:
- Modifications to validated SOP:
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: yes
- Wavelength: 540 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. if the mean TER value is less than or equal to 5 kΩ and the skin disk is obviously damaged, or if the mean TER value is less than or equal to 5 kΩ, and the skin disc is showing no obvious damage, but the mean disc dye content is greater than or equal to the mean disc dye content of the 10M HCl positive control obtained concurrently.]
- The test substance is considered to be non-corrosive to skin if [complete, e.g. if the mean TER value obtained for the test substance is greater than 5 kΩ, or if the mean TER value is less than or equal to 5 kΩ, and the skin disc is showing no obvious damage, and the mean disc dye content is well below the mean disc dye content of the 10M HCl positive control obtained concurrently.]
- Justification for the selection of the cut-off point(s) if different than recommended in TG 430:
Control samples:
yes, concurrent negative control
yes, concurrent no treatment
yes, concurrent vehicle
Amount/concentration applied:
TEST MATERIAL
- Amount : 16 µL +/- 0.5 µL (treatment or control)
Duration of treatment / exposure:
42 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3 epidermises
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
ca. 93.2
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
ca. 106.9
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
ca. 101.9
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Test validation:
The positive control shows a viability percentage of 2,3% ( lower than 40%) and all criteria are fulfilled, this validates the test.
The negative control shows a viability percentage of 100%

The test item shows a viability percentage of 100,7% (mean value of the 3 experiments) and therefore is considered as non irritant.

Interpretation of results:
GHS criteria not met
Executive summary:

Under the retained experimental conditions and according to the CLP regulation, the test item must not be classified. No symbol, risk phrase, no signal word or hazard statement is required.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19/04/2016-21/04/2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Test item
Description: Yellow liquid
Storage condition: At room temperature
Specific test item requirements
(handling conditions): None
Purity: Not applicable, mixture
Correction factor: No correction factor
Re-test date: 09 February 2017
Disposal of the test item: Destruction (except the archived test item sample) (any remaining test item is kept for at least 6 months after last use in the project and then disposed of according to instructions described in CiToxLAB France in-house procedures)
The test item does not contain surfactants.
Data relating to the characterization of the test item are documented in a test item information sheet (archived with the study files) and a Certificate of Analysis provided by the Sponsor.
The description of the test item was confirmed by the Sponsor in an email dated 04 April 2016 (archived with the study files).
Confirmation of the identity of the test item is the responsibility of the Sponsor.

As the test item is a non-surfactant containing liquid, it was tested undiluted (i.e. in its original form). The test item was a yellow to brownish liquid.
According to CiToxLAB France in-house procedures, the test item was sampled and stored at room temperature until use.
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Origin: bovine eyes obtained from freshly slaughtered cattle at the abattoir EVA, Saint-Pierre-sur-Dives, France.
Age of the cattle: up to 12 months old.
Reason for choice: recommended by regulatory authorities. They are adapted for the evaluation of potential ocular irritants since they are part of the target organ.
Transport from Supplier to CiToxLAB France: the eyes were transported to CiToxLAB France at ambient temperature, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)]. A container with smooth internal surfaces was used for the transport to avoid damage to the corneas
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
yes, concurrent positive control
Amount / concentration applied:
Applied undiluted.
As the test item was a non viscous liquid and could be sampled using a micropipette, a volume of 750 μL (± 8 μL) was applied on each cornea using the closed-chamber method as follows: the test item was introduced into the anterior chamber of the corneal holder, through the dosing holes to cover the epithelial side of the cornea. The dosing holes were then sealed.
Duration of treatment / exposure:
10 minutes (± 30 seconds) (because the test item is a non surfacant liquid)
Duration of post- treatment incubation (in vitro):
Following the 10-minute treatment, the holders were incubated horizontally (corneas placed vertically) for 2 hours (± 10 minutes) in a water bath at +32°C (± 1°C)
Number of animals or in vitro replicates:
3 replicates for each serie (positive control, negative control, test item)
Details on study design:
Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C.
A single experiment was performed using three corneas for each treated series (test item, positive control and negative control).
Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer. The test item, applied undiluted, and the positive and negative controls were evaluated in a single experiment using a treatment time of 10 minutes and using the closed-chamber method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed.
The corneas were then incubated for 2 hours (± 10 minutes) at +32°C before a second opacity measurement was performed.
After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes (± 5 minutes) at +32°C. At the end of the incubation period, the optical density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
ca. 22
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
For the validation of an experiment, the following criteria had to be fulfilled:
- the mean In Vitro Irritancy Score (IVIS) of the positive control corneas should fall within two standard deviations of the historical mean,
- the mean opacity of the negative control corneas should be < 1.8,
- the mean OD490 nm of the negative control corneas should be < 0.0269.
Interpretation of results:
study cannot be used for classification
Executive summary:

The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 22. As the test item-induced a mean IVIS > 3 and ≤ 55, the eye hazard potential of the test item could not be predicted.

