Registration Dossier

Administrative data

Description of key information

The test item containing 39.8% active ingredient was predicted to be irritant to human skin based on the three-dimensional EST 1000 human skin model, showing a cell viability of 1.3% compared to control, which meets the 50% classification threshold). However it was negative for corrosion in the same test model (3 minutes and 1 hour exposure), showing cell viability of 101 and 78.0% versus controls, respectively, which were above threshold value of 50%. CLP classification 2 for skin irritation is proposed for the registered substance. A 5 and 10% solution were previously tested to be non-irritant based on human application to arm skin for 48 hours, and therefore these are concentration limits for non-classification. 
The test item containing 39.8% active ingredient was predicted to be irritant to human eye based on the in vitro HET-CAM model, showing an in vitro irritation score of 19, which is comparable to positive control value. However it was negative for severe eye irritation and damage in the in vitro BCOP model, showing an in vitro irritation score of 21.13, which was below classification threshold of 55.1. CLP classification 2 for eye irritation is proposed for the registered substance. A 20% solution was previously tested to be mildly irritant based on the Draize method in rabbit eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted according to GLP and valid methods and is considered relevant and reliable for classification. It is an in vitro study predicting skin irritation, which is considered adequate in combination with the in vitro corrosivity testing.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Remarks:
EST1000
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified: Batch no. EST-120730-001; CellSytems® Biotechnology GmbH, 53842 Troisdorf, Germany
Justification for test system used:
Skin irritation by cytotoxic action of substances with direct human skin contact refers to the production of reversible damage to the skin following the application of a test item for up to 4 hours. This test method provides an in vitro procedure that, depending on information requirements, may allow determining the cytotoxic potency and skin irritancy of test items as a stand-alone replacement test within a testing strategy, in a weight of evidence approach.
The test method is based on reconstructed human epidermis models, which in their overall design (the use of human derived epidermis keratinocytes as cell source, representative tissue and cyto-architecture) closely mimic the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The Skin model EST1000 was used.
- Tissue batch number(s): Batch no. EST-120730-001; CellSytems® Biotechnology GmbH, 53842 Troisdorf, Germany
- Production date: August 13, 2012
- Shipping date: Not provided.
- Delivery date: Not provided.
- Date of initiation of testing:
Start of the experimental phase: July 18, 2012
Termination of the experimental phase: August 16, 2012

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 21°C
The models were cultivated at 21°C for 20 minutes according to the instructions of the EST1000 supplier CellSystems®.
- Temperature of post-treatment incubation: not specified
Viability measurements were not performed immediately after the exposure to the test item, but after a post-treatment incubation period of the rinsed tissues in fresh medium of 42-hours.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1
At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT solution of 1 mg/mL
- Incubation time: 3 hours (37°C incubation temperature, 5% CO2, 95% humidity)
- Spectrophotometer: Yes, no further details given.
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The preferred assay for determining the magnitude of viability was the MTT. The optical density (OD) of the extracted (solubilised) dye from the tissue treated with the negative control (NC) should be at least 20-fold greater than the OD of the extraction solvent alone. The tissue treated with NC should exhibit stability in culture (provide similar viability measurements) for the duration of the test exposure period.
- Barrier function: Each batch of the epidermal model used meets defined production release criteria, set by the supplier, among which those for viability and for barrier function are the most relevant (MTT, 2 hours Triton X-100: target > 50%; result 58.5%). The barrier properties of the tissues were verified by the supplier.
- Morphology: No. of cornified layers and No. of vital cell layers were in line with the target of 5 and 4 respectively.
- Contamination: The skin model was free of contamination by bacteria, viruses, mycoplasma, or fungi.
- Reproducibility: Associated and appropriate measures of variability between tissue replicates are defined (e.g. if standard deviations should be ≤ 18%).

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Not applicable

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
1 ( at 20 min exposure + 42 h post-treatment incubation)
9 tests in total (Negative control, Test item and Positive control in triplicate at 1 timepoint)


PREDICTION MODEL / DECISION CRITERIA

- The test substance is considered to be irritant to skin with UN GHS category 2 if the tissue viability after exposure and post-treatment incubation was ≤ 50%
- The test substance is considered to have no category if the tissue viability after exposure and post-treatment incubation was > 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 75.3 µL Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts, were applied to the skin model with a surface of 0.6 cm2 to uniformly cover the skin surface. Usually 30 µL are used, however as the supplied test item contains only 39.8% active matter, a correction factor of 2.51 was used.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 75.3 µL
- Concentration (if solution): water for injection Batch no. 120378141, B. Braun Melsungen AG, 34212 Melsungen, Germany

