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Diss Factsheets

Administrative data

Description of key information

- In an in vitro skin irritation study (OECD 439) test item was non irritant to skin (GLP, Rel. 1)

- Serious eye damage/ eye irritant:: Serious eye damage based on the rules of the CLP Regulation for classification of mixtures

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:
2019
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
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
other: non-transformed keratinocytes
Cell type:
non-transformed keratinocytes
Cell source:
other: foreskin
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- 0.50 cm² reconstructed epidermis (Episkin SA, RHE/S/17) were received, and on the same day, the insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The inserts were placed in 6 wells culture plate which had been previously filled with 1 mL of growth medium (Episkin SA) during 2 hours and 35 minutes. Then just before treatment, the inserts were placed in 24 wells culture plate which had been previously filled with 300 µL of maintenance medium (Episkin SA).

TREATMENT
- The test item was applied as supplied, at the approximate dose of 16 mg, on the epidermal surface of 3 living human skin models during 42 minutes at room temperature.
- In the same experimental conditions, a positive control (5% SDS) and a negative control (DPBS) were carried out. The 5% SDS solution was prepared by weighing 0.5 g of SDS in a 10 mL volumetric flask qsp 10 mL of distilled water. Then, the preparation was magnetically stirred, just before the treatment. To ensure a good contact with the epidermises, during all the treatment period, the test item was recovered with a nylon mesh provided by Episkin SA.

REMOVAL OF TEST MATERIAL AND CONTROLS
- 42 minutes after the test item application, the human epidermises were washed with 25 x 1 mL of DPBS. The rinsed tissues were checked for any coloration and noted to be slightly brown instead of being whitish as for the coloration of the negative control tissues. Residual test item with brown coloration was noted on all Reconstructed Human epidermis after the rinse. They were incubated for a 42 hours post-treatment incubation period in fresh medium at 37°C, 5% CO2. Then, the epidermises were put in contact with the MTT solution.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- The cell viability was quantified by the measurement of the cell succinate dehydrogenase activity. This enzyme was responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS No. 298-93-1)] reduction into blue formazan crystal that is quantitatively measured by Optical Density (OD) after extraction from tissues. The measured OD were proportional to the number of living cells.
- The skin samples were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours at 37°C, 5% CO2. The precipitated blue formazan product was then extracted using isopropanol during 2 hours under gentle agitation in the dark, and the concentration of formazan was measured by determining the OD (Optical Density) at 570 nm, just after dilution of the extracts (1:2 in isopropanol).
- The OD of MTT extract was measured in triplicate. The measurement of OD was performed using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

VIABILITY CALCULATION:
- The results were expressed as a viability percentage compared with the negative control: viability % = (mean OD test item / mean OD negative control) x 100
As the test item is identified as a direct MTT reducer, true tissue viability is calculated as follows: True viability % = [(OD of living tissues exposed to test item - OD of killed tissues exposed to test item) / OD of living tissues exposed to negative control] x 100
- Data from individual replicate tissues (OD values and calculated percent tissue viability data for the test item and controls), mean percent tissue viability and standard deviation for each individual test item and control were reported in Table.

PREDICTION MODEL / DECISION CRITERIA
The OD values obtained for each test sample were used to calculate a percentage of viability relative to the negative control, which was arbitrarily set at 100%. The cut-off values for the prediction of irritation associated with the RHE models were as follows:
- The test item is considered to be non-irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is >50%.
- The test item is considered to be irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is ≤ 50% and the result of a skin corrosion test is "non-corrosive". In accordance with Regulation EC No. 1272/2008, the test item has to be classified in Category 2 “Irritant”. The corresponding hazard statement is “H315: Causes skin irritation” with the signal word “Warning”.
- The test item is considered to be irritant or corrosive to skin if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤50% and in absence of information on a skin corrosion test. In accordance with the Regulation (CE) No.1272/2008 and in absence of information on a skin corrosion test, the item has to be classified in Category 2 "Irritant" or in Category 1 "Corrosive". The corresponding hazard statement is respectively, "H315: Causes skin irritation" with the signal word "Warning" or "H314: Causes severe skin burns and eye damage" with the signal word "Danger".
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg
- Concentration (if solution): Undiluted
Duration of treatment / exposure:
42 minutes at room temperature
Duration of post-treatment incubation (if applicable):
42 minutes post-incubation period at 37°C, 5% CO2
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Value:
72.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
MTT VIABILITY ASSAY RESULTS
- The mean percent viability of the treated tissues was 72.1%, versus 1.2% in the positive control (5% Sodium Dodecyl Sulfate).
Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test item was considered as non-irritant to skin. It corresponds to UN GHS No Category. No hazard statement or signal word is required.
Executive summary:

