4-anilinobenzenediazonium hydrogen sulphate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From November 15th to 16th, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)

Version / remarks:

July 28th, 2015

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4-anilinobenzenediazonium hydrogen sulphate

EC Number:

224-756-8

EC Name:

4-anilinobenzenediazonium hydrogen sulphate

Cas Number:

4477-28-5

Molecular formula:

C12H11N3O4S

IUPAC Name:

4-anilinobenzene-1-diazonium hydrogen sulfate

Test material form:

solid: particulate/powder

In vitro test system

Test system:

artificial membrane barrier model

Justification for test system used:

Test system used according to the OECD TG 435.

Vehicle:

unchanged (no vehicle)

Details on test system:

SOURCE AND COMPOSITION OF MEMBRANE BARRIER USED
- Was the Corrositex® test kit used: yes.
- Components: the test system CORROSITEX® is composed of two components: a synthetic macromolecular bio-barrier and a CDS (Chemical Detection System).The membrane barrier (bio-barrier) consists of two components: a proteinaceous macro-molecular aqueous gel and a permeable supporting membrane. The proteinaceous gel should be impervious to liquids and solids but it could be corroded and made permeable. The proteinaceous gel serves as the target for the test item and is placed on the surface of the supporting membrane and is allowed to gel, prior to placing the membrane barrier over the indicator solution. The proteinaceous gel should be of equal thickness and density on the entire surface and with no air bubbles or defects that could affect its functional integrity. The permeable supporting membrane provides mechanical support to the proteinaceous gel during the gelling process and exposure to the test item. The supporting membrane prevents sagging or shifting of the gel and is readily permeable to all test substances. The proteinaceous gel is supplied in the form of a powder to be reconstituted, prepared over the supporting membrane and stored according to the manufacturer’s instructions.
- Apparatus and preparation procedures: CORROSITEX® (Batch: CT120516, Supplier: in vitro INTERNATIONAL (17751 Sky Park East, Ste. G. Irvin - California 92614 - US). The bio-barrier was prepared starting from mixing two components (Biobarrier matrix powder and Biobarrier diluent) and slow dissolution occurred at a maximum temperature of 68 - 70 °C by a magneting stirring hot plate for 20 minutes. After 5 minutes of rest, the solution was pipetted over the supporting membrane ensuring the entire membrane was covered and no bubbles were formed. The solidification occurred after incubation at 2 - 8 °C. The test system was used the day after preparation but it is stable up to 7 days.

WAS THE COMPATIBILITY TEST PERFORMED: yes.
A qualification of the test item was carried out to verify if the CDS undergoes a physical change (Compatibility test - qualification). A Qualify test tube was supplied with the test system, containing the same indicator solution used in the main assay. An aliquot of 100 mg of test item was added to the Qualify test tube (amber solution). The tube was shaken to dissolve the test item and the vial was let stand for 1 minute. Colour change (e.g. from amber to red, orange, lightening) or consistency modification (flaking or precipitation) of the CDS was evaluated. In case no change occurred, the substance would have not been considered idoneous to proceed with the test.

WAS THE TIMESCALE CATEGORY TEST PERFORMED: yes.
A categorising test was then carried out to verify what time scale should have been used in the main experiment (Timescale category test - categorisation). Components of the test item timescale category test (Test Tube A, Test Tube B, Colour Chart and one bottle of Confirm reagent) were supplied with the test system. An aliquot of 100 mg of test item was added to Tube A (yellow solution) and Tube B (clear solution). Tubes were shaken to mix substances. The colour change in each tube was assigned and recorded according to the Colour chart. Each colour in the colour chart was associated to a letter. The substance is classified as Category 1 or 2 (time scale category), on the basis of the colour changes and according to the manufacturer’s instructions. When a colour change is not detected either in Tube A or B, a confirm reagent is added to Tube B to confirm the test system suitability. As the test item is coloured, the timescale category was determined by pH measurement. Essentially, the pH of a 10% w/v aqueous solution of test item was measured and, on the basis of results, the pH of a solution of the test item in either tube A or B was evaluated.

TEMPERATURE USED DURING TREATMENT: room temperature.

MAIN TEST:
A main assay was carried out including the test item, positive and negative controls. The pre-filled CDS vials were kept at room temperature (17 - 25 °C) before use. A cold biobarrier disc, kept on crushed ice on the bench, was added to the top of each vial. A fixed amount of samples was added to the top within 2 minutes from putting the biobarrier at room temperature. Replicates (4 replicates for test item and 1 replicate for negative and positive control) were staggered approximately at 5 minute intervals to ensure correct time recording. Effective starting time was recorded (Start time) using a timer. According to the time category (time category 2 determined in the preliminary test), test item samples were observed continuously at least during the following intervals: 0-5 min, 25-35 min and 55-65 min. Positive control vial was observed up to 18 minutes from start of treatment. Negative control vial was observed at 60 minutes.
Any change of the CDS was recorded. Changes in the CDS (amber liquid) contained in each tube may include change of colour (red, orange, lightening) or consistency (flaking or precipitation). Time of colour change detection was recorded (Detection time).