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% aqueous sodium dodecyl sulphate (SDS) Batch no. 15733; SERVA Electrophoresis GmbH, 69115 Heidelberg, Germany
Duration of treatment / exposure:
20 minutes
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean of 3 replicates; % versus negative control group
Run / experiment:
test group
Value:
1.3
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Time point: 20 minutes, followed by 42h incubation.
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean of 3 replicates; % versus negative control group
Run / experiment:
positive control group
Value:
1.3
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Time point: 20 minutes, followed by 42h incubation.
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: No interactions of the test item with MTT were noted.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The viability of cells treated with the positive reference item, 5% SDS, was 1.3% of the negative controls and below the 50% cut-off value. Hence, 5% SDS is predicted to cause pronounced skin irritation.
All quality criteria required were fulfilled.
No interactions of the test item with MTT were noted.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control (NC): For each assay using valid batches, tissues treated with the NC exhibit OD reflecting the quality of the tissue that followed all shipment and receipt steps and all the irritation protocol process. The OD values of controls should not be below historical established lower boundaries.
- Acceptance criteria met for positive control (PC): Tissues treated with the PC, i.e. 5% aqueous SDS, should reflect the sensitivity retained by tissues and their ability to respond to an irritant substance in the conditions of each individual assay (e.g. viability ≤ 50% for the validated method)
- Acceptance criteria met for variability between replicate measurements: Associated and appropriate measures of variability between tissue replicates are defined (e.g. if standard deviations should be ≤ 18%).

The cell viability was measured by determining the optical density (OD) at a wavelength of 540 nm. An exposure time of 20 minutes was employed.

The test item, Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts,was applied to the model skin surface. Water for injection was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item.

The mean viability of cells exposed to the test item was 1.3% of the negative controls and, hence, below the 50% cut-off value.The test item was considered to be cytotoxic and is predicted to be irritant to skin in accordance with UN GHS category 2.

The viability of cells treated with the positive reference item, 5% SDS,was 1.3% of the negative controls and below the 50% cut-off value. Hence, 5% SDS is predicted to cause pronounced skin irritation.

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Under the present test conditions Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts, tested at an exposure time of 20 minutes, was cytotoxic and, hence, irritant to skin in an experiment employing an artificial three-dimensional model of human skin.

Executive summary:

The purpose of this study was to determine cytotoxic properties to skin cells, which might lead to irritation by Butandioic acid, 2(or 3)-sulfo, 4 - [2 - [(1 -oxo(C12 -C18(even numbered) and C18 unsaturated)alkyl)amino]ethyl], esters, disodium salts to human skin, in an experiment with an artificial three-dimensional model of human skin. The EST1000 model was employed. The test item containing 39.8% active ingredient was applied. The cell viability was measured by determining the optical density (OD) at a wavelength of 540 nm. An exposure time of 20 minutes was employed, followed by refreshment of the medium and further 42 hours incubation. Water for injection was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item. The mean viability of cells exposed to the test item was 1.3% of the negative controls and, hence, below the 50% cut-off value. The test item was considered to be cytotoxic and is predicted to be irritant to skin in accordance with UN GHS category 2.

The viability of cells treated with the positive reference item, 5% SDS, was 1.3% of the negative controls and below the 50% cut-off value. Hence, 5% SDS is predicted to cause pronounced skin irritation.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to GLP and valid methods and is considered relevant and reliable for classification. It is an in vitro study predicting corrosivity, which is considered adequate in combination with the in vitro irritation testing.
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified. Batch no. EST-120730-001; CellSytems® Biotechnology GmbH, 53842 Troisdorf, Germany
Justification for test system used:
Skin corrosion refers to the production of irreversible tissue damage in the skin following the application of a test item [as defined by the Globally Harmonised System for the Classification and Labelling of Chemical Substances and Mixtures (GHS)]. The OECD Guideline 431 does not require the use of live animals or animal tissue for the assessment of skin corrosivity.
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The Skin model EST-1000 was used.
- Tissue batch number(s): Batch no. EST-120730-001; CellSytems® Biotechnology GmbH, 53842 Troisdorf, Germany
- Production date: August 13, 2012
- Shipping date: Not provided.
- Delivery date: Not provided.
- Date of initiation of testing:
Start of the experimental phase: July 18, 2012
Termination of the experimental phase: August 16, 2012

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Not provided
- Temperature of post-treatment incubation (if applicable): No post-treatment incubation

