Sodium [N-[2-[bis(carboxymethyl)amino]ethyl]-N-(2-hydroxyethyl)glycinato(4-)]ferrate(1-)

Endpoint summary

Description of key information

An in vitro skin (OECD 439) and an in vitro eye (OECD 437) irritation test were available.  

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:

August-September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed and reported GLP study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: in vitro

Details on test animals or test system and environmental conditions:

Test system: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 12-EKIN-031).
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Source: SkinEthic Laboratories, Lyon, France.

Type of coverage:

other: not applicable

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

The solid test substance (10.8 to 14.7 mg) was applied directly on top of the skin tissue. HEDTA-Fe(III)Na was spread to match the size of the tissue.

Duration of treatment / exposure:

See below

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

See below

Irritant / corrosive response data:

HEDTA-Fe(III)Na was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that HEDTA-Fe(III)Na did not interact with MTT.
The mean absorption at 570 nm measured after treatment with HEDTA-Fe(III)Na and controls are presented in Table 1.
Table 2 shows the mean tissue viability obtained after 15 minutes treatment with HEDTA-Fe(III)Na compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with HEDTA-Fe(III)Na compared to the negative control tissues was 107%. Since the mean relative tissue viability for HEDTA-Fe(III)Na was above 50% HEDTA-Fe(III)Na is considered to be non-irritant.

The positive control had a mean cell viability after 15 minutes exposure of 4%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range (See APPENDIX 3). The standard deviation value of the percentage viability of three tissues treated identically was less than 17%, indicating that the test system functioned properly.

Other effects:

No

Table1            Mean absorption in the in vitro skin irritation test with HEDTA-Fe(III)Na

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	0.883	1.018	0.969	0.957	±	0.069
HEDTA-Fe(III)Na	1.177	1.033	0.868	1.026	±	0.154
Positive control	0.029	0.035	0.044	0.036	±	0.007

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

 

In this table the values are corrected for background absorption (0.042). Isopropanol was used to measure the background absorption.

 

Table2            Mean tissue viability in thein vitroskin irritation test with HEDTA-Fe(III)Na

	Mean tissue viability (percentage of control)
Negative control	100
HEDTA-Fe(III)Na	107
Positive control	4

 

 

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substance is not irritating in an in vitro test (OECD 439).

Executive summary:

This report describes the ability of HEDTA-Fe(III)Na to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SM^TM)). The possible skin irritation potential of HEDTA-Fe(III)Na was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch CFC-10340 (308H0351) of HEDTA-Fe(III)Na was a red brown powder with a purity of ~81%. Skin tissue was moistened with 5 µl of Milli-Q water and 10.8 to 14.7 mg of HEDTA-Fe(III)Na was applied directly on top of the skin tissue for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with HEDTA-Fe(III)Na compared to the negative control tissues was 107%. Since the mean relative tissue viability for HEDTA-Fe(III)Na was above 50% after 15 minutes treatment HEDTA-Fe(III)Na is considered to be non-irritant.

The positive control had a mean cell viability of 4% after 15 minutes exposure. The absolute mean OD₅₇₀(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 17%, indicating that the test system functioned properly.

Finally, it is concluded that this test is valid and that HEDTA-Fe(III)Na is non-irritant in thein vitroskin irritation test under the experimental conditions described in this report.

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:

August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed and reported GLP study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: in vitro

Vehicle:

physiological saline

Controls:

no

Amount / concentration applied:

A 20% (w/w) solution of HEDTA-Fe(III)Na was prepared in physiological saline (Merck, Darmstadt, Germany).

Duration of treatment / exposure:

See below

Observation period (in vivo):

See below

Number of animals or in vitro replicates:

Not applicable

Details on study design:

Negative control: a negative control, physiological saline (Merck, Darmstadt, Germany) was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.
Positive control: 20% (w/v) Imidazole (Merck Schuchardt DHG, Germany) [CAS Number 288-32-4] solution prepared in physiological saline.

Test System: bovine eyes were used as soon as possible after slaughter on the same day.
Rationale: in the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.
Source: bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
Transport: eyes were collected and transported in physiological saline in a suitable container.

