hydronium;iron(3+);hexahydroxide;disulfate

Endpoint summary

Description of key information

Skin Irritation For the characterisation of the skin irritation potential of members of the iron salt category, the study of Clouzeau (1994) was chosen as the key study. Based on the results of this study, FeSO4 x 7 H2O was classified as skin irritating – Category 2 according to CLP. This classification for skin irritation is proposed for all members of the iron salt category. Other available skin irritation studies with iron salts were assigned as supporting studies. The tests with FeCl3 and Fe2(SO4)3 support the proposed classification as skin irritant. In the case of FeCl2 the classification due to the precautionary principle represents a worst case. No test data was available for FeClSO4, therefore read across from FeCl3 was proposed, i.e. FeClSO4 should be classified into Category 2 according to CLP. Eye Irritation / Corrosion For the characterisation of the eye irritation / corrosion potential of members of the iron salt category, the study of Jeong (2004) was chosen as key study. Based on the results of this study, FeCl2 was classified as causing irreversible effects on the eye – Category 1 according to CLP. This classification for eye irritation / corrosion is proposed for all members of the iron salt category. Other iron salt studies were assigned as supporting studies, where FeCl3 was classified as well into Category 1 according to CLP. An exception was made for FeSO4, which is listed in Annex I of the European Plant Protection Products Directive. This substance is already classified as irritating to eyes (R36) in the Draft Assessment Report for Iron Sulphate (September 2008) and it is proposed to keep this classification for FeSO4 and the submission item . According to CLP, both should be classified into Category 2.

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:

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

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

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.

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.

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)

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

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.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

The Reporting Format for the Chemical Category According to ECHA (2008) Guidance R.6.2.6.2 can be found in the Endpoint Summary of Toxicokinetics, metabolism and distribution.

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

2009

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

Species:

other: an isolated bovine cornea was used in vitro

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- Source: Eyes were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee and placed in Hanks’ Balanced Salt Solution (HBSS), supplemented with Penicillin and Streptomycin, and transported to the laboratory on ice packs. The corneas were refrigerated on arrival and used within 24 hours of receipt.
- Age at study initiation: The eyes were taken from adult cattle

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 32±1

Vehicle:

physiological saline

Controls:

other: 3 replicates were used in the positive control and also in the negative control

Amount / concentration applied:

TEST MATERIAL & VEHICLE
- Amount(s) applied: 0.75 mL of the test item preparation or control items were applied to the appropriate corneas
- Concentration: For the purpose of this study the test item was prepared as a 20 % w/v solution in 0.9 % w/v sodium chloride solution.

VEHICLE
- Lot/batch no: 300999 104
- Purity: 0.9 %

Duration of treatment / exposure:

240 min

Observation period (in vivo):

The results were recorded immediately after exposure as the In Vitro system cannot recover.

Number of animals or in vitro replicates:

3 replicates were used in the treatment group

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: At the end of the exposure period the test item preparation and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM. The anterior and posterior chambers were refilled with fresh complete MEM. RThen a post treatment opacity reading was taken.
- Time after start of exposure: 240 min

SCORING SYSTEM:
The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

TOOL USED TO ASSESS SCORE
- Opacity Measurement: Opacity reading was taken for each cornea using a calibrated opacitometer.
- Permeability Measurement: Optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.

Irritation parameter:

other: In Vitro Irritancy Score

Basis:

mean

Remarks:

of 3 replicates

Time point:

other: 240 minutes

Score:

10.6

Max. score:

14.25

Reversibility:

other: not applicable

Remarks on result:

other: In Vitro Irritancy Score = mean opacity value + (15 · mean OD492 value)

Irritant / corrosive response data:

- Corneal Opacity and Permeability Measurement: Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Table 1.
- Corneal Epithelium Condition: The condition of each cornea post treatment and at the final opacity measurement is given in Table 2. The corneas treated with the test item were slightly cloudy post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.
- In Vitro Irritancy Score of the controls: Negative Control 2.6; Positive Control 90.6

Table 1: Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea number	Opacity				Permeability (OD)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Score Post-Treatment - Pre-Treatment	Corrected Value		Corrected Value
Negative Control	17	2	4	2		0.036
	18	2	4	2		0.050
	19	2	4	2		0.034
				2.0*		0.040#		2.6
Positive Control	14	2	79	77	75.0	1.180	1.140
	15	2	66	64	62.0	1.944	1.904
	16	4	79	75	73.0	1.111	1.071
					70.0§		1.372§	90.6
Test Item	11	3	17	14	12.0	0.190	0.150
	12	3	12	9	7.0	0.147	0.107
	13	3	14	11	9.0	0.024	0.000
					9.3§		0.086§	10.6

