Registration Dossier

Administrative data

Description of key information

Skin corrosion (in vitro, OECD 431): not corrosive

Skin irritation (in vitro, OECD 439): not irritating

Eye irritation (in vivo, OECD 405): not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 Aug 2012 - 10 Jan 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: SkinEthic Laboratories, Lyon, France
Source strain:
other: EPISKIN™ model kit 0.38 cm²; reconstructed three-dimensional human epidermis
Details on animal used as source of test system:
TEST SKIN MODEL
- Source: SkinEthic Laboratories, Lyon, France
- Description: The EPISKIN™ model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

ADAPTATION TO CELL CULTURE CONDITIONS
2.2 mL of maintenance medium, warmed to appr. 37 °C was pipetted into two wells of the first column of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (2 units per plate). A different 12-well plate was used for the test item, control and time point item. The tissues were incubated at 37 °C, 5% CO2 in air overnight. After 24 hours the medium underneath the tissues was refreshed and the tissues were returned to the incubator for a further 24 hours.

INCUBATION CONDITIONS (Incubator)
- Temperature (°C): 37
- CO2 gas concentration (%): 5
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SITE
- Area of exposure: 0.38 cm²

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the exposure period, each tissue was removed from the well and rinsed with phosphate buffered saline (PBS) with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test item. Each rinsed tissue was placed into the third column of the 12-well plate until all tissues were rinsed.
- Time after start of exposure: 3, 60 and 240 min

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed. Therefore, 2.2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into 2 wells of the fourth column of each 12 well plate. The tissues were transferred into the MTT filled wells. The tissues were incubated for 3 h at room temperature in a biological safety cabinet ensuring that the plates were protected form light. At the end of the 3-hour incubation period, each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was taken using a punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into labelled 1.5 mL micro tubes containg 850 µL of acidified isopropanol. Each tube was plugged, mixed thoroughly and stored overnight at room temperature, protected from light, to extract formazan crystals out of the MTT-loaded tissues. The optical density was measured at 540 nm wave length in a plate spectrophotometer.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 20 mg (100 µL of 0.9% w/v sodium chloride was added for wetting of the test item)

NEGATIVE CONTROL
- Negative control: 0.9% w/v sodium chloride solution
- Amount(s) applied: 50 µL

POSITIVE CONTROL SUBSTANCE:
- Positive control substance: glacial acetic acid (50 µL)
Duration of treatment / exposure:
3, 60 and 240 min
(EPISKIN cultures exposed to the control compounds for 240 min serve as the controls for all three exposure periods)
Duration of post-treatment incubation (if applicable):
Not applicable
Number of replicates:
Duplicates for each treatment and control group.
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 2 tissues
Run / experiment:
3 min exposure
Value:
99.4
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 2 tissues
Run / experiment:
60 min exposure
Value:
95.5
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 2 tissues
Run / experiment:
240 min exposure
Value:
97.4
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Table 2: Mean OD540 values and viabilities of the negative control item, positive control item and test item

Item

Exposure period

Mean OD540 of duplicate tissues

Relative mean viability (%)

Negative control

240 min

0.156

100*

Positive control

240 min

0.020

12.8

Test substance

240 min

0.152

97.4

 

60 min

0.149

95.5

 

3 min

0.155

99.4

*: The mean viability of the negative control tissues is set at 100%.

EPISKIN cultures exposed to the control compounds for 240 min serve as the controls for all three exposure periods.