All eyes were carefully examined for defects by holding the eyes submersed in physiological saline. Those exhibiting unacceptable defects, such as opacity, scratches, pigmentation and neovascularization were discarded.

The isolated corneas were stored at 32 +/- 1 degrees C in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont, France) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1 degrees C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1 degrees C.

After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (OP-KIT, MC2, Clermont, France). The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

The medium from the anterior compartment was removed and 750 uL of the negative control, 20% (w/v) Imidazole solution (positive control) or 20% (w/w) test substance solution were introduced onto the epithelium of the cornea. The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 +/- 10 minutes at 32 +/- 1 degrees C. After the incubation the solutions were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation). Possible pH effects of the test substance on the corneas were recorded. The anterior and the posterior compartment were refilled with fresh cMEM and an opacity determination was performed without any further incubation. After the completion of the incubation period each cornea were inspected visually for dissimilar opacity patterns and the opacity determination was performed.

The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each positive control or test substance treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test substance treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck) was evaluated.

The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 5 mg Na-fluorescein/ml cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 +/- 5 minutes at 32 +/- 1 degrees C.

After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 uL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.

The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.

Irritant / corrosive response data:

HEDTA-Fe(III)Na was tested in a 20% (w/w) solution. Table 1 summarizes the opacity, permeability and in vitro irritancy scores of HEDTA-Fe(III)Na and the controls. The individual in vitro irritancy scores for the negative controls ranged from -2.0 to 2.0. The individual positive control in vitro irritancy scores ranged from 124 to 134. The corneas treated with the positive control were turbid after the 240 minutes of treatment.

The corneas treated with HEDTA-Fe(III)Na showed opacity values ranging from 1 to 3 and permeability values ranging from -0.007 to 0.028. The corneas were clear after the 240 minutes of treatment with HEDTA-Fe(III)Na, only a little light spot was observed in the middle of the corneas. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 1.4 to 2.9 after 240 minutes of treatment with HEDTA-Fe(III)Na.

Table1            Summary of opacity, permeability andin vitroscores

Treatment	Mean Opacity^1	Mean Permeability^1	Mean In vitro Irritation Score^1, 2
Negative control	0	0.000	0.0
Positive control	81	3.308	131
HEDTA-Fe(III)Na	2	0.009	2.1

 

1     Calculated using the negative control mean opacity and mean permeability values.

2     In vitroirritancy score (IVIS) = mean opacity value + (15 x mean OD₄₉₀value).

Table 2            Historical control data for the BCOP studies

 

	Negative control			Positive control
	Opacity	Permeability	In vitro Irritancy Score	In vitro Irritancy Score
Range	-5 – 1	-0.03 – 0.059	-4.7 – 1.1	70 – 180
Mean	-0.1	0	0	111
SD	0.73	0.01	0.71	20
n	90	90	90	90

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of January 2009 to January 2012.

 

 

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substances was not irritating in OECD test 437.

Executive summary:

This report describes the ocular irritation properties of HEDTA-Fe(III)Na on an isolated bovine cornea. The possible ocular irritancy of HEDTA-Fe(III)Na was tested through topical application for approximately 240 minutes.  

The study procedures described in this report were based on the most recent OECD and EC guideline.

Batch CFC-10340 (308H0351) of HEDTA-Fe(III)Na was a red brown powderwith a purity of ~81%. The test substance was applied as a 20% (w/w) solution (750 µl) directly on top of the corneas.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas,except the response of one cornea. However since the opacity was just above the upper limit of the range and all other values were less than the upper limits of the laboratory historical range,this deviation in the opacity score had no effect on the results of the study.

The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 131 and within the historical positive controldata range.It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

HEDTA-Fe(III)Na did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 2.1 after 240 minutes of treatment.

Finally, it is concluded that this test is valid and that HEDTA-Fe(III)Na is not severe irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Test results showed that HEDTA-FeNa is not irritating to skin and eyes. Based on read across with comparable metal-chelates like EDTA-FeNa and DTPA-FeHNa no further testing was performed (see also section 13 for read across).

Justification for selection of skin irritation / corrosion endpoint:
Well performed and reported GLP study

Justification for selection of eye irritation endpoint:
Well performed and reported GLP study

Justification for classification or non-classification

Based on the test results, no classification for skin and eye irritation is needed.