Magnesium diniobate

Endpoint summary

Description of key information

Eye irritation: not corrosive/severe irritant to the eyes (OECD 437, GLP)

Eye irritation: not irritating (OECD 492, GLP)

Skin irritation: not irritating (OECD 439, GLP)

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:

2017-07-13 to 2017-08-18

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:

2015-07-28

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2009-07-23

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-09-14

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

Test system:

human skin model

Source species:

human

Cell type:

other: normal, human-derived epidermal keratinocytes

Cell source:

other: humans

Source strain:

other: not applicable

Details on animal used as source of test system:

not applicable

Justification for test system used:

In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.

Vehicle:

other: Dulbecco's phosphate buffered saline (DPBS)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ skin model (source: MatTek Corporation, 82105 Bratislava, Slovakia)
- Tissue lot number: 25837
- Delivery date: 2017-08-15

TEMPERATURE USED FOR TEST SYSTEM
- Temperature of pre-incubation: 37 ± 1.5 °C (23.5 hours)
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 minutes, room temperature for 25 minutes
- Temperature of post-treatment incubation: 37 ± 1.5 °C (41 hours)

REMOVAL OF TEST MATERIAL AND CONTROLS
After the end of the treatment interval the inserts were removed and the tissues were rinsed with DPBS for at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in 150 mL DPBS at least three times. Afterwards the inserts were once again rinsed with DPBS.
The tissues were then transferred into plates with assay medium. Tissues were incubated for nearly 23 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. After incubation, the medium was changed (pre-warmed fresh medium). Thereafter tissues were incubated for another 18 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The complete incubation time was approx. 41 hours.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (300 µL/ well)
- Incubation time with MTT: 3 hours
- Extraction of Formazan: after the incubation period, the tissues were transferred to the MTT-plates, prefilled with 300 µL of MTT and incubated for 3 hours. After MTT incubation the tissues were rinsed three times with DPBS, and carefully dried with blotting paper. The tissues were transferred into new plates containing extractant solution (isopropanol) in each well so that the tissues were covered completely and the plate was sealed to inhibit the evaporation of isopropanol. The formazan salt was extracted for 3 hours while shaking at room temperature.
After the extraction was completed, the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken and the insert was discarded. The 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour.
Per each tissue, 3 × 200 μL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate. The optical density was read in a microplate reader. Mean values were calculated from the 3 wells per tissue.
- Spectrophotometer: Versamax® Molecular Devices
- Wavelength: 570 nm

TEST FOR COLOUR INTERFERENCE
Before the test started, a functional check for colour interference was performed. 25 ± 2 mg of the test item were added to deionised water. The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 60 minutes. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated.

TEST FOR DIRECT MTT REDUCTION
The test item was evaluated for its potential to interfere with MTT assay. To test if a test item directly reduces MTT, 25 ± 2 mg of the test item were added to 1 mL of the MTT-solution (1mg/mL) and was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) for 60 minutes. Untreated MTT medium was used as control.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissues pass analysis for tissue viability
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1- virus, Hepatitis B virus, and Hepatitis C virus
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
The mean optical density (OD) of the three negative control tissues was calculated after blank correction. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
relative viability (%) = (mean OD test item or positive control/ mean OD of negative control) x 100
For the test item and the positive control, the mean relative viability ± relative standard deviation of the three individual tissues was calculated and used for classification according to the following prediction model: if the mean relative tissue viability of three individual tissues is less or equal to 50 % of the negative control, the test item needs to be classified and labelled for its skin irritation potential: Category 2 – irritant, H315 according to Regulation (EC) No 1272/2008.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 25 mg (~ 39 mg/cm²) of the test item, wetted with vehicle

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL DPBS

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of a 5% SLS in deionised water

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

41 hours

Number of replicates:

triplicates

Irritation / corrosion parameter:

% tissue viability

Value:

101.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: Since the MTT solution did not turn blue/purple, the test item was not considered to reduce MTT and an additional test with freeze-killed tissues did not have to be performed in the main experiment.
- Colour interference with MTT: Since the test item did not dye water and did not change colour in the presence of water, an additional test with viable tissues (but without MTT addition) was not necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: after treatment with the negative control, the absorbance values (mean: 1.337) were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval.
- Acceptance criteria met for positive control: treatment with the positive control induced a decrease of the relative absorbance to 4.4 % compared with the negative control (acceptability criterion: positive control is ≤ 20 %).
- Acceptance criteria met for variability between replicate measurements: the standard deviations between the % variability values of the test item, the positive and negative controls in the main test were 2.0, 0.3 and 11.5, respectively (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: < 18%).
Please refer to the field "Any other information on results incl. tables" below

HISTORICAL DATA

Positive Control		Negative Control [OD570]
Mean Viability	4.37%	Mean Absorption	1.74
Rel. Standard Deviation	21.60%	Rel. Standard Deviation	9.40%
Range of Viabilities	2.20 % - 6.78 %	Range of Absorbance	1.34– 2.00
Mean Absorption	0.08
Rel. Standard Deviation	20.12%
Range of Absorbance	0.03 - 0.11

Data of 103studies performed from July 2015 untilMarch2017.

