Disodium naphthalene-1,5-disulphonate

Endpoint summary

Description of key information

No skin or eye irritation was seen in early in vivo studies with limited documentation on the test compound.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

31 Jan - 07 Feb 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study with only minor limitations

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

Analytical purity of test material is slightly below 80%

GLP compliance:

yes

Species:

rabbit

Strain:

other: Albino HC:NZW (New Zealand White)

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated sites of the same animal served as control

Duration of treatment / exposure:

4 h

Observation period:

14 days
Reading time points: 1, 24, 48 and 72 h and 7 days

Number of animals:

3 (females)

Irritation parameter:

erythema score

Basis:

mean

Remarks:

of 3 animals

Time point:

other: mean of 24, 48 and 72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

edema score

Basis:

mean

Remarks:

of 3 animals

Time point:

other: mean of 24, 48 and 72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Executive summary:

No skin irritation was seen in the OECD TG 404 compliant study.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Justification for test system used:

Commercially available test system

Vehicle:

physiological saline

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany).
- Tissue batch number(s): Cat.-No.CS-1001
- Date of initiation of testing: 25 Nov 2015
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: incubator (37 ± 2° C) for 20 min- exposure
- Temperature of post-treatment incubation (if applicable): 37 ± 2° C
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/ml
- Incubation time: 3 hours
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
the optical density of the isopropanol-extracts of 3 insert was determined by duplicate per insert = 6 OD values.
PREDICTION MODEL / DECISION CRITERIA:
- The mean optical density (OD) value obtained with the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100 %.
- A category of UN GHS (Category 2 or Category 1) is predicted if the mean percent tissue viability after exposure and post treatment incubation is less than or equal (≤ ) to 50 %.

The irritating potential of the test item is assessed by determination of its cytotoxic effect on an in vitro reconstructed human epidermis. The test principle is based on the MTT assay reflecting the cell viability after exposure to the topically applied test item.
All tests were performed in triplets for each time point. The test item was applied at a 100% concentration, i.e. 30 µl per insert for 20 min. Cell viability was measured by the amount of MTT reduction (calculated on the basis of optical density of the negative control).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 30 μl
- the test item and the solvent ethyl acetate were tested in parallel

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μl
- Concentration (if solution): 0.9% NaCl

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl
- Concentration (if solution): 0.3% Sodium Dodecyl Sulfate (SDS)

Duration of treatment / exposure:

20 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

result for test material

Value:

ca. 111

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- Reliability of the test was previously confirmed by interlaboratory validation

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

Compound	Cell viability [%]	Evaluation
Armstrongsaure-di-Natriumsalz	110.55	Non-irritant
Positive control	1.02	Irritant
Negative control	100.00	Non-irritant

Executive summary:

A study was performed for the assessment of the skin irritancy of the test item Armstrongsaure-di-Natriumsalz with reconstructed human epidermis (RhE). The experiment

was carried out in vitro using the commercially available test method epiCS®. The study was conducted in accordance with OECD TG 439 and EU Test Method B.46.

The test item was applied undiluted topically to the RhE tissue construct in triplicates and incubated for 20 minutes, followed by a 42 hours post-treatment incubation period.

Cell viability was measured in a photometer by the amount of MTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to

calculate the percentage of viability relative to the negative control, which is set at 100%. The results of the concurrent negative control (NC, 0.9% NaCl) and positive control

(PC, 5 % SDS) demonstrated the viability (NC) and sensitivity (PC) of the test model. The following value of cell viability was recorded for the test item: 111 % (rounded).

In conclusion the results of the assay used show no skin irritant properties of the test item Armstrongsaure-di-Natriumsalz and thus, the test item requires no classification according to UN GHS (Category 2 or Category 1).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

31 Jan - 07 Feb 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study with only minor limitations

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

Analytical purity of test material is slightly below 80%

GLP compliance:

yes

Species:

rabbit

Strain:

other: Albino HC:NZW (New Zealand White)

Vehicle:

unchanged (no vehicle)

Controls:

other: the untreated eye served as control

Duration of treatment / exposure:

24 h (0.1 mL (corresponding to 0.07 g) of test material was instilled in the rabbits eye and removed 24 h after instillation by washing)

Observation period (in vivo):

7 days
Reading time points: 1, 24, 48 and 72 h and 7 days

Number of animals or in vitro replicates:

3 (females)

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

of 3 animals

Time point:

other: mean of 24, 48 and 72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Irritation parameter:

iris score

Basis:

mean

Remarks:

of 3 animals

Time point:

other: mean of 24, 48 and 72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

other: mean of 24, 48 and 72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

other: mean of 24, 48 and 72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

other: mean of 24, 48 and 72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

of 3 animals

Time point:

other: mean of 24, 48 and 72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

Other effects:

Slightly increased lacrimation was observed in two of three animals 1 h after instillation of the test material.

