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Description of key information

The test substance containing 34.4% active ingredient was predicted to be non-irritating  to human skin based on the in vitro EST-1000 human skin model for corrosion (3 minutes and 1 hour exposure) and irritation (20 minutes exposure, followed by 42 hours incubation). The same test substance was predicted to be  irritant to human eye based on the in vitro HET-CAM model, however it was negative for severe eye irritation in the in vitro BCOP model. A 35.8% solution was previously tested to be mildly irritant based on the Draize method  in rabbit eyes.  Finally test substance containing 34.4%  active ingredient was tested to be not irritating in rabbit eyes. In conclusion, a subgroup CLP category 2 classification was proposed, with concentration limit of 34% or less for non-classification. 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2012
Reliability:
2 (reliable with restrictions)
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
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
other: a three-dimensional reconstructed human epidermis model
Strain:
other: The EST1000 model was employed.
Details on test animals or test system and environmental conditions:
Not applicable
Details on study design:
TEST SITE
- Area of exposure: skin model with a surface area of 0.6 cm2

REMOVAL OF TEST SUBSTANCE
- Washing (if done): with Dulbecco’s phosphate buffered saline (D-PBS)
Irritation parameter:
other: cell viability test group
Basis:
mean
Time point:
other: 3 minutes
Score:
92.2
Remarks on result:
other: % versus negative control group
Irritation parameter:
other: cell viability test group
Basis:
mean
Time point:
other: 1 hour
Score:
74.1
Remarks on result:
other: % versus negative control group
Irritation parameter:
other: cell viability positive control group
Basis:
mean
Time point:
other: 3 minutes
Score:
21.6
Remarks on result:
other: % versus negative control group
Irritation parameter:
other: cell viability positive control group
Basis:
mean
Time point:
other: 1 hour
Score:
12.3
Remarks on result:
other: % versus negative control group

The test item, Aspartic acid, N-(3 -carboxy-1 -oxo-sulfopropyl)-N-(C16 -C18(even numbered), C18unsaturated alkyl) tetrasodium salts, was applied to the skin surface. Water for injection was used as the negative control. 8N 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 92.2% after a 3-minute exposure period and 74.1% 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 21.6% (3-minute incubation) and 12.3% (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 LPT 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:
other: predicted non-corrosive
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the present test conditions Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18unsaturated alkyl) tetrasodium 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 Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18unsaturated alkyl) tetrasodium 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, Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18unsaturated alkyl) tetrasodium 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 92.2% after a 3-minute exposure period and 74.1% after a 1-hour exposure.The OD540values were well above the cut-off percentage cellviability 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 21.6% (3-minute incubation) and 12.3% (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:
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
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
other: three-dimensional reconstructed human epidermis model
Strain:
other: The EST1000 model was employed.
Irritation / corrosion parameter:
other: other: cell viability test group
Value:
104.1
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 20 minutes, followed by 42h incubation. Remarks: % versus negative control group. (migrated information)
Irritation / corrosion parameter:
other: other: cell viability positive control group
Value:
< 0.1
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 20 minutes, followed by 42h incubation. Remarks: % versus negative control group. (migrated information)
The cell viability was measured by determining the optical density (OD) at a wavelength of 540 nm. Anexposure time of 20 minutes was employed.
The test item, Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18unsaturated alkyl) tetrasodium 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 the cells exposed to the test item was 104.1% of the mean negative control value. The OD540values were well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of >50% for a 20-minute exposure.
The test item was considered to be non-cytotoxic and predicted to be not irritant to skin.
The viability of cells treated with the positive reference item, 5% SDS,was <0.1% of the negative controls and below the 50% cut-off value. Hence, 5% SDS is predicted to cause pronounced skin irritation.
Interpretation of results:
other: predicted non-irritating
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the present test conditions Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18unsaturated alkyl) tetrasodium salts, tested at an exposure time of 20 minutes, was non-cytotoxic and not 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 Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18unsaturated alkyl) tetrasodium saltsto human skin,in an experiment with an artificial three-dimensional model of human skin. The EST-1000 model was employed.