Under the experimental conditions of this study, the ocular corrosive or severe irritant potential of the test item, Extrait de plantes KEOC Version 3, could not be predicted. The test item could not be identified as inducing serious eye damage (UN GHS Category 1) or as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category). According to OECD Guideline 437, further testing should be conducted for classification and labeling purposes.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10/10/2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to
Guideline:
other: HET CAM
Principles of method if other than guideline:
The principle is based on the observation of the irritant effects (hyperemia, hemorrhage, coagulation) which may occur within five minutes after placing a test item onto the chorio-allantoic membrane (CAM) of an embryonated hen egg, on the tenth day of incubation. Depending on the presence of Mese effects and their appearance lime, a score is established. The mean scores obtained from four eggs allows to note the test item and to classe according to its irritant potential. This study is carried out according to the Official Journal of the Republic of France (N° 300), December 26". 1996.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Aspect : Solid
Color : Yellowish
Storage conditions : Hygroscopic and room temperature
Test item nature : Cosmetic ingredient
Re-analysis date : 24/11/2017
Purity : By sponsor
Species:
chicken
Strain:
other: White Leghorn
Details on test animals or tissues and environmental conditions:
Egg embryonated (White Leghorn strain) with a weight of 50 to 65 g before incubation.
Receipt of eggs was carried out according to the current instruction IL 01.
The eggs cracked or broken were removed, the other eggs were stored at 20°C +/- 5°C, protected from light. until placed in the incubator.
Before incubation, the eggs were weighed: the weight was between 50 and 65 g. The weight of each egg was recorded on the shell and was retranscribed on the scores sheet during the test.
The eggs weighed were incubated with the air pocket upwards (pointed end of egg downwards), at 37.5°C +/- 0.5°C with a relative humidity of 40 to 60% during 10 days in an egg incubator with automatic oscillating plates.
The study started on the tenth day of incubation.
Vehicle:
physiological saline
Remarks:
NaCl 9g/L, stored at room temperature or diluent recommended by the order giver
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
300 mg of the warmed test item (pure) or reference item are deposited gently on the CAM
Duration of treatment / exposure:
20sec
Duration of post- treatment incubation (in vitro):
5 minutes
Number of animals or in vitro replicates:
Treatment test on 4 egges
Reference test on 2 eggs
Details on study design:
The different steps were performed rapidly under sufficient intensity lighting non heat releasing to avoid drying of the CAM (otherwise the humidity level is maintained by misting).
The egg was placed vertically on a support (air pocket upwards).
The shell was cracked at the level of the air pocket taking care not to injure the CAM, then she was removed using forceps or a pair ofscissors to the level of the shell membrane.
The released surface was moistened with saline solution warmed to 37°C.
After removal of saline solution by lifting the egg, the shell membrane was gently detached with forceps and then removed in order to uncover the underlying CAM.
Any egg with a defective CAM or traces of haemorrhage is removed.
The CAM integrity was recorded for every egg on scores sheet during the test. Eggs which do not have a living hen embryo are removed too.
300 mg of the warmed test item (pure) or reference item are deposited gently on the CAM with a pipette and the timer was immediately started.
After 20 seconds of contact, the membrane was rinsed with 5 ml of saline solution at 37°C. avoiding any violent splashing. The rince liquid was removed by tilting the egg. Any irritant effects were observed for 5 minutes. The lime of occurrence of each effect is recorded.
At the end of testing, the eggs are frozen at-80 °C until the day of waste disposal according to the instruction 1G HSE 02.

The observations taken into account in the scoring of the test item were made with naked eye.
The observed effects were identified according Io the law of all or nothing (the presence and not the severity of the effect was recorded).

- Hyperemia: capillaries which were invisible before adding the lest or references items become visible, whereas capillaries which were already visible dilate and become redder. This phenomenon may also affect large diameler vessels.
- Hemorrhage: release of blood escaping from the vessels and or capillaries. may take on different appearances, and particularly « cauliflower », patches. diffuse sheet, or punctate (the blood escapes intermittently at différent places in the membrane).
it must be noted that:
Hemorrhage may take on a transient appearance: this must nevertheless by counted
Masked hyperemia must be counted if massive haemorrhage occurs during the first 30 seconds
- Coagulation (Opacity and/or Thrombosis):
1. Opacity: apparition on a part or all of the membrane, of an opalescent sheet or direct opacification (take tare that this effect is not due to the physico-chemical properties of the test item in aqueous medium. formation of a colloid, precipitate,...).
2. Thrombosis: discontinuation of blood flow producing a segmented appearance of the vessels (alternating areas of strangulation more or less dark turgescent areas). Changes occurring in the capillaries were trot counted.

An evaluation note was assigned to each phenomenon according toits occurrence lime: Note according to the Lime:
Phenomenon
(Time) T .s"30 s 30 s < T s - 2 min 2 < T ç5 min
Hyperemia 5 3 I
Hemorrhage 7 5 3
Coagulation,
opacity and/or thrombosis 9 7 5

The score for each egg is the sum of each phenomenon notes.
The test item notation is the arithmetic mean, rounded to two decimal points, of the scores obtained for ail eggs (maximum rating = 21).
The test item irritation potential is given by the following scale:
Note (N) Classification
N < 1 Practically non irritant
I s'N < 5 Slightly irritant
5 SN < 9 Moderately irritant
N .. 9 Irritant



Irritation parameter:
in vitro irritation score
Remarks:
Addition of Hyperemia / Hemorrhage / Coagulation score
Run / experiment:
1
Value:
ca. 8
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Remarks:
Addition of Hyperemia / Hemorrhage / Coagulation score
Run / experiment:
2
Value:
ca. 6
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Remarks:
Addition of Hyperemia / Hemorrhage / Coagulation score
Run / experiment:
3
Value:
ca. 8
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Remarks:
Addition of Hyperemia / Hemorrhage / Coagulation score
Run / experiment:
4
Value:
ca. 8
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
IL 04. In order to validate the test, it is essential that the test validity criteria are confirmed:
0.05% Lauryl Sulfobetaine: Practically non-irritant or Slightly irritant
0.4% Lauryl Sulfobetaine: Moderately irritant or Irritant
3.2% Lauryl Sulfobetaine: Irritant

Mean of the irritation score (on 4 eggs) : 7.5

Interpretation of results:
Category 2B (mildly irritating to eyes) based on GHS criteria
Executive summary:

The mean score calculated for the test item is 7.5

Under the retained experimental conditions. the irritant potential of the test item pure tested may be classified as moderately irritant according to the adopted scale.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Justification for classification or non-classification