Interpretation of results:
other: not corrosive (in vitro)
Conclusions:
There is regulatory acceptance in the EU that a substance can be considered corrosive (Skin Corrosive Cat.1A) based on a positive result in the human epidermis model test. Negative in vitro corrosivity responses are not conclusive with respect to non-classification or classification as irritant and shall therefore be subject to further evaluation.
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 Sep 2012 - 25 Mar 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
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)
Remarks:
The Department of Health of the Government of the United Kingdom, UK
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: SkinEthic Laboratories, Lyon, France
Source strain:
other: EPISKIN™ reconstructed human epidermis model
Details on animal used as source of test system:
TEST SKIN MODEL
- Source: SkinEthic Laboratories, Lyon, France
- Description: The EPISKIN™ model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

ADAPTATION TO CELL CULTURE CONDITIONS
2 mL of maintenance medium, warmed to appr. 37 °C was pipetted into the first column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

INCUBATION CONDITIONS (Incubator)
- Temperature (°C): 37
- CO2 gas concentration (%): 5
Vehicle:
unchanged (no vehicle)
Details on test system:
REMOVAL OF TEST SUBSTANCE
- Washing: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca2+ and Mg2+. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.
- Time after start of exposure: 15 min

CELL VIABILITY MEASUREMENTS
- Method: colourimetric MTT reduction assay
- Principles of method: Cell viability is measured by enzymatic reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells. The formazan product is extracted from the tissue with acidified isopropanol and the optical density of the extracts is measured at 540 nm using acidified isopropanol as blank.
- Time after exposure: 42 h (MTT loading/Formazan extraction) and on Day 6 (Absorbance/Optical density measurements)

INTERPRETATION OF RESULTS
For the test item, the relative mean tissue viabilities obtained after the 15 min exposure period followed by the 42 h post-exposure incubation period were compared to the mean of the negative control tissues (n = 3). The relative mean viabilities were calculated as follows:

Relative mean viability (%) = (mean OD540 of test item / mean OD540 of negative control) x 100

Classification of irritation potential was based upon relative mean tissue viability following the 15 min exposure period followed by the 42 h post-exposure incubation period according to the following criteria:

Relative mean tissue viability ≤ 50%: Irritant
Relative mean tissue viability ≥ 50%: Non-irritant

The results were evaluated according to Regulation (EC) No. 1272/2008 (CLP).

QUALITY CRITERIA
The results of the assay were considered acceptable if the following assay acceptance criteria are achieved:
- Positive control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was ≤ 40% relative to the negative control treated tissues, and the standard deviation value of the percentage viability is ≤ 18%.
- Negative control: The assay establishes the acceptance criterion for an acceptable test if the mean OD540 for the negative control treated tissues was ≥ 0.6, and the standard deviation value of the percentage viability is ≤ 18%.
- Test item: The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tisues is ≤ 18%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 10 mg (The epidermis surface had previously been moistened with 5 μl of sterile distilled water to improve contact between the solid test item and the epidermis.)

NEGATIVE CONTROL
- Negative control: Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca2+ and Mg2+
- Amount(s) applied: 10 µL

POSITIVE CONTROL
- Positive control substance: Sodium Dodecyl Sulphate (SDS), 5% w/v
- Amount applied: 10 µL
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
The test was performed in triplicates for each treatment and control group.
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean value of 3 tissues
Run / experiment:
15 min exposure
Value:
90.9
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

QUALITY CRITERIA

The relative mean tissue viability for the positive control treated tissues was 5.5% ± 1% relative to the negative control treated tissues. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was 0.743 ± 3.8%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 6.2%. The test item acceptance criterion was therefore satisfied.

Table 1. Mean OD540 values and percentage viabilities for the negative control item, positive control item and test item

Item

OD540 of tissues

Mean OD540 of triplicate tissues

± SD of OD540

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of relative mean viability

Negative control

0.711

0.743

0.028

95.7

100*

3.8

0.751

101.1

0.766

103.1

Positive control

0.034

0.041

0.007

4.6

5.5

1.0

0.040

5.4

0.048

6.5

Test item

0.728

0.675

0.046

98.0

90.9

6.2

0.641

86.3

0.657

88.4

 SD: standard deviation

*: The mean viability of the negative control tissue is set at 100%

Interpretation of results:
other: CLP/EU GHS classification criteria not met, no classification required according to Regulation (EC) No. 1272/2008.
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:
07 Nov 2012 - 10 Jan 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Species:
other: cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST METHOD
The bovine corneal opacity and permeability (BCOP) test is an in-vitro test method used to classify substances as “ocular corrosives and severe irritants”. The potential of a test substance to cause ocular corrosivity or severe irritancy is measured by its ability to induce opacity and increased permeability in isolated bovine corneas. The opacity and permeability assessments of the corneas are combined to derive an in-vitro irritancy score (IVIS), which is used to classify the irritancy level of the test substance.

