Zinc EDDHA

Endpoint summary

Description of key information

Skin irritation

RhE, OECD 439, GLP, positive

Corrositex, OECD 435, negative

Eye irritation

RhCE, OECD 492, GLP, positive

mixture rule calculation, 7.5 % zinc (Eye damage Cat 1) in registered substance, C&L as Eye damage Cat 1

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Feb 2021 to 02 June 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 26. Jun. 2020

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

adopted 06. Jul. 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt Rheinland-Pfalz 15 May 2018

Test system:

human skin model

Remarks:

commercially available EpiDermTM-Kit, procured by MatTek

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis.

Details on animal used as source of test system:

not applicable

Justification for test system used:

This in vitro study was performed in order to evaluate the potential of "zinc-EDDHA sodium salts" to evoke skin irritation in a Reconstructed human Epidermis (RhE) test method.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RhE) TISSUE
- Model used:
EpiDermTM-Kit, procured by MatTek
- Designation of the kit:
EPI-200-SIT
- Tissue batch number(s):
34134
- Delivery date:
2021 March 16
- Date of initiation of testing:
2021 March 15 (pre-tests)
- Date of experimantal completion
2021 March 19

TEMPERATURE USED FOR TEST SYSTEM
Tissues were dosed in 1-minute-intervals. After dosing the last tissue, all plates were transferred into the incubator for 35 minutes at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% rel-ative humidity.

REMOVAL OF TEST MATERIAL AND CONTROLS
1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals.
After rinsing thoroughly with DPBS, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL). The surface of the inserts was then carefully dried with a sterile cotton tipped swab.
Then, the tissues were set in the incubator for 24 hours at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity.
After post-incubation, the tissues were removed from the incubator and shaken for 5 minutes (120 rpm). 0.9 mL assay medium were filled in the lower row of the 6-well-plate. Then the inserts were transferred into the lower row of the 6-well-plate and set into the incubator for 18 hours and 30 minutes for post-incubation at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:
1 mg/mL
- Incubation time:
3 h
- Spectrophotometer:
Microtiter plate photometer Anthos Reader 2010 Flexi (Anthos Microsysteme GmbH)
- Wavelength:
570 nm

NUMBER OF REPLICATE TISSUES:
3

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: MTT QC assay, 4 hours, n=3 -> OD=1.57 ± 0.047 (range 1 - 3 -> passed)
- Barrier function: ET50 assay (1 % Triton) -> ET50=6.28 h (range 4.77 - 8.72 h -> passed)
- Contamination: no contamination

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if the viability after 60 minutes exposure is less than or equal to 50%
- The test substance is considered to be non-irritating to skin if the viability after 60 minutes exposure is greater than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
25.9; 26.4; 26.6 mg

VEHICLE
no vehicle used

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):
30 µL [“Dulbecco’s Phosphate Buffered Saline” (DPBS buffer without CaCl2 and without MgCl2.]
- Concentration (if solution):
100 %

POSITIVE CONTROL
- Amount(s) applied (volume or weight):
30 µL [SDS-solution]]
- Concentration (if solution): 5 %

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42.5 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

37.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

30.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

30.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

32.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
All validity criteria were met.
The values for negative control and for positive control were within the range of historical data of the test facility

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The test item Zinc-EDDHA sodium salts is considered as at least irritant to skin. After the treatment, the mean value of relative tissue viability was reduced to 32.6. This value is below the threshold for skin irritation (50%). The optical density of the negative control was well within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8. The positive control has met the acceptance criterion too, for thus ensuring the validity of the test system. The variation within replicates was within the accepted range for negative control, positive control and test item (required: ≤ 18%). For these reasons, the result of the test is considered valid.

Executive summary:

One valid experiment was performed according to OECD Guideline 439 following GLP with three tissues and two independent MTT measurements.Three tissues of the human skin model EpiDerm^TM were treated with the test item for 60 minutes. The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm²; as indicated by the supplier). DPBS-buffer was used as negative control and 5% SDS solution was used as positive control. After treatment with the negative control, the mean absorbance value was within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.653. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 2.8% (required: ≤ 20%). The variation within the tissue replicates of negative control, positive control and test item was acceptable (required: ≤ 18%). After the treatment with the test item, the mean value of relative tissue viability was reduced to 32.6%. This value is below the threshold for skin irritation potential (50%). Test items that induce values below the threshold of 50% are considered at least irritant to skin. The OECD guideline 439 addresses the human health endpoint skin irritation. Therefore, further testing on skin corrosion potential with another suitable in vitro study (e.g. OECD 431) may be required. Therefore, the test item Zinc-EDDHA sodium salts is considered at least irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 AUG 2021 - 10 NOV 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)

Version / remarks:

adopted 28. Jul. 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

artificial membrane barrier model

Cell source:

other: synthetic biobarrier (Corrositex®) produced by InVitro International, USA, 17751 Sky Park East, Suite G, Irvine, CA 92614 and procured by Romer Labs Deutschland GmbH.

