Ammonium iron bis(sulphate)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11/06/2018 - 20/07/2018

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guideline for the Testing of Chemicals, Version 439, adopted 28. July 2015,“In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”

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:

Commission Regulation (EU) No. 640/2012 amending Regulation (EC) No.440/2008, Annex III, EU method B.46 “IN VITRO SKIN IRRITATION: RECONSTRUCTEDHUMAN EPIDERMIS MODEL TEST”, adopted 06. Jul. 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

7783-83-7

Molecular formula:

NH4Fe(SO4)2 • 12 H2O

Test material form:

solid

Specific details on test material used for the study:

Name: Ammonium iron bis(sulphate) Dodecahydrate; Batch no.: 114123; Appearance: Pale violet lumps; Purity: Reagent grade, 99.6% (test data from supplier); Homogeneity: homogeneous; Expiry date: 05. Oct. 2018; Storage: Room Temperature (20 ± 5°C); Keep away from light;

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: human-derived epidermal keratinocytes

Cell source:

other: EpiDerm tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava. Designation of the kit: EPI-200-SIT; Day of delivery: 17. Jul. 2018; Batch no.: 28634

Details on test system:

Chemicals and Media: MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (=MTT), which can be reduced to a blue formazan. A MTT stock solution of 5 mg/mL in DPBS buffer was prepared and stored in aliquots of2 mL in the freezer (– 20 ± 5 °C). 2 mL of the stock solution were thawed and diluted with 8 mL of medium. This MTTsolution with the resulting concentration of 1 mg/mL was used in the test. For the pre-test (testing the ability of direct MTT reduction), the stock solution was thawed and diluted with serum-free MEM directly before use.For the main test, the stock solution was thawed and diluted with assay medium directly before use.MEM with Phenol Red for Pre-Test: Serum-free MEM (Minimum Essential Medium), procured by Life Technologies GmbH, batch no.: 1880322. Assay Medium: Serum-free DMEM (Dulbecco’s Modified Eagle’s Medium), procured by MatTek In VitroLife Science Laboratories, batch no.: 071218MSA. Isopropanol: CH3-CH(OH)-CH3, p.A., 99.9 %, batch no.: 276245555, used as extracting solvent for formazan. DPBS-buffer: Solution for the rinsing of the tissues and solvent for MTT concentrate, also used as negative control. A subset was procured by MatTek In Vitro Life Science Laboratories; the other subset was prepared by LAUS GmbH. Composition of the subset from MatTek In Vitro Life Science Laboratories (batch no.: 071018MSA): KCl 0.2 g, KH2PO4 0.2 g, NaCl 8.0 g, Na2HPO4 * 7H2O 2.16 g, H2O ad 1 L.Composition of the subset from LAUS GmbH (batch no.: 20171114): KCl 0.2 g, KH2PO4 0.2 g, NaCl 8.0 g, Na2HPO4 * 2H2O 1.44 g, H2O ad 1 L. The buffer which was procured by MatTek Corporation was used as negative control and for rinsing the test item from the tissues. The buffer which was prepared by LAUS GmbH was used as solvent for MTT concentrate and for rinsing the outside of the inserts at the end of the incubation time with MTT.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Tissue 1: 27.1 mg; Tissue 2: 26.8 mg; Tissue 3: 27.3 mg

Duration of treatment / exposure:

1 h

Duration of post-treatment incubation (if applicable):

23 h 20 min

Number of replicates:

1

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 1

Value:

77.6

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 2

Value:

78.1

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 3

Value:

86

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean (tissue 1-3)

Value:

80.6

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Skin irritation potential of the test item is assessed as: if the tissue viability is ≤ 50 % of negative control the test material is regarded as Corrosive/Irritant to skin (Category 1 or 2) according to UN GHS. At tissue viabilities above 50 % of the negative control the test material is regarde as non-irritant to skin.

Any other information on results incl. tables

OD @ 570 nm: Blank isopropanol: 0.038

Designation	Measurement	Negative Control	Test material	Positive control
Tissue 1	1	1.593	1.268	0.080
Tissue 1	2	1.561	1.321	0.096
Tissue 2	1	1.808	1.306	0.090
Tissue 2	2	1.834	1.298	0.084
Tissue 3	1	1.564	1.433	0.088
Tissue 3	2	1.580	1.428	0.088

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The mean value of relative tissue viability of the test item was reduced to 80.6% after the treatment. This value is above the threshold for skin irritation (50%). Therefore, the test item is considered as non-irritant to skin.

Executive summary:

The skin irritation potential of ammonium iron bis(sulphate) dodecahydrate was assessed in a study on reconstructed human epidermis according to EU and OECD guidelines. The test item Ammonium iron bis(sulphate) Dodecahydrate is considered as non-irritant to skin. After the treatment, the mean value of relative tissue viability was reduced to 80.6%. This value is above the threshold for skin irritation (50%). The optical density of the negative control was well within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8. The positive control has met the acceptance criterion too, for thus ensuring the validity of the test system. Variation within replicates was within the accepted range for negative control, positive control and test item (required: ≤ 18%). For these reasons, the result of the test is considered valid.