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1
At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT solution of 1 mg/mL
- Incubation time: 3 hours (37°C incubation temperature, 5% CO2, 95% humidity)
- Spectrophotometer: Yes, type not specified.
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The magnitude of viability was quantified by using MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue ). In this case the optical density (OD) of the extracted (solubilised) dye from the negative control tissue was at least 20-fold greater than the OD of the extraction solvent alone. The negative control tissue has been shown to be stable in culture (provide similar viability measurements) for the duration of the test exposure period.
- Barrier function: The stratum corneum has been shown to be sufficiently robust to resist the rapid penetration of certain cytotoxic maker chemicals (e.g. 1% Triton X-100). This property was estimated by the exposure time required to reduce cell viability by 50% (ET50) (for the EST 1000 model this is >2 hours). MTT, 2hrs Triton X-100 resulted in 58.5% barrier function (> 50% target).
- Morphology: No. of cornified layers and No. of vital cell layers were in line with the target of respectively 5 and 4 layers.
- Contamination: The skin model was free of contamination with bacteria (including mycoplasma) or fungi.
- Reproducibility: The tissue employed has been shown to demonstrate reproducibility over time between laboratories. Three tissue replicates were used for the test item and the negative and positive controls instead of two replicates as stated in the Study Plan to assure reproducibility.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Not applicable

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 (3 minutes exposure and 1 hour exposure)
18 tests in total (test group, NC, PC in triplicate (n=9) at 2 exposure times (total n = 18)


PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 125.5 µL Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts were applied to the skin model with a surface area of 0.6 cm2 to uniformly cover the skin surface. Usually 50 µL are used, however as the supplied test item contains only 39.8% active matter, a correction factor of 2.51 was used.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 125.5 µL
- Concentration (if solution): water for injection Batch no. 112558141, B. Braun Melsungen AG, 34212 Melsungen, Germany

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8 N KOH Batch no. 4174013504C04; FLUKA Feinchemikalien GmbH, 89231 Neu-Ulm, Germany
Duration of treatment / exposure:
1st exposure period: 3 min; 2nd exposure period: 60 min
Duration of post-treatment incubation (if applicable):
Not applicable
Number of replicates:
3
Species:
other: three-dimensional human skin model
Strain:
other: The Skin model EST-1000 was used.
Details on test animals and environmental conditions:
Not applicable
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 125.5 µL Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts were applied to the skin model with a surface area of 0.6 cm2 to uniformly cover the skin surface.


Duration of treatment / exposure:
1st period: 3 min; 2nd period: 60 min
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean; % versus negative control group.
Run / experiment:
test group 3 minutes exposure
Value:
101
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean; % versus negative control group.
Run / experiment:
test group 1 hour exposure
Value:
78
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean; % versus negative control group.
Run / experiment:
positive control group 3 minutes exposure
Value:
4.1
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean; % versus negative control group.
Run / experiment:
positive control group 1 hour exposure
Value:
3.6
Negative controls validity:
valid
Positive controls validity:
valid

The test item, Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts,was applied to the skin surface. Water for injection was used as the negative control. 8 N KOH was used as the positive reference item. Two exposure times of 3 minutes or 1 hour were employed.

In comparison to the negative controls, the mean viability of cells exposed to the test item was 101.0% after a 3-minute exposure period and 78.0% after a 1-hour exposure. The OD540 values were well above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of <50% or <15% for a 3-minute or 1-hour treatment, respectively. Therefore, the test item was non-corrosive in this skin model and is predicted to be non-corrosive to human skin.

The mean viability of cells treated with the positive reference item 8 N KOH were 4.1% (3-minute incubation) and 3.6% (1-hour incubation) of the negative controls and were below the cut-off values and well within the range of historical background data of 3.3 – 47.4% (3-minute incubation) and 0.2 – 4.9% (1-hour incubation) of the negative controls (the last 6 experiments, not audited by the QAU-department). Hence, 8 N KOH caused pronounced corrosion in this skin model and is predicted to be corrosive to human skin.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the present test conditions Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts tested at two exposure times of 3 minutes or 1 hour was non-corrosive to skin in an experiment employing an artificial three-dimensional model of human skin.
Executive summary:

The purpose of this study was to determine cytotoxic properties to skin cells which might lead to corrosion by Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts to human skin, in an experiment with an artificial three-dimensional model of human skin. The EST-1000 model was employed. The test item containing 39.8% active ingredient was applied to the skin surface. Water for injection was used as the negative control. 8 N KOH was used as the positive reference item.Two exposure times of 3 minutes or 1 hour were employed. In comparison to the negative controls, the mean viability of cells exposed to the test item was 101.0% after a 3-minute exposure period and 78.0% after a 1 -hour exposure.The OD540 values were well above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of <50% or <15% for a 3-minute or 1-hour treatment, respectively. Therefore, the test item was non-corrosive in this skin model and is predicted to be non-corrosive to human skin.