Executive summary:

No eye irritation was seen in this OECD TG 405 compliant study.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

2013

GLP compliance:

yes (incl. QA statement)

Details on test animals or tissues and environmental conditions:

Bovine eyes of slaughtered cattle were extracted and transferred in containers with Hank’s balanced salt solution (HBSS) with penicillin/streptomycin solution. For transportation the containers were ice cooled.
Eyes with defects were sorted out and disposed of, eyes without any defects were transferred into fresh HBSS supplemented with penicillin/streptomycin solution and 1 % FBS and stored overnight at 2-8 °C. On the next day (day of testing) the containers with the eyes were placed in an incubator at 32 ° C (± 1 ° C) for about 2 hours before preparation of the corneas.
For the preparation of the cornea the sclera was incised with a scalpel and cut by scissors. A 2-3 mm wide scleral edge was left around the cornea for further handling. The isolated corneas were placed with the epithelium side down into a prepared beaker filled with MEM medium supplemented with 1 % penicillin / streptomycin solution and 1 % FBS. Each cornea was placed into a cornea holder with the endothelial side facing the sealing ring of the posterior chamber. The anterior chamber was then fixed by screws on the other side. The chambers were filled with MEM medium, avoiding air bubbles. The holders were placed for at least 1 hour in the incubator at 32 °C (± 1 °C).

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µl
- the test item and the solvent were tested in parallel

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

not applicable

Number of animals or in vitro replicates:

3 corneas per tested material

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Bovine eyes of slaughtered cattle were extracted and transferred in containers with Hank’s balanced salt solution (HBSS) with penicillin/streptomycin solution. For transportation the containers were ice cooled.
Eyes with defects were sorted out and disposed of, eyes without any defects were transferred into fresh HBSS supplemented with penicillin/streptomycin solution and 1 % FBS and stored overnight at 2-8 °C. On the next day (day of testing) the containers with the eyes were placed in an incubator at 32 ° C (± 1 ° C) for about 2 hours before preparation of the corneas.
For the preparation of the cornea the sclera was incised with a scalpel and cut by scissors. A 2-3 mm wide scleral edge was left around the cornea for further handling. The isolated corneas were placed with the epithelium side down into a prepared beaker filled with MEM medium supplemented with 1 % penicillin / streptomycin solution and 1 % FBS. Each cornea was placed into a cornea holder with the endothelial side facing the sealing ring of the posterior chamber. The anterior chamber was then fixed by screws on the other side. The chambers were filled with MEM medium, avoiding air bubbles. The holders were placed for at least 1 hour in the incubator at 32 °C (± 1 °C).

QUALITY CHECK OF THE ISOLATED CORNEAS
yes

NEGATIVE CONTROL USED: physiological saline

SOLVENT CONTROL USED (if applicable): ethyl acetate

POSITIVE CONTROL USED: NaOH (1%)

APPLICATION DOSE AND EXPOSURE TIME
the test materials were applied pure for 4 hours

TREATMENT METHOD:
closed chamber method

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
- POST-EXPOSURE INCUBATION:

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The measurements of opacity were carried out using an opacitometer (BASF OP3.0). Before each measurement the opacitometer was adjusted to about 1000 LUX and a filter calibration measurement was carried out by using 3 different filters.
- Corneal permeability: The medium in anterior chamber of each holder was replaced by 1ml of fluorescein sodium solution (concentration 5 mg/mL). Afterwards the holders were incubated at 32 °C (± 1 °C) for about 90 minutes. After the incubation period, the medium of the posterior chamber was aspirated by a syringe and filled into a 10 mL tube. Three wells of a 96 well plate were filled with 300 μL of each tube (triplicate determination). In addition, a standard series of 5 mg/mL sodium fluoresceinsolution was prepared and also filled into the 96-well plate, in triplicates. The permeability was determined by measuring the amount of fluorescein sodium which diffused through all cell layers of the cornea. The measurement was carried out at a wavelength of 490 nm (OD490) by an ELISA - Reader).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: Test item formulations that cause an IVIS value > 55 are classified as seriously damaging the eye (UN GHS Cat 1). Test item formulations that cause IVIS values of ≤ 55 are considered as not seriously damaging the eye (not UN GHS Cat 1).

