Registration Dossier

Administrative data

Description of key information

Skin irritation: not irritating based on WoE (BASF SE, 1995 and 2016)

Eye irritation: irritating based on WoE (BASF SE, 1995 and 2016)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2015-01-07 to 2016-09-02
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)
Version / remarks:
26 July 2013
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)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Sample 14/078 from Mollescal HW, charge 10924344R0
- Expiration date of the lot/batch: April 28, 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
other: Dulbecco's modified eagle's medium
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200
- Tissue batch number: 21678
- Date of initiation of testing: 2015-07-01

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: once with sterile PBS
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.976±0.049 (1.0 - 3.0)
- Barrier function: 6.44 hours (4.77 - 8.72 hours)
- Contamination: No contamination (sterile)

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues: Epi-200 tissue that is killed by freezing at –20°C
- N. of replicates : 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 25 µL
- Vehicle applied: 25 µL

VEHICLE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5 %
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
18±2 hours
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Skin irritation
Value:
73.5
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: Due to the ability of the test substance to reduce MTT directly, KC tissues were applied in parallel. The results of the KC tissues indicate an increased MTT reduction (mean viability 0.6 % of NC). Thus for the test substance the final mean viability is given after KC correction.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
Interpretation of results:
GHS criteria not met
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1995-03-06 to 1995-03-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was only conducted at a concentration of ca. 39.5 % test substance in an aqueous solution.
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
adopted July 17, 1992.
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Version / remarks:
Publication no. L 383A, December 29, 1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
testing lab.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- ot/batch No.of test material: 80-9159

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Dr. K. Thomae GmbH, Biberach, Germany
- mean body weight: male 3.03 kg, females 2.9 kg
- Housing: individually (in stainless steel wire mesh cages (floor area: 3000 cm2))
- Diet: ad libitum, standardized animal laboratory diet (about 130 g/animal/day)
- Water: ad libitum, tap water (about 250 mL/animal/day)
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
semiocclusive
Preparation of test site:
shaved
Vehicle:
unchanged (no vehicle)
Controls:
other: untreated skin of the same animal
Amount / concentration applied:
The test patch (2.5 x 2.5 cm) was moistened with a dose of 0.5 mL of a 39.5 % test substance preparation.
Duration of treatment / exposure:
4 hours
Observation period:
72 hours
Number of animals:
3
Details on study design:
The test substance was applied in a single dose to the intact untreated skin. The test substance was removed at the end of the application period with Lutrol and Lutrol/water (1:1).
Application site: upper third of the back or flanks
Readings: 1 h , 24 h, 48 h and 72 h after removal of the test patches.
A check for general observations and dead and/or moribund animals was made twice each working day and once on weekends and on public holidays.
Irritation parameter:
erythema score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritant / corrosive response data:
No signs of irritation of the intact skin were observed.
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:
weight of evidence
Study period:
2015-07-07 to 2015-07-28
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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Sample 14/078 from Mollescal HW, charge 10924344R0
- Expiration date of the lot/batch: April 28, 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Schlachthof Mannheim, Schlachthofstr. 21, 68165 Mannheim, Germany
- Characteristics of donor animals (age): 12 to 60 months
- indication of any existing defects or lesions in ocular tissue samples: No
Vehicle:
water
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL
- Concentration (if solution): 20 % (w/v)

VEHICLE
- Amount applied: 750 µL
Duration of treatment / exposure:
4 hours
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagles’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the equilibration period the medium in both chambers was replaced with fresh prewarmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that showed macroscopic tissue damage or an opacity value < 542 opacity units were discarded.

NUMBER OF REPLICATES
3 corneas were used per treatment group.

SOLVENT CONTROL USED
De-ionized water

POSITIVE CONTROL USED
Imidazole (CAS No. 288-32-4) 20 % (w/v) solution in deionized water for non-surfactant solid test substances

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red)

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: was observed visually. Final corneal opacity readings were taken for each cornea with an opacitometer.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader(OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: Evaluation of results were based on the decision criteria used in OECD guideline 437.
Irritation parameter:
in vitro irritation score
Run / experiment:
test substance
Value:
0.5
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: Not observed

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values: Mean ± SD (Mean-2SD - Mean+2SD)

Historic range NC
Opacity = 11.1 ± 6.9 (-2.7 - 24.9)
Permeability = 0.003 ± 0.001 (0.001 - 0.006)

Historic range PC
Opacity = 76.3 ± 15.1 (46.2 - 106.4)
Permeability = 2.803 ± 0.616 (1.571 - 4.034)
IVIS = 118.3 ± 18.7 (81 - 155.6)

Opacity score of test substance, NC and PC

Cornea No.

