Zinc bis(benzenesulphinate)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 28 to 31, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2009

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Zinc bis(benzenesulphinate)

EC Number:

246-148-1

EC Name:

Zinc bis(benzenesulphinate)

Cas Number:

24308-84-7

Molecular formula:

C12H10O4S2Zn

IUPAC Name:

zinc bis(benzenesulphinate)

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on test system:

The reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Bratislava, Slovakia) consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis. The EpiDerm™ system is manufactured according to defined quality assurance procedures.
The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts and shipped as kits, containing tissues on shipping agarose together with the necessary amount of culture media.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 25 min at room temperature, 35 min at 37 ± 1 °C
- Temperature of post-treatment incubation: 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: two, the first after 1 h of exposure; the second, after 25 h and 14 min of post-incubation period

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Libra S22
- Wavelength: 570 nm
- Blank: isopropyl alcohol
- Filter bandwidth: 2-3 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: one

PREDICTION MODEL / DECISION CRITERIA
Relative cell viability is calculated for each tissue as % of the mean of the negative control tissues viability, which is set at 100 %.
The cut-off values for the prediction of irritation are given below; these values are stated in OECD guideline 439, par. 36:
- in case test chemical is found to be non-corrosive (e.g., based on TG 430, 431 or 435), and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50 %, the test chemical is considered to be irritant to skin in accordance with UN GHS Category 2.
- test chemical may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50 %.
A single testing run composed of three replicate tissues should be sufficient for a test chemical when the classification is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent viability equal to 50 ± 5 %, a second run should be considered, as well as a third one in case of discordant results between the first two runs.

ASSAY ACCEPTANCE CRITERIA
Negative control
The absolute OD of the negative control (NC) tissues (treated with sterile PBS) in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use. The assay meets the acceptance criterion if the mean OD570 of the NC tissues is ≥ 0.8 and ≤ 2.8. OD570 historical negative control range is 1.455-2.347.
Positive control
A 5 % SDS (in H2O) solution is used as positive control (PC) and tested concurrently with the test chemicals. Concurrent means here the PC has to be tested in each assay, but not more than one PC is required per testing day. Viability of the positive control should be within 95±1 % confidence interval of the historical data. The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is ≤ 20 %. The 95% confidence interval of historical positive control is 0-0.190.
Standard deviation (SD)
Each test skin irritancy potential is predicted from the mean viability determined on 3 single tissues, so the variability of tissue replicates should be acceptably low. The assay meets the acceptance criterion if the SD calculated from individual % tissue viabilities of the 3 identically treated replicates is < 18 %.

When any of the acceptance criteria is not met, the experiment has to be repeated.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

25 µl of test substance was dosed directly on tissue moistened with 25 µl of PBS. The substance was spread on the tissue surface. A single experiment, composed of three replicate tissues, was run.

Duration of treatment / exposure:

On the day of receipt, EpiDerm tissues were conditioned by incubation to release transport stress related compounds and debris. Duration of the first pre-incubation was 60 minutes and the second 19 hours and 11 minutes.
After pre-incubations, tissues were topically exposed to the test chemicals for 60±1 minutes (25 minutes at room temperature and the remaining 35 minutes at culture conditions). Three tissues were used for test substance, three for positive (PC) and three for negative (NC) controls. Tissues were then thoroughly rinsed with PBS. Inserts were then transferred to fresh medium.

Duration of post-treatment incubation (if applicable):

After 25 hours and 14 minutes of post-incubation period, the medium was replaced by fresh one. Tissues were incubated for another 18 hours, 12 minutes.

Number of replicates:

3 tissues per test substance, 3 tissues per positive control, 3 tissues per negative control.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

moistened tissues

Run / experiment:

1

Value:

96.7

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

not moistened tissues

Run / experiment:

1

Value:

100.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS
- Direct-MTT reduction: 25 mg of test substance was added to 1.0 ml of MTT medium. Solution was incubated for 1 hour at culture conditions. After incubation, the medium was coloured light green. Medium did not change colour to blue (purple). Test substance did not reduce MTT directly.

ACCEPTANCE OF RESULTS:
The mean OD570 of the NC tissue was 1.949 which meets the acceptance criteria of ≥ 0.8 and ≤ 2.8.
The mean viability of PC tissues expressed as % of negative control tissues was 2.9 % which meets the acceptance criterion of ≤ 20 %.
The SD calculated from individual % tissue viabilities of 3 identically treated replicates was < 18 % in all cases.
All study acceptance criteria were fulfilled.

Any other information on results incl. tables

OD570 values in MTT test, standard deviation % and relative viabilities

	treatment	OD570			mean	SD	mean viability (% NC)
		1	2	3
NC	PBS	1.961	1.946	1.941	1.949	0.008	100.0
	%	100.6	99.8	99.6	100.0	0.436
sample (moistened)	test substance	0.584	1.791	1.978	1.885	0.618	96.7
	%	30.0	91.9	101.5	96.7	4.797
sample (non-moistened)	5 % SDS	1.915	1.992	-	1.954	0.039	100.2
	%	98.2	102.2	-	100.2	1.975
PC	5 % SDS	0.059	0.053	0.056	0.056	0.02	2.9
	%	3.0	2.7	2.9	2.9	0.126

Applicant's summary and conclusion

Interpretation of results:

other: not irritant according to the CLP Regulation (EC 1272/2008)

Conclusions:

Not irritant.

Executive summary:

Method

Test substance was assayed for in vitro skin irritation in the human epidermal model EpiDermTM. The test was performed according to OECD guideline 439.

Direct MTT reduction was assessed; colour interference was not expected as the substance is coloured white.

In main experiment after pre-incubation of tissues, 25 mg of test substance were placed directly on previously moistened tissues and spread on the entire tissue surface. Two other tissues were treated without previous moistening. Exposure length was 60 minutes. Three tissues were used for positive as well as for negative controls.

After removal of test substance, tissues were post-incubated for approximately 42 hours. Three hours incubation with MTT and two hours extraction period with shaking followed then. Optical density (OD570) of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of mean viability of negative control tissues.

Results

No direct MTT reduction was found. 

Under the above-described experimental design mean viability of treated tissues was 96.7 % in moistened tissues and 100.2 % in non moistened tissues, i.e. viability was > 50 % in both cases.