hydronium;iron(3+);hexahydroxide;disulfate

Eye irritation

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

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

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

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 / tissue source

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

Test system

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.

Results and discussion

In vivo

Results

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.