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

1.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

	Mean opacity value	Mean permeability value	In vitro irritancy score (IVIS)
Negative control; isotonic saline solution	-0,6	0,011	-0,5
Positive control; 20 % Imidazole	74,8	1,676	99,9
20 % Armstrongsaure-di-Natriumsalz	1,4	0,019	1,7

Executive summary:

This study was performed to assess the corneal damage potential of the solid test item Armstrongsaure-di-Natriumsalz with the Bovine Corneal Opacity and Permeability test

(BCOP) using fresh bovine cornea. The study was conducted in accordance with international accepted Guidelines (e.g. OECD TG 437).

20% (w/v) concentrations of the test item and the positive control imidazole in isotonic saline solution were tested on 3 bovine corneas each in comparison to the negative control, isotonic

saline solution. For determination of corneal damage opacity as well as tissue permeability was measured after a 4 hour treatment time.

Test items were applied to the epithelial surface ofthe cornea in a special corneal holder. Corneal opacity was measured quantitatively as the amount of light transmission through the cornea before and after treatment with the test item. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea after treatment. The In Vitro Irritancy Score (IVIS) value was calculated based on these data.

In accordance with OECD TG 437 and the study results Armstrongsaure-di-Natriumsalz was characterized by having no potential to seriously damage the eye.

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

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)

GLP compliance:

yes (incl. QA statement)

Species:

other: reconstructed human cornea-like epithelium (RhCE) cell model EpiOcular™. The model used is standardized and commercially available.

Strain:

other: reconstructed human cornea-like epithelium (RhCE) cell model EpiOcular™. The model used is standardized and commercially available.

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

The solid test item was applied topically to the RhCE tissue surface in duplicate for 6 hours, followed by an 18 hour post-treatment incubation period.

Duration of treatment / exposure:

The solid test item was applied topically to the RhCE tissue surface in duplicate for 6 hours, followed by an 18 hour post-treatment incubation period.

Duration of post- treatment incubation (in vitro):

The solid test item was applied topically to the RhCE tissue surface in duplicate for 6 hours, followed by an 18 hour post-treatment incubation period.

Number of animals or in vitro replicates:

2

Details on study design:

The EpiOcular™ RhCE tissue construct consists of 3 viable layers of cells and a nonkeratinized surface as recommended by the test guidelines (see section 4.1). The cell viability and barrier function as well as sterility of each batch of the RhCE tissue construct used is adequate, as has been demonstrated by the supplier.

RhCE tissue viability in EpiOcular™ EIT is measured by enzymatic conversion of the vital dye MTT by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues.

Irritation parameter:

other: cell viability [%]

Value:

ca. 93

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Compound	Final Cell viability [%]	Category
Armstrongsaure-di-Natriumsalz	92.8	No Cat
Positive control	36.1	Cat 1/2
Negative control	100.00	No Cat

Executive summary:

An in vitro study for assessing ocular irritation properties of the test item Armstrongsaure-di- Natriumsalz was performed using the reconstructed human cornea-like epithelium (RhCE) cell model EpiOcular™. The model used is standardized and commercially available. The EpiOcular™ Eye Irritation Test (EIT) was conducted in accordance with OECD 492.

The solid test item was applied topically to the RhCE tissue surface in duplicate for 6 hours, followed by an 18 hour post-treatment incubation period.

Cell viability was measured in a spectrophotometer by assessing the extent of MTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which was set at 100%.

The results of the concurrent negative control (NC, deionized water) and positive control (PC, neat methyl acetate) demonstrated the viability (NC) and sensitivity (PC) ofthe tissue model.

The final mean percent tissue viability recorded for the test item is 93 % (rounded). According to the results ofthis study the test item Armstrongsaure-di-Natriumsalz was identified as not requiring classification for eye irritation according to UN GHS (No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A study was performed for the assessment of the skin irritancy of Armstrongsaure-di-Natriumsalz with reconstructed human epidermis (RhE; OECD TG 439). No skin irritation potential was observed.

In vitro studies for assessing ocular irritation properties of the test item Armstrongsaure-di- Natriumsalz was performed using the reconstructed human cornea-like epithelium (RhCE) cell model EpiOcular™ (OECD TG 492) and the Bovine Corneal Opacity and Permeability test(BCOP) using fresh bovine cornea (OECD TG 437). In both tests Armstrongsaure-di-Natriumsalz was characterized by having no potential to damage the eye.

The in vitro data confirm the early in vivo studies. No skin or eye irritation was observed for Armstrongsaure-di- Natriumsalz.

Justification for classification or non-classification

No classification is warranted because no skin or eye irritation was seen in early in vivo studies with limted docuentation on the test compound. Recent in vitro studies confirm that Armstrongsäure-di-Natrium salt is not irritating to the skin or eye.EMCSP