Reaction products of naphthalene, benzyl alcohol, sulphuric acid, formaldehyde and ammonia

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 25 - 28, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Diammonium 1,1'-methylenebis[(phenylmethyl)naphthalene-2-sulphonate]

EC Number:

284-353-8

EC Name:

Diammonium 1,1'-methylenebis[(phenylmethyl)naphthalene-2-sulphonate]

Cas Number:

84852-41-5

Molecular formula:

C35H38N2O6S2.2H3N

IUPAC Name:

Diammonium 1,1'-methylenebis((phenylmethyl)naphthalene-2-sulphonate)

Constituent 2

Reference substance name:

Benzyl methylene-bis-napthalene-2-sulphonic acid ammonium salt

IUPAC Name:

Benzyl methylene-bis-napthalene-2-sulphonic acid ammonium salt

Constituent 3

Reference substance name:

Isomers of Benzyl methylene-bis-naphthalene trisulphonic acid ammonium salt

Molecular formula:

Not applicable

IUPAC Name:

Isomers of Benzyl methylene-bis-naphthalene trisulphonic acid ammonium salt

Constituent 4

Reference substance name:

Ammonium sulphate

EC Number:

231-984-1

EC Name:

Ammonium sulphate

Cas Number:

7783-20-2

Molecular formula:

(NH4)2SO4

IUPAC Name:

diammonium sulfate

Constituent 5

Reference substance name:

Unidentified isomers formed during the reaction

Molecular formula:

Not applicable

IUPAC Name:

Unidentified isomers formed during the reaction

Constituent 6

Reference substance name:

Formaldehyde

EC Number:

200-001-8

EC Name:

Formaldehyde

Cas Number:

50-00-0

Molecular formula:

C H2 O

IUPAC Name:

formaldehyde

Test material form:

solid

In vitro test system

Test system:

human skin model

Remarks:

Reconstructed Human Epidermis - EpiDerm™ (EPI-200-SIT)

Source species:

human

Cell type:

non-transformed keratinocytes

Source strain:

other: 00267

Justification for test system used:

Reconstructed Human Epidermis - EpiDerm™ (EPI-200-SIT) has been selected as the test system for in vitro skin irritation as it is recommended in OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200-SIT).
- Tissue lot number: 25850.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for 35 ± 1 minutes and the remaining period of the 60 minute exposure within a sterile hood (temperature not specified).
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C for 43 hours.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Sterile DPBS was used to rinse the tissue by filling and emptying the tissue insert fifteen times in order to remove any residual test item. The constant stream of DPBS was applied from the nearest distance to the tissue surface. After fifteen rinses, the inserts were completely submerged three times in approximately 50 mL of DPBS and shaken to remove all traces of test item/NC/PC. Finally, each tissue was rinsed once from the inside and once from the outside with sterile DPBS. The excess of DPBS was removed by gentle shaking of the insert and blotting the insert on sterile blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT solution was prepared by thawing the MTT concentrate (MTT-100-CON) of 1 mL (5 mg/mL) and diluting this to 5 mL by adding 4 mL of MTT diluent (MTT-100-DIL) to obtain the final concentration of 1 mg/mL.
- Incubation time: 3 hours ± 5 minutes.
- Spectrophotometer: 96-well plate spectrophotometer.
- Wavelength: 570 nm (OD570).

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Pass - 1.448 ± 0.05.
- Barrier function: Pass - 6.16 hours.
- Morphology: Pass - Tissue viability and the barrier function test indicate appropriate formation of the epidermal barrier, the presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers. Well differentiated epidermis consisting 11 layers. Tissue had an average thickness of 119.2 µm.
- Contamination: Pass - sterile and no contamination.

NUMBER OF REPLICATE TISSUES: Triplicate.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be an irritant to skin in accordance with UN GHS Category 2 if the tissue viability after exposure and post-treatment incubation is less than or equal to 50%.
- The test substance is considered to be a non-irritant to skin in accordance with UN GHS Category 2 if the tissue viability after exposure and post-treatment incubation is more than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg.

NEGATIVE CONTROL
- Amount applied: 30 µl.

POSITIVE CONTROL
- Amount applied: 30 µl.

Duration of treatment / exposure:

60 ± 1 minutes.

Duration of post-treatment incubation (if applicable):

22 hours of incubation and an additional 19 hours after a media change. The bottom of all tissues was then blotted and transferred into MTT solution and incubated for 3 hours. Incubation occurred over 43 hours in total.

Number of replicates:

Triplicate.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60-minute exposure

Value:

3.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No.
- Direct-MTT reduction: Did not reduce MTT.
- Colour interference with MTT: No colour change.

DEMONSTRATION OF TECHNICAL PROFICIENCY: All kit components were examined for integrity. All information about supplied material was recorded and documented. MTT diluent and vial containing the MTT concentrate were stored in the refrigerator (2 - 8 °C) and in the deep freezer (-20 ± 5 °C).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes - Mean OD570 was 1.302, which is within the range of ≥1 and ≤2.5.
- Acceptance criteria met for positive control: Yes - Mean viability of tissues was 6.7 %, which is <20 % of the negative control tissue. The standard deviation of the three tissue replicates was 0.27 (below the 18.27 %).
- Acceptance criteria met for variability between replicate measurements: Yes - The standard deviation calculated from individual % tissue viabilities of the three identically treated replicates was <18.27 %, i.e. in the range of 0.27 - 2.27.

Any other information on results incl. tables

Percentage viability of the negative (DPBS) and positive control (5 % aqueous SDS) was 100 ± 2.27 and 6.7 ± 0.27, respectively.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Aryl sulphonate condensate has been concluded to be an irritant (Category 2) to Reconstructed Human Epidermis (EpiDerm™) under the UN Globally Harmonised System as mean percentage tissue viability was less than 50 % of the negative control.

Executive summary:

An experiment was performed using Reconstructed Human Epidermis - EpiDerm™ (EPI-200-SIT) to ascertain the skin irritation potential of aryl sulphonate condensate according to OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method). Triplicate tissue inserts were incubated prior to the experiment for a 20-hour and 10-minute period before being exposed to 30 µl each of Dulbecco's Phosphate Buffered Saline (DPBS) (negative control) and 5 % Aqueous Sodium Dodecyl Sulfate (SDS) (positive control), and 25 mg of the test item. After 60 minutes of exposure, the tissues were washed with DPBS thoroughly and blotted and transferred to fresh medium. The tissues were then incubated as part of a post-treatment incubation period for an additional 43-hour period, interspersed by an assay medium change and later transfer to MTT solution. An extraction of any resultant purple-blue formazan salt, formed predominantly by mitochondrial metabolism, was achieved using an MTT extraction solvent (MTT-100-EXT). A 96-well plate spectrophotometer was used to measure the optical density of the extracted formazan at 570 nm (OD570) and the viability of the tissues was calculated by entering OD values into a MatTek spreadsheet.

Percentage viability scores of 100 ± 2.27, 6.7 ± 0.27, and 3.1 ± 0.57 were determined for the negative control, positive control, and test item, respectively. All experiment acceptance criteria were satisfied. Given that the percentage viability of the test item was not greater than 50 % of the negative control, aryl sulphonate condensate has been concluded to be an irritant (Category 2) to Reconstructed Human Epidermis under the UN Globally Harmonised System.