Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts

Endpoint summary

Description of key information

The test substance was tested to be non-corrosive and non-irritant to human skin based on the three-dimensional EST 1000 human skin model. Viability values were 78.8% and 74.9% in the corrosivity test and 98.3% in the irritation test (all >50% threshold values), therefore it can be concluded be non-irritant to human skin and no further testing is needed.

Eye irritation data were not available for the registered substance, however read across data were available from a category member, CAS No. 37294 -49 -8 (disodium C-isodecyl sulphonatosuccinate), showing 24 -48h scores of 20.6/80 for cornea, 1.9/10 for iris and 4.1/20 for conjuctiva irritation, with 6/6 eyes positive for corneal opacity and 3/ 6 with iritis. As observations were only performed up to 72 hours, reversibility could not be scored; worst case classification CLP class 1 is proposed.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Remarks:

in vitro

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: The study was conducted according to GLP and valid methods predicting corrosivity. In combination with the in vitro irritation test, it is considered relevant, adequate and reliable for classification.

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

adopted April13, 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

three-dimensional human skin model, comprising at least a reconstructed epidermis with a functional stratum corneum.

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Skin model EST-1000 with BAtch no. EST-120611-001

Source strain:

not specified

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.

Vehicle:

other: 50 µL Dulbecco's phosphate buffered saline (D-PBS)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EST1000 model
- Tissue batch number(s): EST-120611-001
- Production date: 2012-08-21
- Shipping date: Not provided
- Delivery date: Not provided
- Date of initiation of testing (experimental phase): 2012-08-07

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Not provided.
- Temperature of post-treatment incubation (if applicable): Not applicable.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1 washing step; no data on volume used. 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).
- Observable damage in the tissue due to washing: Not provided.
- Modifications to validated SOP: Not applicable.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT solution of 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Yes
- 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. 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). 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%).
- 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%). The barrier properties of the tissues were verified by the supplier. Barrier function result was 63.4% (>50%).
- Morphology: Human keratinocytes were used to construct the epithelium. Multiple layers of viable epithelial cells were present under a functional stratum corneum. The skin model also had a stromal component layer. Stratum corneum was multi-layered with the necessary lipid profile to produce a functional barrier with robustness to resist rapid penetration of cytotoxic markers. The containment properties of the model prevented the passage of material around the stratum corneum to the viable tissue. Passage of test chemicals around the stratum corneum would lead to poor modelling of the exposure to skin.
- Contamination: The skin model was free of contamination by bacteria, viruses, mycoplasma, or fungi.
- Reproducibility: The tissue employed has been shown to demonstrate reproducibility over time between laboratories. Moreover it has been shown to be capable of predicting the corrosive potential of the reference chemicals when used in the testing protocol selected.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
No direct MTT interference (Certificate of Analysis Epidermal Skin Test EST 1000)

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 test sequence of the test, negative control and positive control group in triplicate at 3 minutes and 1 hour with duplicate OD measurements of the three tissues.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the tissue viability after 3 minutes exposure was ≤ 50%, or if the viability after 3 minutes exposure was ≥ 50% and the viability after 1 hour exposure was < 15%.
- The test substance is considered to be non-corrosive for skin : if the tissue viability after 3 minutes exposure≥ 50% and the viability after 1 hour exposure was ≥ 15%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431: Not applicable.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 26.25 mg Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts were mixed with 50 µL Dulbecco's phosphate buffered saline (D-PBS) to a slurry to ensure good contact with the skin. 50 µL containing the 26.25 mg test substance were applied to the skin model with a surface area of 0.6 cm2 to uniformly cover the skin surface. Usually 25 mg are used, however a correction factor of 1.05 was used to correct for the purity of the test item. A minimum of 25 µL substance applied per cm2 is required by the guidelines.

VEHICLE
- Amount(s) applied (volume or weight with unit): 50 µL Dulbecco's phosphate buffered saline (D-PBS)
- Lot/batch no. (if required): Batch no. 1080359

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL Dulbecco's phosphate buffered saline (D-PBS)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): 8N KOH

Duration of treatment / exposure:

Two exposure times of 3 minutes or 1 hour were employed.