The mean viability of cells treated with the positive reference item 8 N KOH were 4.1% (3 -minute incubation) and 3.6% (1 -hour incubation) of the negative controls and were below the cut-off values. Hence, 8 N KOH caused pronounced corrosion in this skin model and is predicted to be corrosive to human skin.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
weight of evidence
Study period:
2012-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted according to GLP and valid methods and is considered relevant and reliable for classification. It is an in vitro study by means of BCOP, predicting severe eye irritation, which is considered adequate in combination with the in vitro irritation testing.
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICCVAM BCOP test method, 2007
Deviations:
no
Qualifier:
according to
Guideline:
other: Note for Guidance on Non-Clinical Tolerance Testing of Medicinal Products, CPMP/SWP/2145/00, adopted March 1, 2001.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: Bovine eyes from cattie in the age range of 6 to 12 months were obtained from Hubert Bahlmann GmbH & Co., Lindern.
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: slaughterhouse: Hubert Bahlmann GmbH & Co. Versandschlachterei Spezialmischfutterwerk KG 49699 Lindern, Germany
- Characteristics of donor animals (e.g. age, sex, weight): cattle in the age range of 6 to 12 months
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): To minimize deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution (HBSS) containing 1% Penicillin/Streptomycin . Upon arrival at the laboratory the eyes were examined for defects such as but not limited to increased opacity, scratches and neovascularisation. Only corneas from eyes free of defects were used.
- Time interval prior to initiating testing: > 1 hour
The corneas were dissected with a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers. Beginning with the posterior chambers, the chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM) , while preventing bubble formation. The corneal holder was equilibrated at 32±1°C for at least one hour.
After the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation, neovascularisation) or an opacity >7 opacity units were discarded. The mean opacity of all equilibrated corneas was calculated by use of an opacitometer (BASF OP-3.0). A minimum of three corneas with opacity values close to the median value for all corneas were selected as negative (or solvent) control corneas. The remaining corneas were then distributed into treatment and positive control groups.
- Indication of any existing defects or lesions in ocular tissue samples: Only corneas from eyes free of defects were used.
- Indication of any antibiotics used: Hanks’ Balanced Salt Solution (HBSS) containing 1% Penicillin/Streptomycin
Vehicle:
physiological saline
Remarks:
0.9% NaCl
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration (if solution): The test item was used as a 3.98-fold dilution in 0.9 % NaCl-solution (in order to obtain a 10% w/v dilution of active ingredient, which complies with the description for surfactants).

VEHICLE
- Concentration (if solution): 0.9% NaCl-solution
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
2 hours . After rinsing, the corneas were incubated at 32 ± 1 °C for two hours. After this post-exposure incubation period, the corneas were examined.
Number of animals or in vitro replicates:
3 corneas for each treatment group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The corneas were dissected with a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers. Beginning with the posterior chambers, the chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM), while preventing bubble formation. The corneal holder was equilibrated at 32±1°C for at least one hour.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation, neovascularisation) or an opacity >7 opacity units were discarded. The mean opacity of all equilibrated corneas was calculated by use of an opacitometer (BASF OP-3.0). A minimum of three corneas with opacity values close to the median value for all corneas were selected as negative (or solvent) control corneas. The remaining corneas were then distributed into treatment and positive control groups.

NUMBER OF REPLICATES 3

NEGATIVE CONTROL USED
0.9% NaCl solution
Batch no. 12371453; B. Braun Melsungen AG, 34212 Melsungen, Germany

SOLVENT CONTROL USED (negative control is also solvent control)

POSITIVE CONTROL USED
1% NaOH solution
Batch no. B 3130; Honeywell Specialty Chemicals Seelze GmbH, 30926 Seelze Germany

APPLICATION DOSE AND EXPOSURE TIME
750 µL (of the test or control items)
Exposure period of 10 minutes

TREATMENT METHOD: [closed chamber / open chamber] Not provided.

POST-INCUBATION PERIOD: yes. The corneal holder was equilibrated at 32±1°C for at least one hour.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At least 4 times.
After the exposure period of 10 minutes the exposure solution was removed from each chamber and the epithelium was washed with EMEM containing phenol red at least three times. Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible. The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber. The chamber was then filled with EMEM without phenol red.