The cell viability was measured by determining the optical density (OD) at a wavelength of 540 nm. Anexposure time of 20 minutes was employed. The test item, Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18unsaturated alkyl) tetrasodium salts,was applied to the modelskin 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 the cells exposed to the test item was 104.1% of the mean negative control value. The OD540 values were well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of >50% for a 20-minute exposure.

The test item was considered to be non-cytotoxic and predicted to be not irritant to skin.

The viability of cells treated with the positive reference item, 5% SDS,was <0.1% of the negative controls and below the 50% cut-off value. Hence, 5% SDS is predicted to cause pronounced skin irritation.

Endpoint:
skin irritation: in vivo
Data waiving:
other justification
Justification for data waiving:
other:
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:
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 eye irritation, which is considered adequate in combination with the in vitro testing for serious eye damage.
Qualifier:
according to guideline
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. QA statement)
Species:
other: fertile chicken eggs
Strain:
other: White legghorn
Details on test animals or tissues and environmental conditions:
Not applicable
Vehicle:
physiological saline
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 index
Remarks:
mean
Run / experiment:
test group
Value:
10
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: maximum score = 21
Irritation parameter:
other: irritation index
Remarks:
mean
Run / experiment:
0.1% NaOP positive control group
Value:
18.3
Remarks on result:
other: maximum score = 21
Irritation parameter:
other: irritation index
Remarks:
mean
Run / experiment:
1% SDS positive control group
Value:
10
Remarks on result:
other: maximum score = 21
Irritation parameter:
other: irritation index
Remarks:
mean
Run / experiment:
0.9% NaCl negative control group
Value:
0
Remarks on result:
other: maximum score = 21

The purpose of this study was to determine the eye irritancy potential of Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18­unsaturated alkyl) tetrasodium 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 Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18unsaturated alkyl) tetrasodium 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 Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18unsaturated alkyl) tetrasodium salts revealed aneffect with an irritation index (IS) of 10.0. The test item was considered to be a strong irritant.

The positive control items 0.1% NaOH or 1% SDS caused the expected effect with irritation indices (IS) of 18.3 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:
other: predicted irritating
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the present test conditions, Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18unsaturated alkyl) tetrasodium salts was a strong irritant to eyes in an experiment employing the chorioallantoic membrane of hens' eggs (HET-CAM) as model.
Executive summary:

The purpose of this study was to determine the eye irritancy potential of Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18­unsaturated alkyl) tetrasodium 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 Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18unsaturated alkyl) tetrasodium 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 Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18unsaturated alkyl) tetrasodium salts revealed an effect with an irritation index (IS) of 10.0. The test item was considered to be a strong irritant.

