Ammonium iron bis(sulphate)

Endpoint summary

Description of key information

Skin Irritation: The substance does not need to be classified because the GHS criteria for classification are not met.

Eye Damage: The substance should be classified as Eye Dam. 1 (H318).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

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

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)

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;

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

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 1

Value:

77.6

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 2

Value:

78.1

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 3

Value:

86

Negative controls validity:

valid

Positive controls validity:

valid

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.

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study will be available 31/10/2018

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

OECD Guideline for the Testing of Chemicals, Method No. 437, adopted09. Oct. 2017: “Bovine Corneal Opacity and Permeability Test Method for Identifyingi) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classificationfor Eye Irritation or Serious Eye Damage”

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

Commission Regulation (EU) 2017/735 amending Regulation (EC) No. 440/2008, EUMethod B.47 Bovine Corneal Opacity and Permeability Test Method for Identifying (i)Chemicals Inducing Serious Eye Damage and (ii) Chemicals Not Requiring Classificationfor Eye Irritation or Serious Eye Damage, adopted 14. Feb. 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals, Series on Testing and Assessment No. 160

Version / remarks:

OECD Guideline for the Testing of Chemicals, Series on Testing and AssessmentNo. 160: “GUIDANCE DOCUMENT ON “THE BOVINE CORNEAL OPACITY ANDPERMEABILITY (BCOP) AND ISOLATED CHICKEN EYE (ICE) TEST METHODS:COLLECTION OF TISSUES FOR HISTOLOGICAL EVALUATION AND COLLECTIONOF DATA ON NON-SEVERE IRRITANTS”; 25. Oct. 2011

Deviations:

no

Principles of method if other than guideline:

This in vitro study was performed to assess the corneal damage potential of Ammonium iron bis(sulphate) Dodecahydrate by quantitative measurements of changes in opacity and permeability in a bovine cornea. The study was performed for regulatory purposes. The BCOP test method is an organotypic model that provides short-term maintenance ofnormal physiological and biochemical function of the bovine cornea in vitro. In this testmethod, damage by the test item is assessed by quantitative measurements of changes incorneal opacity and permeability. Both measurements are used to calculate an “In VitroIrritancy Score (IVIS)”, which is used to classify the test item in the UN Globally HarmonisedSystem (GHS). The BCOP test method uses isolated corneas from the eyes of freshly slaughtered cattle. Corneal opacity is measured quantitatively as the amount of light transmission through thecornea. Permeability is measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea, as detected in the medium in the posterior chamber. Test item is applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder.

GLP compliance:

yes (incl. QA statement)

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. Preparation: The test item is a solid non-surface-active substance. In a non-GLP pre-test, the solubility of the test item was determined in Hank’s Balanced Salt Solution (HBSS) 10-fold concentrated, diluted in demin. water (1:10). The test item was sufficiently soluble in a concentration of 20% in HBSS. The solution was prepared freshly on the day of the assay.

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Bos primigenius Taurus (fresh bovine corneas): Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 μg/mL) in a suitable cooled container within 1 hour 5 minutes.

Vehicle:

Hank's balanced salt solution

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µl test item solution (20 % in HBSS)

Duration of treatment / exposure:

4 h

Duration of post- treatment incubation (in vitro):

90 min

Number of animals or in vitro replicates:

3 per treatment group

Details on study design:

Preparations: After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C. On the day of the assay, the minimum essential medium (MEM) without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C. The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate. After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C. Method description: After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used. For each treatment group (negative control solution, test item solution and positive control solution), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 μL negative control solution, 750 μL test item solution and 750 μL positive control solution were applied to each replicate. Open Chamber Method: In order to apply the test item, the nut was unscrewed to remove the glass disc. 750 μL of the test item were tested as solution at 20% concentration in HBSS. The test item was given directly on the epithelium in such a manner that as much as possible of the cornea was covered with test item. Exposure time on the corneas was 4 hours at 32 ± 1 °C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, both chambers were filled with cMEM without phenol red, and the final opacity value of each cornea was recorded. Permeability Test: After the recording of the final opacity value, the cMEM without phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution was added to the front chamber for the detection of permeability of the corneas. For the open chamber method, a sodium fluorescein solution with a concentration of 5 mg/mL was used.The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, its optical density at 492 nm was measured with the microtiter plate photometer.