OD = Optical density

* = Mean of the post-treatment - pre-treatment values ¨

# = Mean permeability

§ = Mean corrected value

Table 2: Corneal Epithelium Condition Post Treatment

Treatment	Cornea Number	Observation
		Post Treatment
Negative Control	17	clear
	18	clear
	19	clear
Positive Control	14	cloudy
	15	cloudy
	16	cloudy
Test Item	11	slightly cloudy
	12	slightly cloudy
	13	slightly cloudy

Interpretation of results:

other: The test item was considered not to be an ocular corrosive or severe, but this does not permit conclusion that the test item does not require classification for eye irritation.

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The result of this study has identified the test item as not causing serious eye damage, but poentially being an eye irritant.

Executive summary:

The in vitro eye irritation potential of the test item Hydronium Jarosite to the isolated bovine cornea was investigated in a GLP-compliant study according to the OECD TG 437 (2009) protocol. 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 test system consisted of eyes taken from adult cattle. Three replicates each were exposed during 240 min at 32±1 °C to 0.75 mL of the test item preparation or control items. The test item was prepared as a 20 % w/v solution in 0.9 % w/v sodium chloride solution, which served without test item as negative control (physiological saline). The positive control item, Imidazole, was used as a 20 % w/v solution in 0.9 % w/v sodium chloride solution. A test item that induces an In Vitro Irritancy Score ≥ 55.1 is defined as an ocular corrosive or severe irritant and will be labelled EU DSD (67/548/EEC) R41 and EU CLP/UN GHS Category 1. In absence of effects a substance can be considered no classifiable.

The In Vitro Irritancy Score of the negative control was 2.6, while the positive control attained 90.6. Therefore the usability of the test system was confirmed. The mean score of the test item was 10.6 (max. 14.25). The corneas treated with the test item were slightly cloudy post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

In conclusion, based on this study alone, the test item was considered not to be an ocular corrosive or severe, but this does not permit conclusion that the test item does not require classification for eye irritation.

• 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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

This endpoint is covered by the category approach for dissociating, inorganic and non-toxic iron compounds (please see the section on toxicokinetics, metabolism and distribution for the category justification/report format).

Skin Irritation

• Animal data

Key studies

- FeSO4 is considered to be a skin irritating - it is classified as skin irritating Category 2 according to CLP. The result from a fully reliable study conducted according to OECD 404 and GLP in rabbits using FeSO4x7H2O (Clozeau 1994) showed that erythema are formed to an extent that warrant a respective classification for this substance. As in anhydrous FeSO4 the relative amount of iron and sulphate is even higher, it can be assumed that anhydrous iron sulphate is also a skin irritant. The study of Clozeau (1994) is considered as a key study for the iron salt category representing the worst case scenario. Therefore, Category 2 will be assigned to all pure and concentrated iron salts.

CLP (regulation EC No 1272/2008 of the European parliament and of the council, 5th ATP), Annex I, Table 3.2.3 provides the generic concentration limits to be used to determine if the mixture is considered to be an irritant or a corrosive to the skin. Accordingly a mixture, e.g. with water, would need classification if a Category 2 classified component is present in a concentration of ≥10 % unless experimental data on the mixture or a comparable mixture (enabling bridging) exist. Experimental results on watery solution are available from a fully reliable study (Johnson 2003) on 24.9 % (w/w) of FeSO4x7H2O in water. This concentration was no longer a skin irritant and produced no effects. It is thus suggested to replace the generic concentration limit by 25 % (w/w) and to consider aqueous solutions of iron salts as irritant, Category 2, from concentrations of 25 % (w/w) on.

Supporting information

- FeCl3 is deemed skin irritating - Category 2. The result was available from the two supporting studies BASF 1977a and BASF 1977b where rabbits have been exposed to either a 40 % (w/w) solution of FeCl3 in water or to pure, anhydrous FeCl3 for 20 h. In the latter study slight necrosis, desquamation, strong oedema and remnant substance at the test site was seen 24 h post exposure. 8 days after exposure still scabbing, necrosis and desquamation were found. No information is available on the effects 14 days post dosing. Following the precautionary principle based on the available results the substance is deemed a skin irritant Category 2 according to CLP. The results in the study (with 40 % (w/w) solution in water) are comparable but of less severity. The proposed classification as Category 2 skin irritant thus is supported.