Initial opacity

Final opacity

Opacity change

Corrected opacity change

Mean

SD

test substance

13

5.1

11.8

6.6

0

0.5

0.8

14

6

9

3

0

15

4.1

12.7

8.6

1.4

NC

1

5.4

11.7

6.3

NA

7.3

3.3

2

5.1

9.8

4.6

NA

3

4.1

15.1

10.9

NA

PC

4

3.5

107

103.4

96.1

70.2

25.4

5

3.4

79.9

76.5

69.2

6

3

55.6

52.6

45.4

Permeability score of test substance, NC and PC

Cornea No.

Mean OD490

Dilution factor

Mean corrected OD490*

Mean

SD

test substance

13

0.008

1

0.004

0.004

0.001

14

0.009

1

0.005

15

0.008

1

0.003

NC

1

0.004

1

NA

0.005

0.001

2

0.004

1

NA

3

0.006

1

NA

PC

4

0.699

5

3.492

2.992

0.533

5

0.611

5

3.052

6

0.487

5

2.432

 In Vitro Irritancy score (IVIS) of the test substance, NC and PC

Cornea No.

Opacity per cornea

Permeability per cornea

IVIS

Per cornea

Mean

SD

test substance

13

0

0.004

0.1

0.5

0.8

14

0

0.005

0.1

15

1.4

0.003

1.4

NC

1

6.3

0.004

6.3

7.3

3.3

2

4.6

0.004

4.7

3

10.9

0.006

11

PC

4

96.1

3.492

148.5

115.1

33.3

5

69.2

3.052

115

6

45.4

2.432

81.2

Interpretation of results:
GHS criteria not met
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2015-07-07 to 2015-07-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Sample 14/078 from Mollescal HW, charge 10924344R0
- Expiration date of the lot/batch: April 28, 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
Species:
human
Strain:
other: three-dimensional non-keratinized tissue construct
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 µL
Duration of treatment / exposure:
6 hours
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
2
Details on study design:
- Details of the test procedure used
Several test substances were tested in parallel within the present test using the same control tissues (NC and PC). Two tissues were treated with each, the test substance, the PC and the NC. In addition two killed tissues were used for each, the test substance and the NC, in order to detect direct MTT reduction.

Pre-incubation of the tissues
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at 37 °C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours.

Pretreatment of the tissues
After the pre-incubation, the tissues were pre-treated with 20 μL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.

Application of the test substance
Using a sharp spoon, a bulk volume of ca. 50 μL of the test material was applied covering the whole tissue surface. Control tissues were concurrently applied with 50 μL of sterile de-ionized water (NC, NC KC) or with 50 μL of methyl acetate (PC) or test substance (KC). After application, the tissues were placed into the incubator until the total exposure time of 6 hours was completed.

Removal of the test substance and postincubation period
To remove the test substance, the tissues were washed with sterile PBS. For this purpose the tissues were immersed and swiveled three times in each of three beakers filled with PBS. Washed tissues were immediately immersed into 12-well plates, pre-filled with 5 mL/well prewarmed medium (post-soak immersion) in order to remove residual test substance. After 25 minutes of post-soak immersion, each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium. Subsequently, the tissues were incubated at standard culture conditions for 18 hours (postincubation period).

MTT incubation
After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol at room temperature overnight or for at least 2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

- RhCE tissue construct used, including batch number:
Tissue model: OCL-200
Tissue Lot Number: 21559 (Certificate of Analysis is attached to the study report)
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
Principle
The OD570 values determined for the various tissues are measures of their viability. The ratio of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether or not a test material was an irritant.

Calculation of individual and mean optical densities
The corrected measured OD570 value for each individual tissue was calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of two tissues treated in the same way was calculated.

Tissue viability
The quantification of tissue viability is presented as the ratio of the mean OD570 divided by the respective OD570 NC value in percent.

- Positive and negative control means and acceptance ranges based on historical data
The negative control OD570 mean value is 1.654 with a range of 1.341 to 1.967. For the positive control a mean OD570 value of 0.398 is given with a range of 0.208 to 0.589. An average viability of 23.9 % with a range of 14.6 to 33.3 % is stated for the positive control.