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

120.12

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

135.78

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

97.13

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

mean (replicas 1-3)

Value:

117.68

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: standard deviation: 16.52 %

Other effects / acceptance of results:

According to the guideline, the test is considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean. The mean IVIS of the negative control has to show an IVIS ≤ 3. Values for negative and positive controls were within the range of historical data of the test facility. Therefore, the test system was acceptable.

Calculation of Opacity:The change of opacity value of each treated cornea with test item, positive control and negative control was calculated by subtracting the initial basal opacity from the post treatment opacity reading for each cornea. The average change in opacity of the negative control cornea was calculated and this value was subtracted from the change in opacity of each treated cornea with test item and positive control to obtain a corrected opacity.푂푝푎푐푖푡푦= [(퐼0/퐼)-b]/a.

a = 0.0251 and b = 0.9894 being Opacitometer-specific empirically determined variables. I0 = the empirically determined illuminance through a cornea holder with windows and Medium, here: Io= 1075.64; I = the measured illuminance (unit: LUX).Calculation of Permeability:The corrected OD492 value of each cornea treated with test item and positive control was calculated by subtracting the mean negative control cornea value from the original permeability value for each cornea. The mean OD492 value for each treatment group (test item, positive control and negative control) was determined by averaging the final corrected OD492 values of the treated corneas for one treatment group.Calculation of IVIS (In VitroIrritancy Score):The IVIS of each replicate of the negative control was calculated from the following equation: IVIS = opacity difference + (15 x corrected OD492 value).

The IVIS of each replicate of the positive control and of the test item were calculated from the following equation: IVIS = (opacity difference – mean opacity difference of the negative control) + [15 x (OD492– mean OD492 of the negative control)].

IVIS:

Test group	replica 1	replica 2	replica 3	mean (replica 1-3)	std. deviation / %
Test item	120.12	135.78	97.13	117.68	16.52
negative control	3.69	2.14	1.24	2.36	52.38
positive control	131.02	111.14	128.27	123.48	8.72

According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 55 induces serious eye damage, that should be classified as UN GHS Category I. In the negative control, no signs of eye irritation were observed. The positive control induced serious eye damage, which would be classified as GHS category 1. The test item Ammonium iron bis(sulphate) Dodecahydrate induced serious eye damage on the cornea of the bovine eye. The calculated IVIS is 117.68. The experiment is considered as sufficient for the classification of the test item, because all three replicates of the test item lead to the same assessment for the test item.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The test item Ammonium iron bis(sulphate) Dodecahydrate induced serious eye damage on the cornea of the bovine eye. The calculated IVIS is 117.68. The test material should be classified as Eye damage Category 1 based on GHS criteria.

Executive summary:

Thisin vitrostudy was performed to assess corneal damage potential of Ammonium iron bis(sulphate) Dodecahydrate by quantitative measurements of changes in opacity and permeability in a bovine cornea. The test item Ammonium iron bis(sulphate) Dodecahydrate was brought onto the cornea of a bovine eye which previously had been incubated with complete minimum essential medium (cMEM) without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been determined. The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured. The test item was tested as 20% solution in Hank’s Balanced Salt Solution (HBSS). Under the conditions of this test, the test item Ammonium iron bis(sulphate) Dodecahydrate induced serious eye damage on the cornea of the bovine eye. The calculated in-vitro irritation score (IVIS) is 117.68. According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 55 induces serious eye damage, that should be classified as UN GHS category I. The negative control (HBSS) and the positive control (20% imidazole solution) have met the validity criteria. No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In a study according to EU and OECD guidelines, the irritation potential of ammonium iron bis(sulphate) on reconstructed human epidermis was investigated. The test material did not show adverse effects.

A study according to OECD and EU guidelines was conducted to assess the corneal damage potential of Ammonium iron bis(sulphate) dodecahydrate. Under the conditions of this test, the test item Ammonium iron bis(sulphate) Dodecahydrate induced serious eye damage on the cornea of the bovine eye. The calculated in-vitro irritation score (IVIS) is 117.68. According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 55 induces serious eye damage, that should be classified as UN GHS category 1