- POST-EXPOSURE INCUBATION:
After rinsing, the corneas were incubated at 32±1°C for two hours.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer resulting in opacity values measured on a continuous scale.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry / visible light spectrophotometer using a standard 1 cm path length (OD492)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) = mean opacity value + (15 x mean permeability OD492 value)

DECISION CRITERIA:
A test item that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.
As stated in the OECD guideline, if the test substance is not identified as an ocular corrosive or severe irritant, additional testing should be conducted for classification and labelling purposes. The BCOP test method has an overall accuracy of 79% (113/143) to 81% (119/147), a false positive rate of 19% (20/103) to 21% (22/103), and a false negative rate of 16% (7/43) to 25% (10/40), when compared to in vivo rabbit eye test method data classified according to the EPA, EU, or GHS classification systems. When substances within certain chemical (i.e., alcohols, ketones) or physical (i.e., solids) classes are excluded from the database, the accuracy of BCOP across the EU, EPA, and GHS classification systems ranges from 87% (72/83) to 92% (78/85), the false positive rates range from 12% (7/58) to 16% (9/56), and the false negative rates range from 0% (0/27) to 12% (3/26).
Irritation parameter:
in vitro irritation score
Remarks:
mean
Run / experiment:
test item
Value:
21.9
Negative controls validity:
valid
Remarks:
mean IVIS 0.03
Positive controls validity:
valid
Remarks:
mean IVIS 87.43
Remarks on result:
other: A test item that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative or solvent/vehicle control responses should result in opacity and permeability values, that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control. Upper limit of acceptance for opacity 1.259; Upper limit of acceptance for permeability 0.049.
- Acceptance criteria met for positive control: A test is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean.
Lower limit of acceptance 76.8; Upper limit of acceptance 133.2.

Table 1. In vitro irritancy score (IVIS)

 

Cornea No.

Opacity

Permeability

IVIS

Per Cornea

Per Group

Mean

SD

NaCl 0.9% NC

1

-0.066

0.0160

0.2

0.03

0.15

2

-0.235

0.0130

0.0

3

-0.262

0.0130

-0.1

NaOH 1%

4

53.497

1.851

81.3

87.43

9.11

5

65.173

2.181

97.9

6

51.047

2.137

83.1

Active ingredient 1:10#

7

0.628

1.128

17.5

21.90

3.83

8

2.295

1.479

24.5

9

4.854

1.256

23.7

 

NC: negative control

SD: standard deviation

#: tested as 10% concentration in 0.9% NaCl solution

Interpretation of results:
GHS criteria not met
Conclusions:
Under the present test conditions Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts, tested in the in vitro BCOP test method, had an IVIS value of < 55.1 and consequently it is not classified as a severe irritant and is not corrosive.
Executive summary:

The purpose of this study was to determine if the test item can be classified as “ocular corrosive and severe irritant” employing an in vitro system. The BCOP test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item was assessed by quantitative measurements of changes in corneal opacity and permeability in isolated corneas from bovine eyes. The corneas of the eyes were dissected and the remaining sclera was mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers.

Corneal opacity was measured quantitatively as the amount of light transmission through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea, as detected in the medium in the posterior chamber. The measurements were used to calculate an in vitro irritancy score (IVIS), which was used to assign an in vitro irritancy hazard classification category for prediction of the in vivo ocular irritation potential of the test item.

Three corneas were used for each treatment group (test item, negative and positive controls). The test item was used as a 3.98-fold dilution in 0.9% NaCl-solution in order to obtain a 10% w/v dilution of active ingredient, which complies with the guideline requirements for surfactants. 0.9% NaCl solution was used as the negative control and 1% NaOH solution as the positive control item. The test item and the controls were applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder for an exposure time of 10 minutes.

The optical density (OD492) or absorbance values were measured at a wavelength of 492 nm. An opacity value of 2.592 and a permeability value of 1.288 compared to the negative control were determined. An IVIS 21.90 was calculated. Hence, the test item was not classified as a severe irritant and not corrosive, based on the results of this test.

The corneas treated with the positive control item 1% NaOH solution revealed an opacity value of 56.572 and a permeability value of 2.056 compared to the negative control. The IVIS value of 87.43 was well above the cut-off value of 55.1 and, hence, the acceptance criteria for the test were fulfilled. 1% NaOH solution was a severe irritant and corrosive to eyes.