An in vitro skin irritation test using the Reconstructed Human Epidermis (SkinEthic RHE® model) was performed according to the OECD Guideline 439 and in compliance with GLP to predict the acute skin irritation potential of the test item.

The test item was applied as supplied, at the dose of 16 mg, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes, followed by a rinse with 25 mL of PBS and a 42 hours post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

The mean corrected percent viability of the treated tissues was 72.1 %, versus 1.2% in the positive control (5% Sodium Dodecyl Sulfate).

Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test item was considered as non-irritant to skin. It corresponds to UN GHS No Category. No hazard statement or signal word is required.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation, other
Remarks:
Classification based on calculation rules for mixtures of the CLP Regulation
Type of information:
calculation (if not (Q)SAR)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
accepted calculation method
Qualifier:
no guideline required
Principles of method if other than guideline:
Classification based on calculation rules for mixtures of the CLP Regulation
Irritation parameter:
other: Classification
Remarks on result:
other: Eye damage category 1
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Executive summary:

The NCS is composed of several identified constituents and in that, it can be considered as a mixture according to the definition of the CLP Regulation. The decision logic for classification of mixtures from the ECHA Guidance on the Application of the CLP Criteria (2017) was used to determine the serious eye damage/ eye irritant hazard of the registered substance. The decision of classification as Eye damage category 1 is based on existing data on constituents (additivity principles) the registered substance has more than 3% of its constituents classified as Eye damage Category 1 and should be classified as a Eye damage Category 1 without further testing according to the rules for classification of mixtures of Regulation (EC) No 1272/2008.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

An in vitro skin irritation test using the Reconstructed Human Epidermis (SkinEthic RHE® model) was performed according to the OECD Guideline 439 and in compliance with GLP to predict the acute skin irritation potential of the test item.

The test item was applied as supplied, at the dose of 16 mg, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes, followed by a rinse with 25 mL of PBS and a 42 hours post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

The mean corrected percent viability of the treated tissues was 72.1 %, versus 1.2% in the positive control (5% Sodium Dodecyl Sulfate).

Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test item was considered as non-irritant to skin. It corresponds to UN GHS No Category. No hazard statement or signal word is required.

Eye irritation/ Corrosion:

The NCS is composed of several identified constituents and in that, it can be considered as a mixture according to the definition of the CLP Regulation.

The decision logic for classification of mixtures from the ECHA Guidance on the Application of the CLP Criteria (2017) was used to determine the Eye irritation/corrosion hazard of the registered substance. The decision of classification as eye corrosive was based on existing data on constituents (additivity principles) the registered substance has more than 3% classified as Eye damage Category 1 and should be classified as serious eye damage without further testing according to the rules for classification of mixtures of Regulation (EC) No 1272/2008

Constituent

 

CAS

Classification

Source

Benzoic acid

65 -85 -0

H318

Harmonised classification

Source: ECHA disseminated dossiers or self classification

Justification for classification or non-classification

Harmonized classification:

The registered substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available information and typical composition provided by the Lead Registrant, the registered substance is not classified for skin irritantation : but is classified as Serious eye damage Category 1 (H318: Causes serious eye damage) according to the criteria of the Regulation (EC) No. 1272/2008 (CLP).