- Reference to historical data of the RhCE tissue construct
Viability: 1.416±0.209 (1.1 - 3.0)
Barrier function: 29.89 min (12.2 - 37.5 min)
Contamination: No contamination (sterile)

- Positive and negative control means and acceptance ranges based on historical data
- Acceptable variability between tissue replicates for positive and negative controls
The absolute OD570 of the NC-tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is equal to or above 1.0. The mean OD570 of the NC should not exceed 2.5.
Methyl acetate used as PC usually leads to a tissue viability of approx. 25%. A viability of < 60% is acceptable.

- Acceptable variability between tissue replicates for the test chemical
Two tissues were treated under the same conditions. A variability between the tissues is considered to be acceptable if the relative difference of the viability is < 20%.
The OD570 of the killed control tissues treated as negative control should be equal to or below 0.35. The value for direct MTT-reduction of a test substance should be equal to or below 30% of the NC.
Irritation parameter:
other: tissue viability [%]
Value:
2.6
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Results

Test substance

 

 

Tissue 1

Tissue 2

Mean

Inter tissue variability [%]

NC

viable tissues

mean OD570

1.476

1.518

1.497

 

viability [% of NC]

98.6

101.4

100

2.8

KC tissues

mean OD570

0.039

0.033

0.036

 

viability [% of NC]

2.6

2.2

2.4

0.4

Test substance

viable tissues

mean OD570

0.039

0.04

0.04

 

viability [% of NC]

2.6

2.7

2.6

0.1

KC tissues*

mean OD570

0

0

0

 

viability [% of NC]

0

0

0

0

Mean viability of tissues [% of NC]:

2.6

 

PC

viable tissues

mean OD570

0.296

0.352

0.324

 

viability [% of NC]

19.7

23.5

21.6

3.8

* Negative values are set to zero for further calculation

Due to the ability of the test substance to reduce MTT directly, KC tissues were applied in parallel. However, the results of the KC tissues did not indicate an increased MTT reduction. Thus the KC was not used for viability calculation.

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1995-03-06 to 1995-03-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was only conducted at a concentration of ca. 39.5 % test substance in an aqueous solution.
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
adopted February 24, 1987
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
Publication no. L 383A, December 29, 1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
testing lab.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- ot/batch No.of test material: 80-9159

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Dr. K. Thomae GmbH, Biberach, Germany
- Weight at study initiation: male 2.91 kg; females 2.81 kg
- Housing: individually in stainless steel wire mesh cages (floor area: 3000 cm2)
- Diet (e.g. ad libitum): ad libitum, standardized animal laboratory diet (about 130 g/animal/day)
- Water (e.g. ad libitum): ad libitum, tap water (about 250 mL/animal/day)
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 -70
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
unchanged (no vehicle)
Controls:
other: untreated eye of the same animal
Amount / concentration applied:
0.1 mL of a 39.5 % test substance preparation
Duration of treatment / exposure:
24 hours
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
3
Details on study design:
Single application to the conjunctival sac of the right eyelid; the test substance was washed out with tap water about 24 hours after instillation.
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
other: 24, 48, 72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
mean
Time point:
other: 24, 48, 72 h
Score:
0
Max. score:
2
Irritation parameter:
chemosis score
Basis:
mean
Time point:
other: 24, 48, 72 h
Score:
0
Max. score:
4
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
other: 24, 48, 72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
other: 24, 48, 72 h
Score:
0
Max. score:
3
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
other: 24, 48, 72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 hours
Irritant / corrosive response data:
A very slight redness of the conjunctivae was observed in two animals. The redness resolved within 48 hours. No other signs of irritancy were observed.
Interpretation of results:
study cannot be used for classification
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

Two studies (in vitro and in vivo) were evaluated in a weight of evidence approach. Based on the below findings it can be concluded that the test substance does not have any skin irritating properties.

The objective of the in vitro testing was to assess the potential for corrosive activity and skin irritation of Disodium (sulphonatothio)acetate; dried. Using the currently available methods a single in vitro assay may not always be sufficient to cover the full range of skin irritating/corrosion potential. Therefore, usually two in vitro assays are part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT).

However, in the current case for Disodium (sulphonatothio)acetate; dried the results derived with SIT according to OECD guideline 439 together with the findings in the below described in vivo test were sufficient for a final assessment. Therefore further testing in SCT was waived.

