Magnesium, EDTA cobalt copper iron manganese zinc complexes

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study; evaluation of the in vitro protocol (EpiOcular) is in progress

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Principles of method if other than guideline:

There are no official national or international guidelines for the EpiOcularTM test yet; however,
the study was performed according to the methods described in the following publications:
- MatTek Corporation, Ashland, MA 01721, USA: EpiOcularTM human cell construct:
Procedure details, Version 3.1a of February 10, 2010.
- Harbell J.W. et al. (2009): COLIPA Program on Optimization of Existing In Vitro Eye
Irritation Assays for Entry into Formal Validation: Technology Transfer and Intra/Inter
Laboratory Evaluation of EpiOcular Assay for Chemicals. Poster # 378, Society of
Toxicology, March 2009.

In addition the study follows the testing strategy for determination of eye irritation/corrosion
as given in the following OECD guideline:
- OECD Guideline for Testing of Chemicals No. 405, October 2, 2012 (“Acute Eye
Irritation/Corrosion”)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

13/0195-1

IUPAC Name:

13/0195-1

Constituent 2

Reference substance name:

Disodium [[N,N'-ethylenebis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON,ON']cuprate(2-)

EC Number:

237-864-5

EC Name:

Disodium [[N,N'-ethylenebis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON,ON']cuprate(2-)

Cas Number:

14025-15-1

IUPAC Name:

14025-15-1

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): Test-substance No.: 13/0195-1
- Physical state: solid
- Analytical purity: The test substance is not fully characterized analytically.
- Lot/batch No.: 0008609840

Test animals / tissue source

Species:

other: in vitro test system (EpiOcularTM): reconstructed three dimensional human cornea model.

Details on test animals or tissues and environmental conditions:

CO2 incubator: Heraeus BBD 6220, Standard incubation conditions: about 37 °C, 5% CO2, 90-95 % humidity

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

50 µL bulk volume (about 35 mg)

Duration of treatment / exposure:

90 Minutes followed by a 18-hours post-incubation period

Observation period (in vivo):

not applicable (in vitro test)

Number of animals or in vitro replicates:

not applicable (in vitro test)

Details on study design:

The EpiOcularTM model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinozytes used to model the human corneal epithelium.
Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colourimetric test.

Method:

1. Direct MTT reduction
To assess the ability of the test material to directly reduce MTT a pretest was performed. Thereby, the test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 55 to 65 minutes. A negative control (de-ionized water) was tested concurrently.
If the MTT solution color or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT.

2. Basic procedure:
Two EpiOcularTM tissues were incubated with the test substance, the PC or NC, respectively for 90 minutes.

CONTROLS
- Negative control (NC): 50 µL De-ionized water, sterile
- Positive control (PC): 50 µL methyl acetate

• Tissues were transferred to sterile plates with 1 mL assay medium and preconditioned at standard culture conditions for 16 – 24 hours (pre-incubation).
• Subsequently, the tissues were pre-treated with 20 μL of PBS and incubated at standard culture conditions for 30 minutes.
• Afterwards, the whole tissue surface was covered with a bulk volume of 50 µL of the undiluted test substance. After application, the tissues were incubated until the total exposure time of 90 minutes was completed.
• Tissues were washed with sterile PBS. After 12 minutes of post-soak immersion with medium, each tissue was dried on absorbent paper and transferred to fresh plates with pre-warmed medium. Subsequently, the tissues were incubated at standard culture conditions for 2 hours (post-incubation period).
• After the post-incubation period, the assay medium was replaced by MTT solution and the tissues were incubated for 3 hours. The formazan that was metabolically produced by the tissues was extracted by isopropanol and the optical density was determined spectrophotometrically at a wavelength of 570 nm (OD570) of the extracts.

Tissue viability: The quantification of tissue viability is presented as the quotient of the mean OD570 divided by the respective OD570 NC value in percent

ACCEPTANCE CRITERIA
In case one of the below given acceptance criteria is not covered, repetition of the test was considered.
- Assay acceptance criteria for the NC: Tissue viability is acceptable if the mean OD570 of the NC is ≥ 1.0<2.5.
- Acceptance criteria for the PC: A viability of < 50% is acceptable.
- Assay acceptance criteria for tissue variability: Two tissues were treated under the same conditions. Variability between the tissues is considered to be acceptable if the difference of the viability is ≤ 20%.

EVALUATION OF RESULTS
The irritation potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile water. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%.
At present no prediction is performed if the mean relative tissue viability with a test material is > 50 ≤ 60% as the cut off value is currently being evaluated to lie in this range.

Results and discussion

In vivo

Irritant / corrosive response data:

The mean viability of the test-substance treated tissues was 89 %.

Other effects:

The test substance is able to reduce MTT directly. However, this ability of direct MTT reduction did not impair the study result as demonstrated by the concurrently performed exposure of control tissues inactivated by freezing.

Any other information on results incl. tables

EpiOcular Test (Summary of results)

Test substance		Tissue 1	Tissue 2	Mean	Inter-tissue variablilty (%)
NC	Mean OD570	1.836	1.700	1.768
	Viability (% of NC)	103.8	96.2	100	7.7
Test substance	Mean OD570	1.593	1.539	1.566
	Viability (% of NC)	90.1	87.1	89	3.0
PC	Mean OD570	0.347	0.239	0.306
	Viability (% of NC)	21.1	13.5	17	7.6

 

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information

Conclusions:

Based on the observed mean viability of 99% and applying the evaluation criteria, it was concluded that the test substance does not show an eye irritation potential in the EpiOcularTMeye irritation test under the test conditions chosen.

Based on this result classification for eye irritation is not warranted.

Executive summary:

The potential of Librel RMX 8 to cause ocular irritation was assessed by a single topical application of 50 μL bulk volume (about 35 mg) of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcular™).

Two EpiOcular™ tissue samples were incubated with the test substance for 90 minutes followed by an 18-hours post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a

tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The EpiOcular™ eye irritation test showed the following results:

The test substance is able to reduce MTT directly. However, this ability of direct MTT reduction did not impair the study result as demonstrated by the concurrently performed exposure of control tissues inactivated by freezing.

The mean viability of the test-substance treated tissues was 89 %.

Based on the observed results and applying the evaluation criteria it was concluded, that the test substance does not show an eye irritation potential in the EpiOcular™ eye irritation test under the test conditions chosen. (BASF 2013)