IDENTIFICATION OF THE SOURCE OF THE EYES, STORAGE AND TRANSPORT CONDITIONS
- Source: local abattoir as by-product from freshly slaughtered animals
- Transport medium and temperature conditions: Hank’s balanced salt solution (HBSS) supplemented with penicillin/streptomycin, transported on ice paks

PREPARATION OF THE EYES (BEFORE EXPOSURE)
- Eyes free of defects (scratches, neovascularisation): yes
- Dissection of the eyes and treatment: cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris were peeled away from the cornea. The isolated corneas were immersed (epithelial side uppermost) in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
- Test medium and temperature conditions used in the cornea holder: complete Minimum Essential Medium (MEM); holders were incubated at 32 ± 1 °C for 60 min
- Quality check of the equilibrated corneas: free of macroscopic defects

DETERMINATION OF THE INITIAL OPACITY
- Method: corneal opacity was determined by the amount of light transmission through the cornea using an opacitometer
- Specification of the device: Opacitometer TX307
Vehicle:
other: 0.9% w/v sodium chloride solution
Controls:
other: negative control: sodium chloride solution; positive control: imidazole
Amount / concentration applied:
TEST MATERIAL
- Concentration (if solution): 20%

NEGATIVE CONTROL
0.9% w/v sodium chloride solution

POSITIVE SUBSTANCE
- Substance: imidazole
- Concentration (if solution): 20% in 0.9% sodium chloride solution
Duration of treatment / exposure:
240 min at 32 ± 1 °C
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
number of corneas for each the test item and controls: 3
Details on study design:
TEST CONDITIONS
- Short description of the method used: closed-chamber method
The MEM was removed from the anterior chamber of the BCOP holder and the test item preparation or control items were applied to the cornea. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 °C for 240 minutes.

POST-EXPOSURE TREATMENT
- Removal of the test substance: at the end of the exposure period, corneas were rinsed three times with fresh complete MEM containing phenol red followed by a final rinsing with complete MEM without phenol red. Afterwards, the anterior and posterior chamber were filled with fresh complete MEM.
- Medium for washing the corneas: complete MEM containing phenol red
- Medium for final rinsing: complete MEM

DETERMINATION OF THE FINAL OPACITY
- Method: corneal opacity was determined by the amount of light transmission through the cornea using an opacitometer
- Time of determination: after the post-exposure treatment and visual inspection of each cornea

DETERMINATION OF THE CORNEAL PERMEABILITY:
- Method: Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced wit 1 mL sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 °C for 90 minutes. After incubation, the medium in the posterior chamber of each holder was decanted and retained. 360 µL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm was measured. If values greater than 1.5 OD492 were obtained, a 1 in 5 dilution of the medium in complete MEM was performed and the measurement repeated. The modified value was multiplied by 5 to reflect the 1 in 5 dilution.
The corneas were retained after testing for possible conduct of histopathology.
Irritation parameter:
in vitro irritation score
Value:
<= 55

Table 1: Individual and mean corneal opacity and permeability measurements

Treatment

Cornea number

Opacity

Permeability

In vitro irritancy score

Pre-treatment

Post-treatment

Post-treatment - Pre-treatment

Corrected value

 

Corrected value

 

Negative control

1

4

6

2

 

0.048

 

 

2

4

7

3

 

0.067

 

 

3

3

9

6

 

0.065

 

 

 

 

 