The positive control items 0.1or 1% SDS caused the expected effect with irritation indices (IS) of 18.3 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:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
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 predicting serious eye damage, which is considered adequate in combination with the in vitro testing for irritation.
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Qualifier:
according to guideline
Guideline:
other: ICCVAM BCOP test method, 2007
Qualifier:
according to guideline
Guideline:
other: Note for Guidance on Non-Clinical Tolerance Testing of Medicinal Products, CPMP/SWP/2145/00, adopted March 1, 2001.
GLP compliance:
yes (incl. QA statement)
Species:
other: Bovine eyes from cattle 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:
Not applicable
Vehicle:
physiological saline
Controls:
other: Negative control item: 0.9% NaCI solution; Positive control item: 1% NaOH solution
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750µL
- Concentration (if solution): The test item will be used as a 3.44-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
Duration of treatment / exposure:
10 minutes
Observation period (in vivo):
After rinsing, the corneas were incubated at 32 ± 1 °C for two hours. After this post-exposure incubation period, the corneas were examined.
Details on study design:
Corneal injury was assessed by evaluating the opacity and permeability of the cornea.
Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with an opacitometer resulting in opacity values measured on a continuous scale.
Irritation parameter:
in vitro irritation score
Remarks:
mean
Run / experiment:
test group
Value:
20.67
Positive controls validity:
valid
Remarks on result:
other: A test item that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.
Interpretation of results:
other: predicted non corrosive
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the present test conditions Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18unsaturated alkyl) tetrasodium 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 3.44-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 2.133 and a permeability value of 1.235 compared to the negative control were determined. An IVIS 20.67 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 92.985 and a permeability value of 1.795 compared to the negative control. The IVIS value of 119.90 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 Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18unsaturated alkyl) tetrasodium 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:
key study
Study period:
2013
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.
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
Himalayan
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: LPT Laboratory of Pharmacology and Toxicology GmbH & Co. KG, branch Löhndorf, 24601 Löhndorf/¬Post Wankendorf Germany
- Age at dosing: Approx. 8 months
- Weight at dosing: Animal no.1: 3.11 kg; Animal no. 2: 2.83 kg; Animal no. 3: 2.76 kg
- Fasting period before study: Not provided
- Housing: For 8 hours following test item application, the animals were kept singly in restrainers which allowed free movement of the head but prevented a complete body turn, wiping of the eyes with the paws and excluded irritation of the eye by excrements and urine.
During the acclimatisation period and after the 8-hour period in restrainers, the animals were kept singly in cages with dimensions of 380 mm x 425 mm x 600 mm (manufacturer: Dipl. Ing. W. EHRET GmbH, 16352 Schönwalde, Germany)
- Diet (e.g. ad libitum): Commercial diet, ssniff® K-H V2333 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany), served as food. The food was available ad libitum before and after the exposure period.
- Water (e.g. ad libitum): Tap water was offered ad libitum before and after the exposure period.
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C ± 3°C (maximum range)
- Humidity (%): 30% - 70% (maximum range)
- Air changes (per hr): Not provided
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: March 22, 2013 To: April 2, 2013
Vehicle:
unchanged (no vehicle)
Controls:
other: The left eye, which remained untreated, served as a control.
Amount / concentration applied:
- Amount(s) applied (volume or weight with unit): 0.1 mL of the test item were administered into one eye each of three animals.
Duration of treatment / exposure:
Single instillation into the conjunctival sac

Observation period (in vivo):
5 days
Number of animals or in vitro replicates:
3
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): No.
The test item was placed into the conjunctival sac of the right eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second in order to prevent loss of the material.

SCORING SYSTEM:
CORNEA
Opacity: degree of density (area most dense taken for reading)
no ulceration or opacity: 0
scattered or diffuse areas of opacity (other than slight dulling of normal lustre), details of iris clearly visible: 1
easily discernible translucent area, details of iris slightly obscured: 2
nacreous areas, no details of iris visible, size of pupil barely discernible: 3
opaque cornea, iris not discernible through the opacity: 4
IRIS
normal: 0
markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia, or injection, iris reactive to light (a sluggish reac¬tion is considered to be an effect): 1
haemorrhage, gross destruction, or no reaction to light: 2
CONJUNCTIVAE
Redness (refers to palpebral and bulbar conjunctivae, excluding cornea and iris)
normal: 0
some blood vessels hyperaemic (injected): 1
diffuse, crimson colour; individual vessels not easily discernible: 2
diffuse beefy red: 3
CHEMOSIS
Swelling/ refers to lids and/or nictitating membranes
normal: 1
some swelling above normal: 1
obvious swelling with partial eversion of lid: 2
swelling with lids about half-closed: 3
swelling with lids more than half-closed: 4