Under the present test conditions Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts, tested in the in vitro BCOP test method, had an IVIS value of < 55.1 and consequently it is not classified as a severe irritant and is not corrosive.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1962
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented study report which meets basis scientific principles, however the method does not comply to current requirements on limited details were available on materials and methods.
Qualifier:
equivalent or similar to
Guideline:
other: Draize, Calvery and Woodward ( Journ. Pharm. Exp. Therap. 82, 377-390, 1944)
GLP compliance:
no
Species:
rabbit
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Not provided
Vehicle:
other: distilled water
Controls:
other: London tap water
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): 20% active matter (except for the proprietary shampoo and the proprietary baby shampoo which were tested at full strength)

Duration of treatment / exposure:
Test solution was instilled into the conjunctival sac of one eye of each rabbit. No washing was done after instillation.
Observation period (in vivo):
7 days
Number of animals or in vitro replicates:
3
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): No


SCORING SYSTEM: Draize scoring method

TOOL USED TO ASSESS SCORE: visually and with the aid of an ophtalmoscope
Irritation parameter:
other: total cornea score (opacity and area involved)
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
80
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
other: total conjunctivae score
Basis:
mean
Time point:
24/48/72 h
Score:
1.77
Max. score:
20
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
other: total conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
3.33
Max. score:
20
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
other: total conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.67
Max. score:
20
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
other: total conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1.33
Max. score:
20
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
other: total iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
10
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
2

Table 1. Results Rabbit 3.G : Eye irritation study with [Trade name] (20% active matter)

 

Hours

Days

 

1

2

4

6

24

48

72

4

7

CORNEA

 

 

 

 

 

 

 

 

 

Opacity

0

0

0

0

0

0

0

0

0

Area

0

0

0

0

0

0

0

0

0

Score

0

0

0

0

0

0

0

0

0

IRIS

 

 

 

 

 

 

 

 

 

Values

0

0

0

0

0

0

0

0

0

Score

0

0

0

0

0

0

0

0

0

CONJUNCTIVAE

 

 

 

 

 

 

 

 

 

Redness

2

3

3

3

2

1

0

0

0

Chemosis

1

2

2

2

1

0

0

0

0

Discharge

2

2

2

2

1

0

0

0

0

Score

10

14

14

14

8

2

0

0

0

 

 

 

 

 

 

 

 

 

 

Total Score

10

14

14

14

8

2

0

0

0

 

 

Table 2. Results Rabbit 3.V : Eye irritation study with [Trade name] (20% active matter)

 

Hours

Days

 

1

2

4

6

24

48

72

4

7

CORNEA

 

 

 

 

 

 

 

 

 

Opacity

0

0

0

0

0

0

0

0

0

Area

0

0

0

0

0

0

0

0

0

Score

0

0

0

0

0

0

0

0

0

IRIS

 

 

 

 

 

 

 

 

 

Values

0

0

0

0

0

0

0

0

0

Score

0

0

0

0

0

0

0

0

0

CONJUNCTIVAE

 

 

 

 

 

 

 

 

 

Redness

2

3

3

3

1

0

0

0

0

Chemosis

2

3

3

2

0

0

0

0

0

Discharge

2

2

2

2

0

0

0

0

0

Score

12

16

16

14

2

0

0

0

0

 

 

 

 

 

 

 

 

 

 

Total Score

12

16

16

14

2

0

0

0

0

 

Table 3. Results Rabbit 3.R : Eye irritation study with [Trade name] (20% active matter)

 

Hours

Days

 

1

2

4

6

24

48

72

4

7

CORNEA

 

 

 

 

 

 

 

 

 

Opacity

0

0

0

0

0

0

0

0

0

Area

0

0

0

0

0

0

0

0

0

Score

0

0

0

0

0

0

0

0

0

IRIS

 

 

 

 

 

 

 

 

 

Values

0

0

0

0

0

0

0

0

0

Score

0

0

0

0

0

0

0

0

0

CONJUNCTIVAE

 

 

 

 

 

 

 

 

 

Redness

2

2

2

2

1

0

0

0

0

Chemosis

2

2

3

2

0

0

0

0

0

Discharge

2

2

2

2

1

0

0

0

0

Score

12

12

14

12

4

0

0

0

0

 

 

 

 

 

 

 

 

 

 

Total Score

12

12

14

12

4

0

0

0

0

 

Interpretation of results:
GHS criteria not met
Conclusions:
mild irritation reversible within 3 days.
Executive summary:

The objective of the study was to determine the potential eye irritation properties of different samples of which the chemical nature was not disclosed. The method used was generally that of Draize, Calvery and Woodward (Journ. Pharm. Exp. Therap. 82, 377 -390, 1944) and later described in "Appraisal of Safety of Chemicals in Foods, drugs and Cosmetics" (F.D.A. Div. Pharmacology, Draize J.H., 1959). The test solutions were made up by diluting the submitted samples of declared content of active matter with distilled water so that the content of active matter was reduced to 20% in each case. The tap water used for the control was London Tap Water. 0.1mL of the test solution (20% act. ingr.) was instilled into the conjunctival sac of one eye of each rabbit, the other eye serving as a control. In no case was the eye washed after instillation. For the sample of [Trade name], 3 rabbits were used. Readings were made visually and with the aid of an ophthalmoscope at 1, 2, 4, 6, 24, 48, 72 hours and 4 and 7 days after instillation and evaluated according to the Draize scoring method.
For the sample of [Trade name], mild irritation was observed which was reversible within 3 days.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted according to GLP and valid methods and is considered relevant and reliable for classification. However it is an in vitro study only predicting eye irritation, and in isolation, not adequate for classification. It is considered adequate in combination with the BCOP test.
Qualifier:
according to
Guideline:
other: ICCVAM-Recommended Test Method Protocol: Hen’s Egg Test – Chorioallantoic Membrane (HET-CAM) Test Method, published 2010.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: fertile chicken eggs
Strain:
other: White legghorn
Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 300 µL/egg (used undiluted)
Duration of treatment / exposure:
20 seconds
Observation period (in vivo):
5 minutes
Irritation parameter:
other: Irritation score
Remarks:
mean
Run / experiment:
test item
Value:
18.6
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: maximum score 21.0
Irritation parameter:
other: irriatation score
Remarks:
mean
Run / experiment:
0.1% NaOH positive control
Value:
19.4
Remarks on result:
other: maximum score 21.0
Irritation parameter:
other: irritation score
Remarks:
mean
Run / experiment:
1% SDS positive control
Value:
10
Remarks on result:
other: maximum score 21.0
Irritation parameter:
other: irritation score
Remarks:
mean
Run / experiment:
negative control
Value:
0
Remarks on result:
other: maximum score 21.0
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The HET-CAM assay is considered acceptable if the negative controls each induce a response that falls within the classification of nonirritating.
- Acceptance criteria met for positive control: The HET-CAM assay is considered acceptable if the positive controls each induce a response that falls within the classification of severely irritating, respectively.
- Range of historical values: Historical control studies demonstrate that using 1% SDS and 0.1 N NaOH as positive controls results in IS values of approximately 10 and 18, respectively.

The eggs treated with Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)­alkyl))amino]­ethyl]­esters, disodium salts revealed a pronounced effect with an irritation index (IS) of 18.6. The test item was considered to be strong irritant.

The positive control items 0.1% NaOH or 1% SDS caused the expected effect with irritation indices (IS) of 19.4 or 10.0, respectively and, hence, were well within the historical data-range. No effects were observed in the negative control 0.9% NaCl solution. Hence, the HET-CAM assay is considered to be valid.

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
Under the present test conditions, Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts was strong irritant to eyes in an experiment with the chorioallantoic membrane of hens' eggs (HET-CAM).
Executive summary:

The purpose of this study was to determine the eye irritancy potential of Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts by means of the chorioallantoic membrane of hens' eggs (HET-CAM). Eye irritation caused by external contact with chemical substances is characterized by corneal damage and/or conjunctival injury and/or iris defects. The CAM of fertile eggs incubated for 9 days is a vital vascular membrane with a blood vessel complex.

Three eggs each were treated with 300 µL Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts/egg and with the control items. 0.9% NaCl solution was used as the negative control item. 0.1 N Sodium hydroxide (NaOH) and 1% aqueous sodium dodecyl sulphate (SDS) were used as the positive control items. The administration volume for the control items was 300 µL per egg.

After administration of the test item blood vessels including the capillary system and the albumen were examined and scored for irritant effects (haemorrhage, coagulation and lysis) during 5 minutes.

The eggs treated with Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1- oxo(C12-C18(even numbered) and C18unsaturated)­alkyl))amino]­ethyl]­esters, disodium salts revealed a pronounced effect with an irritation index (IS) of 18.6. The test item was considered to be strong irritant.

The positive control items 0.1or 1% SDS caused the expected effect with irritation indices (IS) of 19.4 or 10.0, respectively and, hence, were well within the historical data-range. No effects were observed in the negative control 0.9% NaCl solution. Hence, the HET-CAM assay is considered to be valid.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Assessment of skin irritation was based on a weight-of-evidence approach:

- In an in vivo (human) study, a 1 cm2 of unmedicated white lint was soaked in the 5 and 10% test item solution in comparison with toilet soaps, and applied to the skin of the left arm of 4 volunteers for 48 hours under waterproof adhesive dressing, followed by an observation period of 72 hours (Baker Research Laboratories limited, 1962a). No reactions were detected.

- In a first in vitro study, the corrosive properties of the registered substance were studied in an experiment with a three-dimensional EST-1000 human skin model (Flügge, 2013a). The test item (containing 39.8% active ingredient) was applied for exposure times of 3 minutes or 1 hour. The mean viability of cells exposed to the test item was 101.0% after a 3-minute exposure period and 78.0% after a 1-hour exposure. These values were well above the cut-off percentage cell viability values distinguishing corrosive from non-corrosive test items of <50% or <15% for a 3-minute or 1-hour treatment, respectively. Therefore, the test item was non-corrosive in this human skin model and is predicted to be non-corrosive to human skin.