3.7*

 

0.060§

 

4.6

Positive control

4

2

67

65

61.3

1.449

1.389

 

5

3

65

62

58.3

3.420

3.360

 

6

3

64

61

57.3

1.570

1.510

 

 

 

 

 

59.0#

 

2.086#

90.3

Test item

10

2

64

62

58.3

0.112

0.052

 

11

1

58

57

53.3

0.052

0.000

 

12

1

54

53

49.3

0.048

0.000

 

 

 

 

 

53.7#

 

0.017#

53.9 

§: Mean permeability

*: Mean of the post incubation - pre-treatment values

#: Mean corrected value

Interpretation of results:
other: non-corrosive
Conclusions:
There is regulatory acceptance in the EU that a substance can be considered as severe eye irritant (Serious eye damage Category 1) based on a positive result in the Bovine Corneal Opacity and Permeability (BCOP) test. Negative in-vitro corrosivity responses are not conclusive with respect to non-classification or classification as irritant (Category 2) and shall therefore be subject to further evaluation.
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Hoechst AG
- Age at study initiation: 3 - 5 month
- Weight at study initiation: 2.4 - 3.6 kg
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: approximately 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 12 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Vehicle:
unchanged (no vehicle)
Controls:
other: the untreated eye served as control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 100 mg

Duration of treatment / exposure:
24 hours
Observation period (in vivo):
1, 24, 48 and 72 h and 7 days
Number of animals or in vitro replicates:
3
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: yes
- Time after start of exposure: 24 hours

SCORING SYSTEM: Draize scoring system

TOOL USED TO ASSESS SCORE: hand-slit lamp, fluorescein
Irritation parameter:
cornea opacity score
Basis:
animal #1
Remarks:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
other: no irritation observed
Irritation parameter:
cornea opacity score
Basis:
animal #2
Remarks:
mean
Time point:
24/48/72 h
Score:
1.67
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
cornea opacity score
Basis:
animal #3
Remarks:
mean
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
iris score
Basis:
animal #1
Remarks:
mean
Time point:
24/48/72 h
Score:
0.67
Max. score:
2
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
iris score
Basis:
animal #2
Remarks:
mean
Time point:
24/48/72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 7 days
Irritation parameter:
iris score
Basis:
animal #3
Remarks:
mean
Time point:
24/48/72 h
Score:
0.67
Max. score:
2
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
conjunctivae score
Basis:
animal #1
Remarks:
mean
Time point:
24/48/72 h
Score:
2.7
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Basis:
animal #2
Remarks:
mean
Time point:
24/48/72 h
Score:
3
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Basis:
animal #3
Remarks:
mean
Time point:
24/48/72 h
Score:
2.7
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #1
Remarks:
mean
Time point:
24/48/72 h
Score:
2.3
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #2
Remarks:
mean
Time point:
24/48/72 h
Score:
2.3
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #3
Remarks:
mean
Time point:
24/48/72 h
Score:
1.67
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritant / corrosive response data:
From 1 hour up to 3 days after administration the conjunctivae of the animals showed swellings above normal up to swellings with lids about half closed and definitely injected blood vessels up to a diffuse beefy red colour. The iris was reddened in two animals up to day 2 and in the remaining animal until day 3 p.a..
Interpretation of results:
other: Eye Irrit Cat. 2 (H319) according to Regulation (EC) No. 1272/2008.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion

In a GLP-compliant study according to OECD Guideline 431, the skin corrosion potential of Ethanesulfonic acid, 2 -(methylamino)-, N-coco acyl derivs., sodium salts was evaluated using the EPISKIN™ reconstructed human epidermis model after a treatment period of 3, 60 and 240 min (Croda, 2013a). The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item (20 mg/tissue) by means of the colourimetric MTT reduction assay. Cell viability was measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls (treated with 0.9% sodium chloride solution). Positive control tissues were treated with 50 µL glacial acetic acid.