TOOL USED TO ASSESS SCORE: hand-slit lamp/ fluorescein
The eyes were examined ophthalmoscopically with a slit lamp prior to the administration and 1, 24, 48, 72 hours, 4 to 5 days after the administration. The eye reactions were observed and registered.
24 hours after administration, fluorescein was applied to the eyes before being examined to aid evaluation of the cornea for possible lesions.
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
mean
Time point:
24/48/72 h
Score:
1.11
Max. score:
3
Reversibility:
fully reversible within: 5 days
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.67
Max. score:
3
Reversibility:
fully reversible within: 72h
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.33
Max. score:
3
Reversibility:
fully reversible within: 4 days
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1.33
Max. score:
3
Reversibility:
fully reversible within: 5 days
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
0.45
Max. score:
4
Reversibility:
fully reversible within: 4 days
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48h
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 4 days
Irritant / corrosive response data:
Cornea opacity (grade 1) was observed in all animals 60 minutes after instillation.
Conjunctival redness was observed in all animals:
- animal no. 1: 60 minutes to 48 hours (grade 1) after instillation;
- animal no. 2: 60 minutes and 24 hours (grade 2), 48 hours and 72 hours (grade 1) after instillation;
- animal no. 3: 60 minutes and 24 hours (grade 2), 48 hours to 4 days (grade 1) after instillation.
Chemosis was observed in all animals 1 hour, in animal no. 2 until 24 hours and in animal no. 3 until 72 hours after instillation.
The fluorescein tests performed 24 hours after instillation revealed corneal staining in animal no. 2 (1/2 to 3/4 of the surface) and in animal no. 3 (up to 1/4 of the surface).
The irises were not affected by instillation of the test item.

Table 1. Examination of the treated eye (Animal no. 1/2/3)

Time after administration

Cornea

opacity

Iris

Conjunctivae

Redness

Chemosis

Before dosing

0/0/0

0/0/0

0/0/0

0/0/0

60 minutes

1/1/1

0/0/0

1/2/2

1/1/1

24 hours

0/0/0

0/0/0

1/2/2

0/1/1

48 hours

0/0/0

0/0/0

1/1/1

0/0/1

72 hours

0/0/0

0/0/0

0/1/1

0/0/1

4 days

-/-/0

-/-/0

-/-/1

-/-/0

5 days

-/-/0

-/-/0

-/-/0

-/-/0

24 hours fluorescein test:

animal no. 1: no pathological findings
animal no. 2: corneal staining (1/2 to 3/4 of the surface)
animal no. 3: corneal staining (up to 1/4 of the surface)

Interpretation of results:
not irritating
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
According to the EC-Commission directive 67/548/EEC and its subsequent amendments on the approximation of the laws, regulations and administrative provision relating to the classification, packaging and labelling of dangerous substances and the results obtained under the present test conditions, Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18unsaturated alkyl) tetrasodium salts is non-irritating to eyes, hence, no labelling is required.
Also, according to the EC-Regulation 1272/2008 and subsequent regulations, the test item is non-irritating to eyes; classification and labelling of the substance is not necessary.
Executive summary:

Under the present test conditions, a single instillation of 0.1 mL Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18unsaturated alkyl) tetrasodium salts per animal into the conjunctival sac of the right eye of three rabbits caused the following changes:

Cornea opacity (grade 1) was observed in all animals 60 minutes after instillation.

Conjunctival redness was observed in all animals:

- animal no. 1: 60 minutes to 48 hours (grade 1) after instillation;

- animal no. 2: 60 minutes and 24 hours (grade 2), 48 hours and 72 hours (grade 1) after instillation;

- animal no. 3: 60 minutes and 24 hours (grade 2), 48 hours to 4 days (grade 1) after instillation.

Chemosis was observed in all animals 1 hour, in animal no. 2 until 24 hours and in animal no. 3 until 72 hours after instillation.

The fluorescein tests performed 24 hours after instillation revealed corneal staining in animal no. 2 (1/2 to 3/4 of the surface) and in animal no. 3 (up to 1/4 of the surface).

The irises were not affected by instillation of the test item.

All lesions observed had disappeared at 72 hours or 5 days after instillation.

There were no systemic intolerance reactions concerning behaviour, body weight and food consumption.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

Assessment was performed by a weight-of-evidence approach.

- A first in vitro study was conducted with registered substance to determine potential corrosive properties in an artificial three-dimensional EST-1000 human skin model (Flügge, 2012a). The test item containing 34.4% active ingredient, was applied to the skin surface during two exposure times of 3 minutes and 1 hour. The cell viability was measured by determining the optical density (OD) at a wavelength of 540 nm. In comparison to the negative controls, the mean viability of cells exposed to the test item was 92.2% after a 3-minute exposure period and 74.1% after a 1-hour exposure.The values were well above the cut-off percentage cellviability 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.