Details on animal used as source of test system:

Commercially available Corrositex®-Kit was used.
The test system consists of two components: a proteinaceous macromolecular gel (=synthetic biobarrier) on a permeable membrane and a fluid, the Chemical Detection System (CDS).

Justification for test system used:

This test system was chosen as a suitable follow up test of the available RhE Test to clarify the C&L of the substance.

Vehicle:

unchanged (no vehicle)

Details on test system:

SOURCE AND COMPOSITION OF MEMBRANE BARRIER USED
- Was the Corrositex® test kit used: yes
- Components: a proteinaceous macromolecular gel (=synthetic biobarrier) on a permeable membrane and a fluid, the Chemical Detection System (CDS).
- Apparatus and preparation procedures: The test system consists of two components: a proteinaceous macromolecular gel (=synthetic biobarrier) on a permeable membrane and a fluid, the Chemical Detection System (CDS). The biobarrier was produced one day prior to the assay was performed. The membrane discs with the biobarrier were stored in the fridge (2-8 °C) overnight before use.

Corrositex®-Kit was produced by InVitro International, USA, 17751 Sky Park East, Suite G, Irvine, CA 92614 and procured by Romer Labs Deutschland GmbH.
Day of delivery: 15. Jul. 2021
Batch Corrositex®-Kit: CT051021

WAS THE TIMESCALE CATEGORY TEST PERFORMED: yes. The categorisation screen was used to choose the appropriate scoring scale. As the test item has a strong colour itself, the test item colour covered the colour change in the test tubes. Therefore, a dilution of 10% of the test item in demin. water was prepared and the pH value was determined. A pH value of 8 was determined.
Based on the pH value, 150 µL of the 10% test item solution were added in test tube B and the content was mixed. Then the pH value was measured again; the value was pH 6.
Because this value was ≤ 9, the test item was classified as category 2. Since the test item was classified as a category 2 substance, the vials had to be observed for 60 minutes (test item of category 2).

METHOD OF DETECTION
The evaluation was performed visually: break-through causes a change in the colour of the solution; the stop watch was operated accordingly.

METHOD OF APPLICATION: pipetting

NUMBER OF REPLICATES: 4

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if ≤ 60 min elapsed between application of the test substance to the membrane barrier and barrier penetration.
- The test substance is considered to be non-corrosive to skin if > 60 min elapsed between application of the test substance to the membrane barrier and barrier penetration.
- Justification for the selection of the cut-off point(s): prediction model for Category 2 test chemicals (test chemicals with high acid/alkaline reserve) according to OECD TG 435

Control samples:

yes, concurrent negative control

yes, concurrent no treatment

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
Vial 1: 497.8 mg
Vial 2: 490.2 mg
Vial 3: 493.7 mg
Vial 4: 494.4 mg
(used as solid)

NEGATIVE CONTROL
500 µL 10% citric acid solution

POSITIVE CONTROL
111.9 mg of sodium hydroxide (used as solid)

Duration of treatment / exposure:

Blank and negative control test item : 61 minutes
Positive control : 12 minutes

Number of replicates:

4

Irritation / corrosion parameter:

penetration time (in minutes)

Run / experiment:

Vial 1-4

Value:

> 60

Vehicle controls validity:

valid

Remarks:

Blank for CDS

Negative controls validity:

valid

Remarks:

10% citric acid solution

Positive controls validity:

valid

Remarks:

sodium hydroxide (used as solid)

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No break-through

DEMONSTRATION OF TECHNICAL PROFICIENCY: The demonstration of proficiency was performed using proficiency chemicals under non-GLP conditions, but within the GLP-environment at LAUS GmbH. All of the listed proficiency chemicals except one met the right classification according to OECD 435. Selenic acid gave discrepant classification compared to the OECD guideline 435. This result can be seen as outlier and therefore as uncritical.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, negative control showed a clear negative result; no break-through of the negative control within 60 minutes
- Acceptance criteria met for positive control: the break-through time of the positive control falls within ± 2 standard deviations of the current historical mean value
- Acceptance criteria met for variability between replicate measurements: No break-through of the test item was observed in any replicate.

