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Description of key information

In vitro skin and eye irritation studies did not show irritating properties of EDTA-MnNa2. In addition, in vivo studies with EDTA-FeNa, a structurally realted compound, did not show irritating properties (see also the read across document in section 13).

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:
June 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to GLP
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: Draft OECD guideline - in vitro skin irritation: Reconstructed human Epidermis (RhE) test method
Qualifier:
according to
Guideline:
other: Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.46 “In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test "
GLP compliance:
yes (incl. certificate)
Species:
other: reconstructed human skin
Details on test animals and environmental conditions:
No test animals used.
Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: negative and positive control
Amount / concentration applied:
Skin tissue was moistened with 5 µl of Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact to the tissue and at least 10 mg of the solid test substance was applied directly on top of the skin tissue. EDTA-MnNa2 was spread to match the size of the tissue
Duration of treatment / exposure:
15 min
Observation period:
42 h
Number of animals:
3 samples each for negative and positive control and for test sample

Negative control: Phosphate buffered saline (PBS, Invitrogen Corporation, Breda, The Netherlands).
Positive control: 5% (aq) Sodium dodecyl sulphate (SDS, Sigma Aldrich, Zwijndrecht, The Netherlands) [CAS Number 151-21-3].
Details on study design:
Tissues: On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 24 hours at 37°C. The level of Maintenance Medium was just beneath the tissue (Figure 1). Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Nice, France.

MTT medium: MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration
0.3 mg/ml).

Environmental conditions: All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 63 - 86%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 37.4 - 37.8°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

Test for reduction of MTT by the test substance: EDTA-MnNa2 was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, approximately 10 mg of test substance was added to a 12 well plate filled with 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for approximately 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently.

Application/Treatment of the test substance: The test was performed on a total of 3 tissues per test substance together with negative and positive controls. Ten µl of the undiluted test substance was added into 12-well plates on top of the skin tissues. Three tissues were treated with 10 µl PBS (negative control) and 3 tissues with 10 µl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully. The skin tissues were kept in new 12-well plates on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

Cell viability measurement: After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-medium (0.3 mg/ml). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 µl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the Multiskan Spectrum (Thermo Labsystems). Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.
Irritation / corrosion parameter:
other: overall irritation score
Value:
72
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 42 h. Max. score: 100.0. Remarks: negative control was set at 100%; test sample 72%, positive control 9%. (migrated information)
Irritant / corrosive response data:
The relative mean tissue viability obtained after 15 minutes treatment with EDTA-MnNa2 compared to the negative control tissues was 72%. Since the mean relative tissue viability for EDTA-MnNa2 was above 50% EDTA-MnNa2 is considered to be non-irritant. The positive control had a mean cell viability after 15 minutes exposure of 9%.
Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
The relative mean tissue viability obtained after 15 minutes treatment with EDTA-MnNa2 compared to the negative control tissues was 72%. Therefore, EDTA-MnNa2 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report

Executive summary:

This report describes the ability of EDTA-MnNa2 to induce skin irritation on a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-SMTM)). The possible skin irritation potential of EDTA-MnNa2 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 9380 of EDTA-MnNa2 is an off-white powder. Skin tissue was moistened with 5 µl of Milli-Q water and at least 10 mg of EDTA-MnNa2 was applied directly on top of the skin tissue for 15 minutes. After a 42 hour 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 EDTA-MnNa2 compared to the negative control tissues was 72%. Since the mean relative tissue viability for EDTA-MnNa2 was above 50% after 15 minutes treatment EDTA-MnNa2 is considered to be non-irritant. The positive control had a mean cell viability after 15 minutes exposure of 9%. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was according to the acceptability criteria of the assay. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly. It was concluded that this test is valid and that EDTA-MnNa2 is non-irritant in the in vitro skin 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:
June 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to GLP
Qualifier:
according to
Guideline:
other: OECD 437
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: bovine eyes from a slaughterhouse
Details on test animals or tissues and environmental conditions:
No animals used
Vehicle:
physiological saline
Controls:
other: negative and positive controls were used
Amount / concentration applied:
A 20% (w/w) solution of EDTA-MnNa2 was prepared in physiological saline (Merck, Darmstadt, Germany).

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.
Duration of treatment / exposure:
240 min
Observation period (in vivo):
no observation period
Number of animals or in vitro replicates:
3 eyes each were used for the negative and positive control and for the test sample
Details on study design:
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 3 were not used. Three corneas were selected at random for each treatment group.

The medium from the anterior compartment was removed and 750 µl 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). 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 was 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 dgerees 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 µl 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 (Multiskan spectrum, Thermo labsystems, Breda, The Netherlands). 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.
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: 240 min
Score:
2.4
Max. score:
146
Reversibility:
other: not tested
Remarks on result:
other: max score of 146 based on historical control data
Irritant / corrosive response data:
The negative control responses of the opacity and permeability values were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 119 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. EDTA-MnNa2 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 2.4 after 240 minutes of treatment.

Table            Summary of opacity, permeability and in vitro scores

 

Treatment

Mean

Opacity

Mean

Permeability

Mean In vitro Irritation Score1, 2

Negative control

0

0.000

0.0

Positive control

71

3.186

119

EDTA-MnNa2

2

0.025

2.4

 

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

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

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: other:
Conclusions:
Finally, it is concluded that this test is valid and that EDTA-MnNa2 is not a severe irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.
Executive summary:

This report describes the ocular irritation properties of EDTA-MnNa2 on an isolated bovine cornea. The possible ocular irritancy of EDTA-MnNa2 was tested through topical application for 240 ± 10 minutes. The study procedures described in this report were based on the most recent OECD guideline.

Batch CFC 9380 of EDTA-MnNa2 was an off-white powder.The test substance was applied as a 20% (w/w) solution (750 µl) directly on top of the corneas. The negative control responses of the opacity and permeability values were less than the upper limits of the laboratory historical rangeindicating that the negative control did not induce irritancy on the corneas.The mean in vitro irritancy score of the positive control (20% w/v Imidazole) was 119 and within the historical positive controldata range.It was therefore concluded that the test conditions were adequate and that the test system functioned properly. EDTA-MnNa2 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 2.4 after 240 minutes of treatment. It was concluded that this test is valid and that EDTA-MnNa2 is not a 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 adverse effect observed (not irritating)

Additional information

In vitro skin and eye irritation studies did not show irritating properties of EDTA-MnNa2; in vitro studies were carried to to confirm the absence of skin and eye irritation as already had been found with a structurally related analag EDTA-FeNa and several other chelates (see read across document in section 13). In addition, it is not expected that EDTA-MnNa2 will irritate the respiratory tract except for general dust irritation when exposed to very high concentrations.

Justification for selection of skin irritation / corrosion endpoint:

The results of this in vitro study were confirmed by in vivo skin irritation studies with other chelating agents (see also read across docuemnt in section 13)

Justification for selection of eye irritation endpoint:

The results of this in vitro study were confirmed by in vivo skin irritation studies with other chelating agents (see also read across docuemnt in section 13)

Justification for classification or non-classification

Because EDTA-MnNa2 did not show skin or eye irritation, no classification is needed.