- A second in vitro study with registered substance was performed to determine potential irritating properties in an artificial three-dimensional EST-1000 human skin model (Flügge, 2012b). The test item containing 34.4% active ingredient, was applied to the skin surface during 20 minutes, followed by refreshment of the medium and incubation for 42 hours. The cell viability was measured by determining the optical density (OD) at a wavelength of 540 nm.The mean viability of the cells exposed to the test item was 104.1% of the mean negative control value. The value was well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of >50% for a 20-minute exposure. The test item was considered to be non-cytotoxic and predicted to be not irritant to skin.

- 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 negative result in the human skin model for irritation does not need to be confirmed by additional testing.

- In conclusion, the test substance was predicted to be non-corrosive and non-irritant to human skin based on the three-dimensional EST 1000 human skin model, therefore no classsifciation is needed.

Eye irritation

- There was a supporting in vivo eye irritation study with test substance containing 35.8% active ingredient tested in three albino rabbits (Tusing, 1955). A single application of 0.05 mL of the undiluted test item was placed in the conjunctival sac of the left eye of each rabbit, while the untreated right eye served as a control. Within 24 hours following eye application, there was a mild degree of eye irritation characterized by erythema, vascularization of the sclera and nictitating membrane, and edema of the lids and nictitating membrane, accompanied by lacrimation and exudate. The eye irritative signs gradually subsided within the next day or two and by the fourth day, and daily thereafter, the eyes of all animals appeared normal. There were no scorings, therefore the study was supportive.

- In a supporting in vitro study, severe eye irritation potential and corrosivity potential of the test item containing 34.4% 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 3.44-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 2.133 and a permeability value of 1.235compared to the negative control were determined. An IVIS 20.67 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 92.985 and a permeability value of 1.795 compared to the negative control. The IVIS value of 119.90 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. The test item, containing 34.4% active ingredient, tested in the in vitro BCOP test method, had an IVIS value of < 55.1 and consequently it was not classified as inducing severe eye damage.

- In another supporting in vitro study the eye irritancy potential was tested with registered substance by means of the chorioallantoic membrane of hens' eggs (HET-CAM) method with a test item containing 34,4% active ingredient (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 an effect with an irritation index (IS) of 10, compared to IS of 19 or 10 for 0.1% NaOH and 0.1% SDS positive controls and no effects in the negative control 0.9% NaCl solution. The test substance was predicted to be irritating.

-In an in vivo eye irritation study (Hansen 2013), a single instillation of 0.1 mL test substance containing 34.40% act. ingr. per animal into the conjunctival sac of the right eye of three rabbits resulted in the following mean 24 -72h scores: 0/4 for cornea, 0/2 for iris, 1.11/3 for conjunctiva and 0.45/4 for chemosis. The fluorescein tests revealed corneal staining in animal no. 2 animals. All findings were reversible within 5 day. There were no systemic intolerance reactions concerning behaviour, body weight and food consumption. Based on the negative findings, the substance can be concluded to be non-irritating to eyes, hence, no labelling is required.

- In conclusion, the registered substance was found to be non-corrosive and non-irritating at tested concentration of 34.4% active ingredient. Nevertheless a subgroup CLP category 2 classification is proposed, however with concentration limit of 34% or less for non-classification.

Conclusion:

- No classification is needed for skin irritation.
- A subgroup CLP category 2 classification was proposed for eye irritation, with concentration limit of 34% 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:
Key study

Effects on eye irritation: slightly irritating

Justification for classification or non-classification

The substance does not need to be classified for skin irritation according to CLP regulation (No. 1272/2008 of 16 December 2008).

For the eye, the substance is classified as irritating to eyes. According to CLP regulation (No. 1272/2008 of 16 December 2008), the substance is classified as Category 2, with signal word 'warning' and hazard statement: H319-Causes serious eye irritation.  However, a concentration limit of 34% for non-classification can be applied.