hydronium;iron(3+);hexahydroxide;disulfate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 24 June 2014; Experimental Completion Date: 30 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Valid and conclusive guideline study under GLP; Relevant and adequate for this endpoint

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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:

2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

as described in Commission Regulation (EC) No. 761/2009, of 23 July 2009, amending, for the purpose of its adaption to technical progress, Regulation (EC) No. 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Statement of compliance in accordance with Directive 2004/9/EC, Department of Health of the Government of the U.K., 12 September 2014, inspection date 12 to 14 March 2014

Test material

Test material information

Constituent 1

Reference substance name:

Automatically generated during migration to IUCLID 6, no data available

IUPAC Name:

Automatically generated during migration to IUCLID 6, no data available

Test material form:

solid - liquid: suspension

Details on test material:

- Name of test material (as cited in study report): Hydronium Jarosite
- Substance type: Inorganic chemical
- Physical state: Light brown solid
- Analytical purity: > 97 %
- Storage condition of test material: Room temperature in the dark

Test animals

Species:

human

Strain:

other: not applicable, in vitro test using Reconstructed Human Epidermis (EpiSkin™ model)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Not applicable, as a reconstructed human epidermis model was used in vitro.
- EpiSkin™ Tissues (0.38 cm²) lot number : 14-EKIN-023
- Maintenance Medium lot number : 14-MAIN3-027
- Assay Medium lot number : 14-ESSC-024
- Source: SkinEthic Laboratories, Lyon, France
- Preincubation (tissues, Day 0, Tissue Arrival): Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:
Tissues Satisfactory : Yes
Temperature Indicator Colour Satisfactory : Yes
Agar Medium Colour Satisfactory : Yes
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5 % CO2 in air overnight.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37
- Atmosphere: 5 % CO2 in air
- Photoperiod: The test was conducted in the dark.

Test system

Type of coverage:

open

Preparation of test site:

other: 5 μL of sterile distilled water was topically applied to the epidermal surface in order to improve further contact between the test item and the epidermis. 10 mg (26.3 mg/cm²) of the test item was then applied to the epidermal surface.

Vehicle:

water

Remarks:

(sterile distilled)

Controls:

other: Untreated MTT solution was used as a control.

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 10 mg (26.3 mg/cm² per tissue
- Concentration: The amount 10 mg test item in of 5 μL of sterile distilled water exceeds its water solubility by far.

VEHICLE
- Amount(s) applied (volume or weight with unit): 5 μL

Duration of treatment / exposure:

15 min of treatment at room temperature, then rinsing, followed by 42 h of post-incubation period at 37 °C

Observation period:

After a 42 hour post-exposure incubation period, determination of the cytotoxic (irritancy) effect was performed. The incubation for the cell viability measurement was made during 3 h.

Number of animals:

The test was performed on a total of 3 tissues (i.e. three replicates of treatment, negative and positive control)

Details on study design:

TEST SITE
- Area of exposure: In the in vitro study 0.38 cm² of the skin tissue (EPISKIN Standard Model™) was exposed in well plates
- % coverage: The skin tissue (EPISKIN Standard Model™) was completely covered by the liquid test item, no wrap was therefore used

REMOVAL OF TEST SUBSTANCE
- Washing: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5 % CO2 in air for 42 hours.
- Time after start of exposure: 15 min of treatment at room temperature, then rinsing

SCORING SYSTEM:
Cell viability (cytotoxicity) was measured after rinsing, followed by 42 h of post-incubation period at 37 °C. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan (CAS 504-65-4) production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, CAS 298-93-1) at the end of the treatment by OD570 (optical density at 570 nm) determination. Non-irritancy has to be considered according to the test guideline if the treatments show > 50 % of the mean viability of the negative controls.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: relative mean viability determined as % MTT Reduction

Value:

88.5

Remarks on result:

other:

Remarks:

Basis: mean. Time point: after a 15-Minute exposure period and 42 hours post-exposure incubation period. Max. score: 84.5. (migrated information)

Any other information on results incl. tables

Direct MTT Reduction

The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

Test Item, Positive Control Item and Negative Control Item

The individual and mean OD562 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Table 1. The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in Table 1.

Table 1: Mean OD562 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD562 of tissues	Mean OD562 of triplicate issues	± SD of OD562	Relative individual tissue viability [%]	Relative mean viability [%]	± SD of Relative mean viability [%]
Negative Control Item	0.945	1.018	0.067	92.8	100*	6.6
	1.031			101.3
	1.077			105.8
Positive Control Item	0.069	0.069	0.012	6.8	6.8	1.2
	0.080			7.9
	0.057			5.6
Test Item	0.947	0.901	0.044	93.0	88.5	4.3
	0.860			84.5
	0.896			88.0

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100%

OD562 = Optical Density

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Not classified (UN GHS Category 3 cannot be determined)

Executive summary:

The in vitro skin irritation potential of the test item Hydronium Jarosite to Reconstructed Human Epidermis tissues (RHE, EpiSkin™ model) was investigated in a GLP-compliant study according to the OECD TG 439 (2013) and EU B.46 (2012) protocols. The experiment can be considered valid, relevant and adequate for final conclusion on the presence or absence of corrosive (UN-GHS category 1) and/or irritant (UN-GHS category 2) properties. Therefore it can be deemed conclusive for presence of absence of non-irritancy and was rated „reliable without restrictions“, i.e. “Klimisch 1” according to the scale of Klimisch et al. (1997).

The purpose of this test is to evaluate the skin irritation potential of the test item using the EPISKIN™ reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-Hour post-exposure incubation period may also be determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point can be used to either confirm a non-irritant result or will be used to override the non-irritant result.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 562 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The quality criteria required for acceptance of results in the test were satisfied confirming the suitability of the test system. The relative mean viability of the test item treated tissues was 88.5 % after the 15-Minute exposure period and 42 hours post-exposure incubation period.

In conclusion the test item was classified as non-irritant. The following classification criteria apply based on the results of this study alone:

EU DSD & CLP - Not classified for Irritation.

UN GHS - Not classified for Irritation (but category 3, which is not implemented in CLP, cannot be determined).

• Klimisch HJ, Andreae M, Tillmann U (1997). A Systematic Approach for Evaluating the Quality of Experimental Toxicological and Ecotoxicological Data. DOI 10.1006/rtph.1996.1076 PMID 9056496 Regul Toxicol Pharmacol 25:1-5.