Skin irritation test

The potential of Disodium (sulphonatothio)acetate; dried to cause dermal corrosion/irritation was assessed by a single topical application of ca. 25 μL bulk volume (ca. 18 mg) of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). (BASF SE,69V0318/14A253, 2016)

The irritation test was performed with three EpiDerm™ tissue samples, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The EpiDerm™ skin irritation test showed the following results:

The test substance is able to reduce MTT directly. Therefore an additional MTT reduction control KC (freeze-killed control tissues) was introduced.

The final mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 72.9 %.

Based on the observed results and applying the evaluation criteria as detailed in the endpoint study record it was concluded, that Disodium (sulphonatothio)acetate; dried does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen. This result is further supported by an in vivo study performed at a lower concentration.

In vivo test

In a primary dermal irritation study according to OECD guideline 404, young adult New Zealand White rabbits (one male and two females) were dermally exposed to 0.5 mL of the test substance in water (39.5 %) for 4 hours under a semiocclusive dressing (BASF SE, 18H0045/952018, 1995). After the exposure period, the test substance was removed with Lutrol and Lutrol/water (1:1). Animals then were observed for 72 hours. No erythema or edema is observed on any of the treated animals. Under the test conditions chosen and considering the described findings the test substance does not give indication of irritation either by edema or erythema on the intact rabbit skin at a concentration of 39.5 % in water.

Eye irritation

Three studies (2 in vitro and 1 in vivo) were evaluated in a weight of evidence approach. The results below show that the test substance is irritating to the eyes. The in vivo test does not show any irritating properties but as it was conducted with a 39.5 % solution of the test substance only, eye irritation cannot be excluded.

The objective of the present study was the determination of a possible eye irritating potential of Disodium (sulphonatothio)acetate; dried (BASF SE, 71V0318/14A254, 2016). Using the currently available methods a single in vitro assay may not always be sufficient to cover the full range of eye irritating potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test.

BCOP

The potential of Disodium (sulphonatothio)acetate; dried to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 μL of a 20 % test-substance preparation to the epithelial surface of isolated bovine corneas.

Three corneas were treated with the test-substance preparation for an exposure period of 4 hours. In addition to the test substance a negative control (NC; de-ionized water) and a positive control (PC; 20 % imidazole in de-ionized water) were applied to three corneas each. Corneal opacity was measured quantitatively as the amount of light transmitted through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance. The following results were obtained in the BCOP Test:

Table 1: BCOP - Mean values for opacity, permeability and IVIS of the test substance, NC and PC

Test

substance

Mean

Opacity

Value

Mean

Permeability

Value

Mean In

Vitro

Irritancy

Score

14/0318-2

0.5

0.004

0.5

NC

7.3

0.005

7.3

PC

70.2

2.992

115.1

 

EpiOcular

The potential of Disodium (sulphonatothio)acetate; dried to cause ocular irritation was assessed by a single topical application of ca. 50 μL bulk volume (about 23 mg) of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcular™). Two EpiOcular™ tissue samples were incubated with the test substance for 6 hours followed by 18-hours post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability.

The EpiOcular™ eye irritation test showed the following results: The test substance is able to reduce MTT directly. Therefore an additional MTT reduction control KC (freeze-killed control tissues) was introduced. The final mean viability of the test-substance treated tissues was 2.6 %.

Summary of the individual test results of the in vitro eye irritation turnkey testing strategy:

Table 2: BCOP and EpiOcular - Summary of individual test results and test strategy evaluation

Test

Method

Test Result

Test Evaluation

Evaluation Test

Strategy

BCOP Test

The mean IVIS of the test-substance treated corneas was 0.5.

not identified as corrosive or severe irritant

ocular irritant

EpiOcular

Mean viability of the test-substance treated tissues was 2.6%.

irritant

 

In vivo Test

In a primary eye irritation study according to OECD guideline 405, 0.1 mL of the test substance in water (33.2 %) was instilled into the conjunctival sac of young adult New Zealand White rabbits (one male and two females) for 24 hours (BASF SE, 11H0045/952019, 1995). The eyes were washed at the end of the treatment period. Animals were then observed for 72 hours. The animals did not show any reactions of the iris or the cornea. No chemosis of the conjunctivae was observed in any of the three animals. Two animals showed a very slight conjunctival redness at the 24 hour reading which had resolved after 48 hours. The test substance is not considered as irritating to the eyes at a concentration of 33.2 % in water.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance needs to classified and labelled as irritating to the eyes (H319) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776. No skin irritating properties were observed.