Interpretation of results:

other: Category 2 (irritant) based on EU GHS criteria

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item possesses no skin corrosion potential and is considered as a non-corrosive substance. Therefore, Zinc-EDDHA sodium salts is considered as non-corrosive to skin in the Corrositex® - Test. The test item is therefore not to be classified for corrosivity to skin according to GHS. The negative control showed no break-through and therefore no corrosive effects. The measured time of the positive control was 12 minutes and thus ensuring the validity of the test system. For these reasons, the result of the test is considered valid.

Executive summary:

The study was performed according to OECD Guideline 435 under GLP compliance using Corrositex® kit. A blank and 10% citric acid solution served as negative control and sodium hydroxide was the positive control. The test item was applied as a solid (fine powder). As the test item was classified as a category 2 substance, the test item vials were observed for 60 minutes. No break-through of the test item was observed within 61 minutes. Thus, it can be stated that in this study and under the experimental conditions reported, the test item possesses no skin corrosion potential and is considered as a non-corrosive substance. The negative control showed no break-through and therefore no corrosive effects. The measured time of the positive control was 12 minutes and thus ensuring the validity of the test system. For these reasons, the result of the test is considered valid.

Therefore, Zinc-EDDHA sodium salts is considered as non-corrosive to skin in the Corrositex® - Test. The test item is therefore not classified for corrosivity to skin according to GHS.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation, other

Remarks:

classification based on mixture rules calculation

Type of information:

mixture rules calculation

Adequacy of study:

supporting study

In the ECHA disseminated dossiers the two zinc salts ZnSO₄ and ZnCl₂, which serve also as source substances for read-across are classified as Eye damage Cat 1 (H318 and H314). Based on the rules on classification for mixtures as specified in Regulation (EC) No 1272/2008 (Table 3.3.3), the threshold for classification of a mixture as Eye damage Cat 1 is a concentration of ≥ 3 % of a constituent classified as Eye damage Cat 1. The registered substance contains zinc at a concentration of 7.5 %. Thus, it is classified as Eye damage Cat 1 (H318).

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The registered substance contains zinc at a concentration of 7.5 %. Thus, it is classified as Eye damage Cat 1 (H318) according to Regulation (EC) No 1272/2008.

Executive summary:

In the ECHA disseminated dossiers the two zinc salts ZnSO₄and ZnCl₂, which serve also as source substances for read-across are classified as Eye damage Cat 1 (H318 and H314). Based on the rules on classification for mixtures as specified in Regulation (EC) No 1272/2008 (Table 3.3.3), the threshold for classification of a mixture as Eye damage Cat 1 is a concentration of ≥ 3 % of a constituent classified as Eye damage Cat 1. The registered substance contains zinc at a concentration of 7.5 %. Thus, it is classified as Eye damage Cat 1 (H318).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 FEB 2021 - 2 JUN 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 18. Jun. 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt Rheinland-Pfalz 15 May 2018

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method (e.g. ICE, EIT, RhCE) and considerations regarding applicability: This study was performed in order to evaluate the eye hazard potential of ManganeseEDDHA sodium salts in a Reconstructed human Cornea-like Epithelium (RhCE) model in an in vitro study (e.g. EpiOcularTM Eye Irritation Test). The EpiOcular™ Eye Irritation Test (EIT) predicts the acute eye hazard potential of chemicals by measurement of tissue damage caused by cytotoxic effects in the reconstructed
human cornea-like tissue model. Within a testing strategy, the EpiOcular™ EIT can be used as a replacement of the in vivo Draize Eye Irritation Test. It is utilized for the classification and labelling of chemicals concerning their eye hazard potential. The EpiOcular™ EIT can be used to identify chemicals that do not require classification for eye irritation or serious eye damage according to the UN GHS classification system. A limitation of this guideline is that it neither allows discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1), nor between eye irritants (optional Category 2A) and mild eye irritants (optional Category 2B). For these purposes, further testing with other suitable test methods is required The solid test item was applied topically to a three-dimensional RhCE tissue construct in duplicate for an exposure time of 6 hours. Eye hazard materials are identified by their ability to produce a decrease in cell viability as determined. The cell viability is measured by dehydrogenase conversion of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide), present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percentage reduction of cell viability in comparison of untreated negative controls is used to predict the eye irritation potential. The test substance falls within the applicaility domain of this model.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: No bacteria, yeast and other fungi were detected, Tissue viability and Barrier function were within the acceptance criteria
- Cell line used, its source, passage number and confluence of cells used for testing: Keratinocyte strain 4F1188
- RhCE tissue or hCE cell construct used, including batch number: EpiOcular™ Tissue (Lot No. 34901)