- For Fe2(SO4)3 a clear classification based on the available data is not possible. The results from OECD TG 404 and GLP compliant study ICI 1991b suggest that the substance is not irritating. Nevertheless the available methodological information is so scarce that these results are not reliable. In addition the reported findings are surprising as a solution of ferric sulphate has a very low pH value, comparable to a FeCl3 solution with the same concentration of iron. Accordingly here a read across from FeCl3 is applied and the substance is deemed a skin irritant Category 2 according to CLP.

- FeCl2 is deemed not irritating. A fully reliable in vivo study in rabbits (Park 2004) conducted according to OECD 404 and GLP shows that the effects inflicted by FeCl2 (pure, moistened with distilled water) on the rabbit skin at all time-points are clearly below the threshold values that would trigger a classification as skin irritant. According to precaution reasons and category approach, we recommend the worst case scenario and to assign FeCl2 to the Category 2.

- For FeClSO4 no test data is available. As the substance has the same oxidation state as FeCl3 the latter is use as the template for read across. Accordingly FeClSO4 is deemed a skin irritating - Category 2 according to CLP.

Futher testing for this endpoint has been waived in accordance with Annex XI, 1.2 restrictions, based on the existing test data on the submission item and considering its pH.

• Human data

No relevant human data on skin irritation are available. An overview of existing observation is available from the OECD (2007, p 45) SIDS Initial Assessment Report (SIAR).

• OECD Organisation for Economic Co-operation and Development (2007). SIDS Initial Assessment Report for SIAM 24. Chemical Category: Iron Salts. Self-published, Paris, France 138 p.

• Summary:

According to the worst case scenario, all pure and concentrate iron salts (>25 %) should be assigned to the skin irritation Category 2.

Eye Irritation / Corrosion

• Animal data

Key study

- FeCl2 is deemed corrosive to the rabbit eye - Category 1 according to CLP based on the results from a fully reliable study (Jeong 2004, according to OECD TG 405 and GLP). Irritation was observed in the cornea, iris and conjunctiva reaching threshold values that clearly trigger the classification. This study was selected as the key study and considered as the worst case scenario for all iron salts included in this category.

Supporting information

- FeCl3 is deemed corrosive to the rabbit eye - Category 1 (according to CLP). The result was available from the two supporting studies BASF 1977a and BASF 1977b where rabbits have been exposed to either 50 µL of a 40 % (w/w) solution of FeCl3 in water or to 50 mg of pure, anhydrous FeCl3. In the latter study at 1 h after the application of the test item to the rabbit eye the palpebral conjunctiva was yellowish stained and slight oedema, strong corneal opacity and discharge was seen. All signs increased in severity after 24 h and 8 days post dosing leading to corneal ablation and suppurative inflammation of the eye. The results in the study conducted with 40 % aqueous solution, are comparable but of less severity than in the study with the pure compound.

- For Fe2(SO4)3 a clear classification based on the available data is not possible. Information is available from only one unreliable secondary source (Grant 1986). No numerical data is given. As a solution of ferric sulphate has a very low pH value, comparable to a FeCl3 solution with the same concentration of iron, here a read across from FeCl3 is applied and the substance is deemed corrosive to the rabbit eye - Category 1 according to CLP.

- For FeClSO4 no test data is available. As the substance has the same oxidation state as FeCl3 the latter is used as the template for read across. Accordingly FeClSO4 is deemed to be corrosive to the rabbit eye - Category 1 according to CLP.

Supplementary information

- According to an EFSA (2008) Draft Assessment Report for Iron Sulphate, FeSO4 is classified as irritating to eyes (R36). A fully reliable study is available (Bayer AG 1992, conducted according to OECD 404 and GLP), using FeSO4 x 7 H2O as test item and indicating that this substance is slightly irritating to the rabbit eye. Due to the water content the data are not fully indicative for the irritation/corrosion potential of anhydrous FeSO4.

Based on the already available documents, FeSO4 is an exception of iron salt category and classified as irritating to eyes - Category 2 according to CLP.

• EFSA European Food Safety Authority ed (2008) Draft Assessment Report (DAR) – public version – Initial risk assessment provided by the rapporteur member state U.K. for the existing active substance Iron Sulphate of the fourth stage of the review programme referred to in Article 8(2) of Council Directive 91/414/EEC. September 2008. Volumes 1 to 3.

• Human data

No human data on eye irritation are available. Existing information is available from the OECD (2007, p 45) SIDS Initial Assessment Report (SIAR).