Duration of post-treatment incubation (if applicable):

Not applicable

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean test group versus negative control group

Run / experiment:

3 minutes

Value:

78.8

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

meann test group versus negative control group

Run / experiment:

1 hour

Value:

74.9

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean positive control group versus negative control group

Run / experiment:

3 minutes

Value:

0.5

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean positive control group versus negative control group

Run / experiment:

1 hour

Value:

< 0.1

The test item, Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts,was applied to the skin surface. Dulbecco’s phosphate buffered saline (D-PBS) 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 78.8% after a 3-minute exposure period and 74.9% 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 0.5% (3-minute incubation) and <0.1% (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.

Interpretation of results:

other: predicted non-corrosive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the present test conditions the test itemapplied at two exposure times of 3 minutes or 1 hour was predicted 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 test item to human skin, in an experiment with an artificial three-dimensional model of human skin. The EST-1000 model was employed. The test item was applied to the skin surface. Dulbecco’s phosphate buffered saline (D-PBS) was used as the negative control.8 N KOHwas 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 78.8% after a 3-minute exposure period and 74.9% 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 thepositive reference item 8 N KOH was 0.5% (3-minute incubation) and <0.1% (1-hour incubation) of the negative controls and 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.

Under the present test conditions the test item containing >95% active ingredient, 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.

Reference 2

Endpoint:

skin corrosion: 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: The study was conducted according to GLP and valid testing guidance. In combination with the in vitro corrosion test, it is considered relevant, adequate and reliable for classification.

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified: EST1000 model Batch number EST-120730-001 (Product number: CS-1001)

Source strain:

not specified

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. The procedure described under this test method also allows the hazard identification of irritant substances in accordance with UN GHS category 2.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EST1000 supplier CellSystems®
- Tissue batch number(s): EST-120730-001 (Product number: CS-1001)
- Production date: 2012-08-13
- Shipping date: Not provided
- Delivery date: Not provided
- Date of initiation of testing: 2012-08-07

- Temperature used during treatment / exposure: The models were cultivated at 21°C for 20 minutes according to the instructions of the EST1000 supplier CellSystems®.
- Temperature of post-treatment incubation (if applicable): 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 (no data on temperature). This period allows both for recovery from weakly irritant effects and for appearance of clear cytotoxic effects. Each skin sample was placed in a MTT solution of 1 mg/mL (37°C incubation temperature, 5% CO2, 95% humidity) for 3 hours.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1 washing step; no data on volume used. 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).
- Observable damage in the tissue due to washing: Not provided.
- Modifications to validated SOP: Not applicable.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT solution of 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Yes, not specified
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Standard deviations should be within the one sided 95% tolerance interval of standard deviations calculated from historical data. However, in this study there is a slight deviation from the criteria as given in the guideline as the SD of 0.133 of the test item is outside this range, while the SD of 0.067 of the control is within this range. The 95% tolerance interval of standard deviations of historical control data of the year 2012 was calculated as 0.024 - 0.067 (n = 17). The study director regards this deviation as negligible due to the fact that the test item revealed a clear negative effect and, hence, the deviation does not affect the validity of the study.
- Barrier function: The barrier function is assessed either by determination of the concentration at which a marker substance reduces the viability of the tissues by 50% (IC50) after a fixed exposure time, or by determination of the exposure time required to reduce cell viability by 50% (ET50) upon application of the marker substance at a specified, fixed concentration. 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%). The barrier properties of the tissues were verified by the supplier. Barrier function result was 58.5% (>50%).
- Morphology: Normal human keratinocytes were used to construct the epithelium. Multiple layers of viable epithelial cells (basal layer, stratum spinosum, stratum granulosum) were present under a functional stratum corneum. Stratum corneum was multilayered containing the essential lipid profile to produce a functional barrier with robustness to resist rapid penetration of the cytotoxic marker substance sodium dodecyl sulphate (SDS).- Contamination: The skin model was free of contamination by bacteria, viruses, mycoplasma, or fungi.
- Reproducibility:

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
No direct MTT interference (Certificate of Analysis Epidermal Skin Test EST 1000)

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
duplicate OD measurements of the three tissues.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin in accordance 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 for skin irritation the tissue viability after exposure and post-treatment incubation was > 50%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 439: Not applicable.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 31.5 mg Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts were applied to the skin model with a surface area of 0.6 cm2. 50 µL Dulbecco's phosphate buffered saline (D-PBS) were applied to ensure good contact with the skin to uniformly cover the skin surface. Usually 30 mg test item are used, however a correction factor of 1.05 was used to correct for the purity of the test item. A minimum of 25 mg substance applied per cm2 is required by the guidelines.