After the exposure period, the relative mean tissue viability for the test item compared to the control was 99.4, 95.5 and 97.4% for the 3, 60 and 240 min exposure periods, respectively. The relative mean tissue viability for the positive control treated tissues compared to the control was 12.8% after an exposure period of 240 min.

Based on the study results, the test item was considered to be non-corrosive in vitro.

In a GLP-compliant study according to OECD Guideline 439, the skin irritation potential of Ethanesulfonic acid, 2 -(methylamino)-, N-coco acyl derivs., sodium salts was evaluated using the EPISKIN™ reconstructed human epidermis model after a treatment period of 15 min followed by a post-exposure incubation period of 42 h (Croda, 2013b). The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item (10 mg/tissue) by means of the colourimetric MTT reduction assay. Cell viability was measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls (treated with Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca2+ and Mg2+). Positive control tissues were treated with Sodium Dodecyl Sulphate (SDS), 5% w/v.

After the 15 min exposure period, the relative mean tissue viability for the test item and positive control treated tissues compared to the control was 90.9% (± SD = 6.2%) and 5.5% (± SD = 1.0%), respectively.

Based on the study results, the test item was considered to be non-irritant in vitro.

Eye irritation/corrosion

The potential of Ethanesulfonic acid, 2 -(methylamino)-, N-coco acyl derivs., sodium salts to cause ocular corrosivity or severe irritation was assessed by conducting a bovine corneal opacity and permeability test (BCOP) according to OECD Guideline 437 and GLP (Croda, 2013c). Freshly prepared bovine corneas were obtained from slaughtered cattles.

The test substance (20% in 0.9% w/v sodium chloride solution) was incubated on the cornea for 240 minutes at 32 ± 1 °C. After removal of the test item and 90 min post-incubation, opacity and permeability values were measured.

The calculated IVIS (in vitro irritancy score) was 53.9. The positive control (imidazole, 20% in 0.9% sodium chloride solution) induced severe irritation of the cornea (IVIS score: 90.3), and the negative control (the solvent control) showed no irritating effect the cornea. On the basis of the test findings it can be concluded that the test item under the experimental condition displayed no corrosion or severe irritation potential.

The primary eye irritation potential of the test substance was furthermore evaluated according to OECD Guideline 405 in the New Zealand albino rabbit (Clariant, 1987c). Only animals without ocular abnormalities were used for the study. 100 mg of undiluted test material was applied once to the conjunctival sac of the left eye of three rabbits. The untreated eyes served in each case as a control. The exposure period was 24 hours. 24 hours after instillation and at all the designated examination times at which a corneal examination with fluorescein sodium solution took place, the treated eyes were washed out thoroughly with isotonic saline at approx. 37 °C. The eyes were examined 1, 24, 48 and 72 hours after application of the test substance. At 24 and 72 hours the eyes were also examined for corneal lesions under UV light after instillation of one drop of 0.01% fluorescein-sodium solution. From 1 hour up to 3 days after administration the conjunctivae of the animals showed swellings above normal up to swellings with lids about half closed and definitely injected blood vessels up to a diffuse beefy red colour. The iris was reddened in two animals up to day 2 and in the remaining animal until day 3 p.a. The cornea of two animals showed scattered areas of opacity up to easily discernible translucent areas. Over the 24, 48 and 72 h observation period, a mean corneal opacity score of 0.67, a mean iris score of 0.78, a mean conjunctival redness score of 2.8 and a mean chemosis score of 2.1 was observed. All signs of irritation were reversible within 7 days post exposure. Based on the results of this study, the test substance is considered to be irritating to rabbit eyes.

Justification for classification or non-classification

The available data on skin irritation/ corrosion do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 and are therefore conclusive but not sufficient for classification.

The available data on eye irritation/ corrosion do meet the criteria for classification according to Regulation (EC) No. 1272/2008. Therefore the substance is classified as Eye irritant (Cat. 2, H319).