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50.5mg

Duration of treatment / exposure:

6 h

Duration of post- treatment incubation (in vitro):

18 h

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used :
The solid test item was applied topically to a three-dimensional RhCE tissue construct in duplicate for an exposure time of 6 hours. Eye hazard materials are identified by their ability to produce a decrease in cell viability as determined. The cell viability is measured by dehydrogenase conversion of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide), present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percentage reduction of cell viability in comparison of untreated negative controls is used to predict the eye irritation potential
- Doses of test chemical and control substances used :
test item: 50 mg, positive and negative control: 50 µL
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable) : 6 h exposure 37 ± 1 °C, 18 h post-exposure incubation at 37 ± 1 °C
- Number of tissue replicates used per test chemical and controls (positive control, negative control) : 2
- For hCE cells: number of runs and of hCE models used within each run : 1 run, 2 tissues per test item/controls
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer) : 570 nm
- Description of the method used to quantify MTT formazan, if applicable : The inserts were removed from the 6-well plate and discarded. 1 mL isopropanol was added and the content of each well was thoroughly mixed in order to achieve homogenisation.
From each well, two replicates with 200 µL solution (each) were pipetted into a 96-wellplate. The plate was read in a plate spectrophotometer at 570 nm. In addition, eight wells of the 96-well-plate were filled with 200 µL isopropanol each, serving as blank
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model : according to OECD TG 492
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria :
negative control OD: historical range 1.047 - 2.340 (mean 1.660) - study 1.618
positive control viability: historical range 20.4 - 47.8% (mean 34.1%) - study 26.4%
- Complete supporting information for the specific RhCE tissue construct or hCE cells used :
EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, 82105 Bratislava, Slovakia.
Designation of the kit: OCL-200-EIT
Day of delivery: 16. Mar. 2021
Batch no.: 34901
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals : All 15 proficiency chemicals of the OECD TG 492 were correctly classified.
- Acceptable variability between tissue replicates for positive and negative controls : <20%
- Acceptable variability between tissue replicates for the test chemical: <20%

Irritation parameter:

percent tissue viability 

Run / experiment:

Tissue 1

Value:

43.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent tissue viability 

Run / experiment:

Tissue 2

Value:

40.4

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
The pre-test showed, that the test item was intensely coloured (OD > 0.08), but the mean viability of the tissues treated with test item was 42.2% (≤ 60%) and therefore the additional test was not necessary.

DEMONSTRATION OF TECHNICAL PROFICIENCY: demonstrated on the 15 proficiency chemicals indicated in the OECD TG 492

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: demanded OD > 0.8 < 2.8 - found OD 1.618
- Acceptance criteria met for positive control: demanded < 50 % of negative control - found 26.4 %

Table 1: Absorbance Values Negative Control, Positive Control and Test Item (OD at 570 nm)

Designation	Measurement	Negative Control	Positive Control	Test Item
Tissue 1	1	1.740	0.442	0.744
	2	1.750	0.443	0.745
Tissue 2	1	1.561	0.479	0.687
	2	1.558	0.482	0.688

Table 2: Mean Absorbance Negative Control, Positive Control and Test Item

Designation	Negative Control	Positive Control	Test Item
Mean – blank (Tissue 1)	1.711	0.409	0.711
Mean – blank (Tissue 2)	1.526	0.447	0.654

Table 3: % Viability Positive Control and Test Item

Designation	Positive Control	Test Item
% Viability (Tissue 1)	25.2%	43.9%
% Viability (Tissue 2)	27.6%	40.4%
% Viability Mean	26.4%	42.2%

Interpretation of results:

study cannot be used for classification

Conclusions:

After treatment with the test item, the mean value of relative tissue viability was 42.2%. This value is below the threshold for eye irritation potential (≤ 60%). Test items that induce values below the threshold are considered either eye irritant or inducing serious eye damage.