VEHICLE
- Amount(s) applied (volume or weight with unit): 50 µL Dulbecco's phosphate buffered saline (D-PBS)
- Lot/batch no. (if required): Batch no. 1080359

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL water for injection

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): 5% aqueous sodium dodecyl sulphate (SDS)

Duration of treatment / exposure:

20-minute exposure.

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Remarks:

test group mean versus negative control

Run / experiment:

time point 20 minutes exposure followed by 42 h incubation

Value:

98.3

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean positive control versus negative control group

Run / experiment:

time point 20 minutes exposure followed by 42 h incubation

Value:

1.2

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: Not provided.
- Direct-MTT reduction: No

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
For each assay using valid batches, tissues treated with the negative control 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. Similarly, tissues treated with the positive control, 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). Associated and appropriate measures of variability between tissue replicates are defined (e.g. if standard deviations are used they should be within the one sided 95% tolerance interval of standard deviations calculated from historical data).
However, in this study there is a slight deviation from the criteria as given in the guideline as the SD of 0.133 of the test item is outside this range, while the SD of 0.067 of the control is within this range. The 95% tolerance interval of standard deviations of historical control data of the year 2012 was calculated as 0.024 - 0.067 (n = 17). The study director regards this deviation as negligible due to the fact that the test item revealed a clear negative effect and, hence, the deviation does not affect the validity of the study.

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

The test item, Butanedioic acid, sulfo-, 1-C12-18-alkyl 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 the cells exposed to the test item was 98.3% 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 1.2% of the negative controls and below the 50% cut-off value. Hence, 5% SDS is predicted to cause pronounced skin irritation.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the present test conditions the test item, applied at an exposure time of 20 minutes, was non-cytotoxic and not irritant to skin in an experiment with 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 the test item to human skin,in an experiment with an artificial three-dimensional model of human skin. The EST1000 model was employed. 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 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 98.3% 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 1.2% of the negative controls and below the 50% cut-off value. Hence,5% SDS is predicted to cause pronounced skin irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

See attached read-across information

Reason / purpose for cross-reference:

read-across source

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

19.7

Max. score:

80

Reversibility:

not fully reversible within: 72h

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

1.9

Max. score:

10

Reversibility:

not fully reversible within: 72h

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

4.1

Max. score:

20

Reversibility:

not fully reversible within: 72h

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

10

Max. score:

80

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

8.3

Max. score:

80

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

18.3

Max. score:

80

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

cornea opacity score

Basis:

animal #4

Time point:

24/48/72 h

Score:

33.3

Max. score:

80

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

cornea opacity score

Basis:

animal #5

Time point:

24/48/72 h

Score:

8.3

Max. score:

80

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

cornea opacity score

Basis:

animal #6

Time point:

24/48/72 h

Score:

40

Max. score:

80

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

10

Reversibility:

other: no effects at 24 h

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.67

Max. score:

10

Reversibility:

fully reversible within: 48 h

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

10

Reversibility:

other: no effects at 24 h

Irritation parameter:

iris score

Basis:

animal #4

Time point:

24/48/72 h

Score:

5

Max. score:

10

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

iris score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0

Max. score:

10

Reversibility:

other: no effects at 24 h

Irritation parameter:

iris score

Basis:

animal #6

Time point:

24/48/72 h

Score:

5

Max. score:

10

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

4

Max. score:

20

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

20

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

3.33

Max. score:

20

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Basis:

animal #4

Time point:

24/48/72 h

Score:

6

Max. score:

20

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Basis:

animal #5

Time point:

24/48/72 h

Score:

3.33

Max. score:

20

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

conjunctivae score

Basis:

animal #6

Time point:

24/48/72 h

Score:

6

Max. score:

20

Reversibility:

not fully reversible within: 72 h

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Remarks on result:

other: not reported

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Remarks on result:

other: not reported

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Remarks on result:

other: not reported

Irritation parameter:

chemosis score

Basis:

animal #4

Time point:

24/48/72 h

Remarks on result:

other: not reported

Irritation parameter:

chemosis score

Basis:

animal #5

Time point:

24/48/72 h

Remarks on result:

other: not reported

Irritation parameter:

chemosis score

Basis:

animal #6

Time point:

24/48/72 h

Remarks on result:

other: not reported

Other effects:

6 of 6 eyes were positive for corneal opacity; 3 of 6 had irritis.