- In a second in vitro study, the irritant properties of the registered substance to skin cells were also studied in an experiment with a three-dimensional EST-100 human skin model (Flügge, 2013b). The test item (containing 39.8% active ingredient) was applied for an exposure time of 20 minutes, followed by refreshment of the medium and further 42 hours incubation. The mean viability of cells exposed to the test item was 1.3% of the negative controls and, hence, below the 50% cut-off value. The test item was considered to be cytotoxic and is predicted to be irritant to skin in accordance with UN GHS category 2.

- According to ECHA progress report 2010 (p. 32), it is accepted that in vitro methods for skin irritation represent a full replacement of the in vivo method in a tiered testing strategy and in conjunction with in vitro skin corrosivity tests, if necessary. A positive result in the human skin model for irritation does not need to be confirmed by additional testing and shall lead to classification.

 

Assessment of eye irritation was also based on a weight-of-evidence approach:

- In an in vivo study, the potential eye irritation properties of different samples were tested according to the Draize mehod in rabbits (Baker Research Laboratories limited, 1962b). The test solutions were diluted with distilled water so that the content of active matter was reduced to 20% in each case. 0.1mL of the test solution (20% act. ingr.) was instilled into the conjunctival sac of one eye of each rabbit, the other eye serving as a control (3 rabbits were used). Readings were made visually and with the aid of an ophthalmoscope at 1, 2, 4, 6, 24, 48, 72 hours and 4 and 7 days after instillation and evaluated according to the Draize scoring method. Mild irritation was observed which was reversible within 3 days, which was below threshold of classification.

- In a first in vitro study the eye irritancy potential of test item containing 39.8% active ingredient was tested by means of the chorioallantoic membrane of hens' eggs (HET-CAM) method (Haferkorn, 2012). Three eggs each were treated with 300 µL/egg. After administration of the test item, blood vessels including the capillary system and the albumen were examined and scored for irritant effects (haemorrhage, coagulation and lysis) during 5 minutes. The test item treated eggs revealed a pronounced effect with an irritation index (IS) of 18.6, compared to IS of 19.4 or 10.0 for 0.1% NaOH and 1% SDS positive controls and no effects in the negative control 0.9% NaCl solution. The test item was predicted to be irritating.

- In a second in vitro study severe eye irritancy potential and corrosivity potential of the test item containing 39.8% active ingredient was tested by means of the BCOP test method (Leuschner, 2013). Three corneas were used for each treatment group (test item, negative and positive controls). The test item was used as a 3.98-fold dilution in 0.9% NaCl-solution in order to obtain a 10% w/v dilution of active ingredient, which complies with the guideline requirements for surfactants. 0.9% NaCl solution was used as the negative control and 1% NaOH solution as the positive control item. An IVIS score of 21.90 was calculated, hence the test item was not classified as a severe irritant and not corrosive, based on the results of this test. The corneas treated with the positive control item 1% NaOH solution revealed an IVIS score of 87.43 was well above the cut-off value of 55.1 and, hence, the acceptance criteria for the test were fulfilled. The test item, containing 39.8% active ingredient was consequently not classified as a severe irritant and is not corrosive.

- According to Column 2, the criteria for classification are met as irritating to eyes, based on weight of evidence, and further in vivo testing was therefore not needed.

 

Conclusion

- A subgroup CLP category 2 classification was decided for skin irritation, with concentration limit of 10% for non-classification of registered substance.

- A subgroup CLP category 2 classification was decided for eye irritation, with concentration limit of 20% for non-classification of registered substance.

- More information on the subgroup classification is provided in the read-across justification, separately attached in Section 13.

 

Justification for selection of skin irritation / corrosion endpoint: Although the in vitro study for irritation was selected, the corrosion study was equally valuable in a weight-of-evidence approach. 

Justification for selection of eye irritation endpoint: Although the BCOP study was selected, the HET-CAM and in vivo studies were equally valuable in a weight-of-evidence approach. 

Justification for classification or non-classification

The registered substance is classified for skin irritation according to the CLP regulation (No. 1272/2008 of 16 December 2008) as Category 2, with signal word 'warning' and hazard statement: H315 - Causes skin irritation. However, a concentration limit of 10% for non-classification can be applied.

For the eye, the test substance is classified as irritating to eyes according to CLP regulation as Category 2, with signal word 'warning' and hazard statement: H319 -Causes serious eye irritation. However, a concentration limit of 20% for non-classification can be applied.