OligoActiv EDDHA-Mn

Endpoint summary

Description of key information

Skin irritation

RhE, OECD439, GLP, negative

Eye irritation

RhCE, OECD 492, GLP, positive

mixture rule calculation, 7.5 % manganese (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

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Feb 2021 to ... July 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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 "manganese-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:
1) EPI-200-SIT
2) EPI-218_SIT
- Tissue batch number(s):
1) 34134
2) 34162
- Delivery date:
1) 2021 March 16
2) 2021 May 25
- Date of initiation of testing:
2021 March
- Date of experimantal completion
2021 March

TEMPERATURE USED FOR TEST SYSTEM

- Temperature used during treatment / exposure: 35 minutes at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity and 25 min at room temperature
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C and 5.0 ± 1% CO2 and ≥ 95% relative 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 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 19 hours and 40 minutes in the main test and 18 hours in the additional test 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

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Batch 34134
- Viability: MTT QC assay, 4 hours, n=3 -> OD=1.624 ± 0.135 (range 1 - 3 -> passed)
- Barrier function: ET50 assay (1 % Triton) -> ET50=4.84 h (range 4.77 - 8.72 h -> passed)
- Contamination: no contamination

Batch 34162
- 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 REPLICATE TISSUES:
3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues
- N. of replicates : 2
- Method of calculation used: Viability treated tissue (corrected) = Viability treated tissue (main test) – Viability treated tissue (additional test, no MTT assay)

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):
main test 26.3; 25.9; 26.3 mg
additional test 26.1; 25.9 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):

43 hours and 40 minutes in the main test and 42 hours in the additional test

Number of replicates:

3 (main test), 2 (additional test)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

91.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

90.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

80.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

87.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no 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:

GHS criteria not met

Conclusions:

The test item Manganese-EDDHA sodium salts is considered as not irritant to skin. After the treatment, the mean value of relative tissue viability was 87.3 %. This value is well above 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 87.3%. This value is well above the threshold for skin irritation potential (50%). Thus, the test item Manganese-EDDHA sodium salts is considered as not irritant to skin. As the test item showed intense coloring in the pre-test, there was the risk to influence the photometric measurement and create a false negative result. Therefore, an additional test for intensely coloured test items was performed.The result of the valid additional test showed, that the test item colour did not critically influence the result of the study and no data correction is necessary.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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. EpiOcular™ 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 mg

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 :
EpiOcular™ 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:

1.1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent tissue viability 

Run / experiment:

Tissue 2

Value:

1.2

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 1.2% (≤ 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	0.052
	2	1.750	0.443	0.052
Tissue 2	1	1.561	0.479	0.054
	2	1.558	0.482	0.054

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	0.018
Mean – blank (Tissue 2)	1.526	0.447	0.020

Table 3: % Viability Positive Control and Test Item

Designation	Positive Control	Test Item
% Viability (Tissue 1)	25.2%	1.1%
% Viability (Tissue 2)	27.6%	1.2%
% Viability Mean	26.4%	1.2%

Interpretation of results:

study cannot be used for classification

Conclusions:

After treatment with the test item, the mean value of relative tissue viability was 1.2%. 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 1.2%. 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™ 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, Manganese-EDDHA sodium salts is considered either eye irritant or inducing serious eye damage in the EpiOcular^TM Eye Irritation Test.

Reference 2

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 manganese salts MnSO₄ and MnCl₂, which serve also as source substances for read-across are classified as Eye damage Cat 1 (H318). 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 manganese 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 manganese 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 manganese salts MnSO₄ and MnCl₂, which serve also as source substances for read-across are classified as Eye damage Cat 1 (H318). 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 manganese at a concentration of 7.5 %. Thus, it is classified as Eye damage Cat 1 (H318).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritaion

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 87.3%. This value is well above the threshold for skin irritation potential (50%). Thus, the test item Manganese-EDDHA sodium salts is considered as not irritant to skin. As the test item showed intense coloring in the pre-test, there was the risk to influence the photometric measurement and create a false negative result. Therefore, an additional test for intensely coloured test items was performed.The result of the valid additional test showed, that the test item colour did not critically influence the result of the study and no data correction is necessary.

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 1.2%. 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, Manganese-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 manganese salts MnSO₄ and MnCl₂, which serve also as source substances for read-across are classified as Eye damage Cat 1 (H318). 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 manganese 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).