Table 1. Rabbit eye irritation after application of 0.1mL of Surfactant E-268 (FHSA procedure)

Time, Hours	Reaction	Rabbit Number, Value						Mean Value
		1	2	3	4	5	6
24	CORNEA IRIS CONJUNCTIVAE	20 0 4	10 5 2	30 0 4	40 5 6	15 0 4	40 5 6	25.8 2.5 4.3
48	CORNEA IRIS CONJUNCTIVAE	5 0 4	10 0 2	20 0 4	30 5 6	5 0 4	40 5 6	18.3 1.7 4.3
72	CORNEA IRIS CONJUNCTIVAE	5 0 4	5 0 2	5 0 2	30 5 6	5 0 4	40 5 6	15.0 1.6 3.7

Interpretation of results:

irritating

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The read-across test item containing +-50% active ingredient was irritating to the rabbit eye (an irritant by FHSA definition). 6 of 6 eyes were positive for corneal opacity; 3 of 6 had iritis.

Executive summary:

6 Male albino rabbits underwent an eye irritation test with 0.1 mL read-across test item containing +-50% active ingredient. According to FHSA procedure. Scoring according to Draize et al. (1944) demonstrated mean 24 -48h score of 20.6/80 for cornea, 1.9/10 for iris and 4.1/20 for conjuctiva irritation, therefore the read-across substance was concluded to be irritating to the rabbit eye (by FHSA definition), with 6 of 6 eyes positive for corneal opacity and 3 of 6 with iritis.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Assessment of skin irritation was tested as a weight-of-evidence approach

-A first in vitro study was conducted to study corrosive properties by means of a three-dimensional EST-1000 human skin model (Flügge, 2013a). The test item containing >95% active ingredient was applied to the skin surface. In comparison to the negative controls, the mean viability of cells exposed to the test item was 78.8% after a 3-minute exposure period and 74.9% after a 1-hour exposure. The 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 concluded to be non-corrosive to human skin.

-A second in vitro study was conducted to determine irritating properties by means of the three-dimensional EST-1000 model of human skin (Flügge, 2013b). The test item containing >95% active ingredient was applied to the skin surface for 20 minutes, followed by refreshment of the medium and a further incubation period of 42 hours. The mean viability of the cells exposed to the test item was 98.3% 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 concluded 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 classification is needed.

 

Eye irritation

Eye irritation data were not available for the registered substance, however read across data were available from a category member, CAS No. 37294 49 -8 (disodium C-isodecyl sulphonatosuccinate). In a key study, 6 Male albino rabbits underwent an eye irritation test with 0.1 mL test item containing +- 50% act. ingr. according to FHSA procedure (Carpenter, 1971). Scoring according to Draize et al. (1944) demonstrated mean 24 -72h scores of 20.6/80 for cornea, 1.9/10 for iris and 4.1/20 for conjuctiva irritation, therefore the substance was concluded to be irritating to the rabbit eye (by FHSA definition), with 6 of 6 eyes positive for corneal opacity and 3 of 6 with iritis. As observations were only performed up to 72 hours, reversibility could not be scored.

In conclusion worst case classification CLP class 1 is proposed.

 

Conclusion

- The test substance was tested to be non-corrosive and non-irritant to human skin based on the three-dimensional EST 1000 human skin model. As the test item for skin and eye irritation contained >95% active ingredient, the registered substance can also be concluded to be non irritating for skin.

- Eye irritation data from read-across substance CAS No. 37294 -49 -8 (disodium C-isodecyl sulphonatosuccinate) indicated a worst case classification CLP class 1, which was also accepted as group classification. Further information supporting the classification CLP 1 for eye damage is provided in the read across justification for the Mono-ester group.

 

Justification for classification or non-classification

Based on these results and according to CLP (No. 1272/2008 of 16 December 2008), the test substance is not classified and has no obligatory labelling requirement for skin irritation.

Based on read across with structural analogues, the test substance needs to be classified to CLP regulation (No. 1272/2008 of 16 December 2008) as Category 1, with signal word 'Danger' and hazard statement: H318 -Causes serious eye damage.