METHOD OF APPLICATION: the test item was used in the form supplied (500 mg).

NUMBER OF REPLICATES: 4 replicates for test item (500 mg of test item per well); 1 replicate for negative control (500 μl per well of 10 % w/v Citric acid solution in sterile water for injection) and 1 replicate for positive control (500 μl per well of Valeroyl chloride).

NUMBER OF INDEPENDENT EXPERIMENTS TO DERIVE FINAL PREDICTION: one experiment

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the time (in minutes) elapsed between application of the test item to the membrane barrier and barrier penetration according to category 2 determined in the preliminary test) is:
0 to 3 min GHS 1A
>3 to 30 min GHS 1B
> 30 to 60 min GHS 1C
- The test substance is considered to be non-corrosive to skin if the time (in minutes) elapsed between application of the test item to the membrane barrier and barrier penetration according to category 2 determined in the preliminary test) is major than 60 minutes.
- Justification for the selection of the cut-off point(s): according to OECD TG 435 time category 2.
Note: CORROSITEX® Time = Detection Time – Start Time.
Means and standard deviations of CORROSITEX® Time were calculated for replicates of the same sample.

ACCEPTANCE CRITERIA:
The assay was considered valid if the following criteria were met:
– the positive control should give the expected penetration response time (historical mean value + 3 SD), based on laboratory historical control values (12.33 ± 6.75 minutes; n=16).
– the negative control should not be corrosive within 60 minutes.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

500 mg of test item per well for test item (4 replicates) ; 500 μl per well of 10 % w/v Citric acid solution in sterile water for injection (negative control, 1 replicate) and 500 μl per well of Valeroyl chloride (positive control, 1 replicate).
NOTE: the volume of test item corresponding to 500 mg was very high, thus it was not possible to add the whole quantity on the membrane surface. Only the amount of substance to the complete membrane coverage was used.

Duration of treatment / exposure:

65 minutes observation time.

Number of replicates:

4 replicates for test item ; 1 replicate for negative control and 1 replicate for positive control.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

penetration time (in minutes)

Run / experiment:

main assay

Value:

> 65

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value for each of the 4 replicates (time category 2 used for the evaluation).

Other effects / acceptance of results:

MAIN ASSAY
The test item did not show the ability to penetrate the biobarrier during the whole observation interval.

PRELIMINARY TESTS
- Compatibility test-qualification: The test item is suitable for this test system since orange colour was recorded after adding the test item (Colour change detected from amber to orange).
- Timescale category test - categorisation: Tube A content changed the colour to orange, while no relevant colour change was observed in Tube B, whose solution became the same colour as the test item. However, since the test item is coloured, the timescale category was determined by pH measurement. pH value of test item suspension in water at 10% (w/v) was 0.93. Based on this result, 100 mg of test item was added to Tube A and the recorded pH was 5.58; hence the test item was assigned to the time category 2.

ACCEPTANCE OF RESULTS:
All the acceptance criteria were fulfilled:
– the positive control Valeroyl chloride gave the expected penetration penetration response time (17.3 minutes) (required: 12.33 ± 6.75 minutes; n=16) equivalent to category 1B GHS
– the negative control was not corrosive within 60 minutes.

Any other information on results incl. tables

Test or control item Sample code	Sample code	Chemical/ physical changes	Breakthrough observation time (min)
			Values observed	Mean ± SD
4-anilinobenzenediazonium hydrogen sulphate	1	No change	> 65.00	N/A
	2	No change	> 65.00	N/A
	3	No change	> 65.00	N/A
	4	No change	> 65.00	N/A
Valeroyl Chloride	+	Colour change	17.3	N/A
10% Citric Acid	-	No change	> 60.00	N/A

Applicant's summary and conclusion

Interpretation of results:

other: not classified as skin corrosive according to the CLP Regulation (EC n.1272/2008).

Conclusions:

4-anilinobenzenediazonium hydrogen sulphate is not classified as skin corrosive according to the CLP Regulation (EC n.1272/2008).

Executive summary:

The skin corrosion potential of the test item 4-anilinobenzenediazonium hydrogen sulphate was assessed with an in vitro membrane barrier assay, using the validated commercial kit CORROSITEX®.

In a preliminary test, the test item was demonstrated to be suitable for this test system, since it induced a colour change in the Chemical Detection System of the kit. Since the test item is coloured, the timescale category was determined by pH measurement. The pH value in Tube A was 5.58, thus the time category 2 was assigned.

In the main assay, the test item was assayed in quadruplicate and observed according to the time scale category 2, as defined in the preliminary test. Valeroyl chloride (classified as GHS 1B substance) and 10% (w/v) citric acid solution were concurrently tested as positive and negative controls, respectively.

The test item did not show the ability to penetrate the biobarrier during the whole observation interval. Positive and negative control results met the acceptance criteria indicating a good functioning of the test system.

According to OECD TG 435, 4-anilinobenzenediazonium hydrogen sulphate is defined as non corrosive to the skin.