Executive summary:

The registered substance was tested for eye irritation in a GLP compliant in vitro study according to OECD Guideline 492. The test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. The solid test item was applied to two tissue replicates. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control and methyl acetate was used as positive control. The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.8, OD was 1.618. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 26.4% (< 50%). The variation within tissue replicates of the controls and the test item was acceptable (< 20%). After treatment with the test item, the mean value of relative tissue viability was 42.2%. This value is below the threshold for eye irritation potential (≤ 60%). Test items that induce values below the threshold are considered either eye irritant or inducing serious eye damage. According to the OECD Guideline 492, the EpiOcular^TM Eye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1). In this case no prediction can be made and further testing with other suitable test methods is required. Under the conditions of the test, Zinc-EDDHA sodium salts is considered either eye irritant or inducing serious eye damage in the EpiOcular^TM Eye Irritation Test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

OECD 439

One valid experiment was performed according to OECD Guideline 439 following GLP with three tissues and two independent MTT measurements.Three tissues of the human skin model EpiDerm^TM were treated with the test item for 60 minutes. The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm²; as indicated by the supplier). DPBS-buffer was used as negative control and 5% SDS solution was used as positive control. After treatment with the negative control, the mean absorbance value was within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.653. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 2.8% (required: ≤ 20%). The variation within the tissue replicates of negative control, positive control and test item was acceptable (required: ≤ 18%). After the treatment with the test item, the mean value of relative tissue viability was reduced to 32.6%. This value is below the threshold for skin irritation potential (50%). Test items that induce values below the threshold of 50% are considered at least irritant to skin. The OECD Guideline 439 addresses the human health endpoint skin irritation. Therefore, further testing on skin corrosion potential with another suitable in vitro study is required. Therefore, the test item Zinc-EDDHA sodium salts is considered at least irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.

OECD 435

The study was performed according to OECD Guideline 435 under GLP compliance using Corrositex® kit. A blank and 10% citric acid solution served as negative control and sodium hydroxide was the positive control. The test item was applied as a solid (fine powder). As the test item was classified as a category 2 substance, the test item vials were observed for 60 minutes. No break-through of the test item was observed within 61 minutes. Thus, it can be stated that in this study and under the experimental conditions reported, the test item possesses no skin corrosion potential and is considered as a non-corrosive substance. The negative control showed no break-through and therefore no corrosive effects. The measured time of the positive control was 12 minutes and thus ensuring the validity of the test system. For these reasons, the result of the test is considered valid.

Therefore, Zinc-EDDHA sodium salts is considered as non-corrosive to skin in the Corrositex® - Test. The test item is therefore not classified for corrosivity to skin according to GHS.

Conclusion

Taken together, based on the positive result of the skin irritation test (OECD Guideline 439) and the negative result in the skin corrosion test (OECD Guideline 435) the substance is concluded to be irritant to the skin.

Eye irritation

OECD 492

The registered substance was tested for eye irritation in a GLP compliant in vitro study according to OECD Guideline 492. The test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. The solid test item was applied to two tissue replicates. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control and methyl acetate was used as positive control. The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.8, OD was 1.618. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 26.4% (< 50%). The variation within tissue replicates of the controls and the test item was acceptable (< 20%). After treatment with the test item, the mean value of relative tissue viability was 42.2%. This value is below the threshold for eye irritation potential (≤ 60%). Test items that induce values below the threshold are considered either eye irritant or inducing serious eye damage. According to the OECD Guideline 492, the EpiOcular^TM Eye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1). In this case no prediction can be made and further testing with other suitable test methods is required. Under the conditions of the test, Zinc-EDDHA sodium salts is considered either eye irritant or inducing serious eye damage in the EpiOcular^TM Eye Irritation Test.

Mixture rule calculation

In the ECHA disseminated dossiers the two zinc salts ZnSO₄and ZnCl₂, which serve also as source substances for read-across are classified as Eye damage Cat 1 (H318 and H314). Based on the rules on classification for mixtures as specified in Regulation (EC) No 1272/2008 (Table 3.3.3), the threshold for classification of a mixture as Eye damage Cat 1 is a concentration of ≥ 3 % of a constituent classified as Eye damage Cat 1. The registered substance contains zinc at a concentration of 7.5 %. Thus, it is classified as Eye damage Cat 1 (H318).

Justification for classification or non-classification

Based on the available information the registered substance is classified as Eye damage Cat 1 (H318) and Skin irritant Cat 2 (H315).