• OECD Organisation for Economic Co-operation and Development (2007). SIDS Initial Assessment Report for SIAM 24. Chemical Category: Iron Salts. Self-published, Paris, France 138 p.

• Summary:

All iron salts in this category are deemed to produce corrosivity in the eye and are classified as follows: irreversible effects on the eyes - Category 1 according to CLP. Exception is FeSO4 which is already classified in the Draft Assessment Report for Iron Sulphate (September 2008) as irritating to eyes - Category 2 according to CLP.

Respiratory irritation

• Animal data

Only one data source is available on this endpoint. The American Conference of Governmental Industrial Hygienists stated in one of their reports from 1980 (ACGIH 1980) that the inhalation of ferric salts as dusts and mists is irritating to the respiratory tract. Quantitative data that would allow a solid decision on the need for classification is not available. The reliability of this information is low.

• Human data:

In a pre-GLP study (Kleinman 1981) the respiratory effects of inhalation exposure to ferric sulphate aerosol were studied in humans (reliability score 2) volunteers (38) were exposed to 75 µg/m³ aerosol of ferric sulphate (= 20 µg/m³ Fe) for 2 hours on each of 5 days. The investigation was conducted as a double blind study. Subjects served as their own control by blind exposure to sham aerosol. The group average results were contrasted between day (sham or exposure) and conditions (pre- and post-exposure) using a two-way analysis of variance with repeated measure. Only five of the 38 volunteers tested showed patterns indicative of a decrement in pulmonary function. Nine of the subjects exhibited significant improvement in function. None of the subjects reported more than slight changes in symptoms during exposure. The authors considered that the tendency for a subject to improve after the ferric sulphate exposure could be related to allergy status, smoking history, or a combination of these factors. Furthermore, they recorded that “it is encouraging to note that at the relatively high aerosol concentration used in this study, there were no significant detrimental changes in the (study) population as a whole.” The study appears to have been well conducted and the conclusions are consistent with the data presented. It may be assumed that ferric sulphate aerosols do not produce any clear pulmonary effects in either normal persons or asthmatics.

• Summary:

The available data are not sufficient to allow decision on classification for the endpoint respiratory irritation.

Justification for selection of skin irritation / corrosion endpoint:
Reliable study conducted according to OECD TG 439 and GLP using the EPISKIN™ Reconstructed Human Epidermis (RHE) model, done with the submission item.

Justification for selection of eye irritation endpoint:
Reliable study conducted according to OECD TG 437 and GLP using the BCOP method, done with the submission item.

Effects on eye irritation: slightly irritating

Justification for classification or non-classification

Skin irritation / corrosion:

Based on the above stated assessment of the skin irritation / corrosion tests of the iron salts, the results from reliable studies or from read across show that the iron salts of this category are skin irritants, except for FeCl2 (Park & Seong 2004). Accordingly the salts belonging to the iron salt category, except FeCl2, need to be classified as “R38 Irritating to skin” according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and as Category 2, “Warning - H315: Causes skin irritation” according to CLP (5th ATP of Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.

Category members except Ferrous chloride and the submission item

Table: Skin corrosion/irritation label elements for category 2 (CLP, 5th ATP, Annex I, 3.2.4.1, Table 3.2.5)

Element		Type
GHS Pictogram		GHS07 exclamation mark
Signal Word		Warning
Hazard Statement		H315: Causes skin irritation
Precautionary Statements	Prevention	P264, P280
	Response	P302 + P352, P321, P332 + P313, P362 + P364 (4th ATP change)
	Storage	none
	Disposal	none

Ferrous chloride and the submission item

No classification for skin irritation/corrosion is required.

Eye irritation / corrosion:

Category member Ferrous sulphate and the submission item

FeSO4 is already classified as irritating to eyes (R36) in the Draft Assessment Report for Iron Sulphate (September 2008) and it is proposed to keep this classification for FeSO4. According to CLP, FeSO4 should be classified into Category 2. The submission item showed experimentally absence of corrosivity, but abverse effects and should thus be classified accordingly

Table: Serious eye damage/eye irritation label elements for category 2 (CLP, 5th ATP, Annex I, 3.3.4.1, Table 3.3.5)

Element		Type
GHS Pictogram		GHS07 exclamation mark
Signal Word		Warning
Hazard Statement		H319: Causes serious eye irritation
Precautionary Statements	Prevention	P264, P280
	Response	P305 + P351 + P338, P337 + P313
	Storage	none
	Disposal	none

Respiratory irritation:

As no reliable data on respiratory irritation is available for iron salts a classification is not possible according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and according to CLP (5th ATP of Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.