RESULTS AFTER TREATMENT WITH MAGNESIUM DINIOBATE AND THE CONTROLS

Dose Group	Tissue No.	Absor-bance 570 nm Well 1	Absor-bance 570 nm Well 2	Absor-bance 570 nm Well 3	Mean Absor-bance of 3 Wells	Mean Absor-bance of three wells blank corrected	Mean Absor-bance of 3 tissues after blank correction	Rel. viability [%] Tissue 1, 2 ,3*	Standard Deviation	Mean Rel. Viability [%]**
Blank		0.038	0.038	0.038	0.038
Negative Control	1	1.349	1.359	1.357	1.355	1.317	1.337	98.6	2.0	100.0
	2	1.415	1.405	1.396	1.405	1.368		102.3
	3	1.374	1.356	1.358	1.363	1.325		99.1
Positive Control	1	0.087	0.092	0.094	0.091	0.053	0.058	4.0	0.3	4.4
	2	0.098	0.098	0.098	0.098	0.060		4.5
	3	0.100	0.099	0.099	0.100	0.062		4.6
Test Item	1	1.233	1.223	1.244	1.233	1.196	1.353	89.5	11.5	101.2
	2	1.396	1.397	1.400	1.398	1.360		101.7
	3	1.534	1.549	1.538	1.540	1.502		112.4

* Relative viability [rounded values]:100 x (absorbance (test item/ positive control/negative control))/ (mean absorbance (negative control))

** Mean relative viability [rounded values]:100 x (mean absorbance (test item/ positive control/negative control))/ (mean absorbance (negative control))

Interpretation of results:

GHS criteria not met

Conclusions:

According to the Regulation (EC) No 1272/2008 and subsequent regulations, magnesium diniobate is not irritating to the skin.

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:

2016-09-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2013-07-26

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997

Version / remarks:

April 1997

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-09-14

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: abattoir (Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany)
- Characteristics of donor animals: at least 9 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue: isolated eyes were transported to the laboratory in Hank’s Buffered Salt Solution (HBSS) supplemented with streptomycin / penicillin at ambient temperature.
- Time interval prior to initiating testing: corneae were isolated and used on the same day after delivery of the eyes

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration: 20 % suspension (w/v) in vehicle

Duration of treatment / exposure:

240 minutes

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

not required

Number of animals or in vitro replicates:

Number of bovine corneae per dose:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- all eyes were examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- cornea was removed from the eye.
- each cornea was mounted in a cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. Both compartments of the holder were filled with incubation medium (MEM*, equivalent to EMEM).
- for equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.

*MEM supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, and 1% fetal calf serum

QUALITY CHECK OF THE ISOLATED CORNEAS
- at the end of the equilibration period, the basal opacity was determined (t0).
- each cornea with a value of the basal opacity > 7 was discarded.

APPLICATION DOSE / EXPOSURE TIME / REMOVAL OF TEST SUBSTANCE
- the anterior compartment received the test item suspension or negative or positive control at a volume of 0.75 mL each on the surface of the corneae.
- corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath (incubation time: 240 minutes).
- after the incubation period, the test item or control items, respectively, were rinsed off from the application side with saline
- fresh incubation medium was added into the anterior compartment and opacity was measured (t240).
- permeability of the cornea was determined

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: the opacitometer (OP_KiT opacitometer (Electro Design)) was calibrated and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
Evaluation of opacity:
- the change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
- the average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of spectrophotometer (Versamax® Molecular Devices) (OD490).
- after the final opacity measurement was performed, the incubation medium was removed from the anterior compartment and replaced by a 0.5% (w/v) sodium fluorescein solution in HBSS.
- corneae were incubated again in a horizontal position for 90 ± 10 minutes in a water-bath at 32 ± 1 °C.
- incubation medium from the posterior compartment were removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer.
Evaluation permeability:
- the corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original
permeability value for each cornea.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula was used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula was used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group was calculated from the IVIS values of each individual treatment and positive control cornea.
Depending on the score obtained, the test item was classified into categories according to OECD guideline 437 (table 1 in the field "Any other information on materials and methods incl. tables" below).

ACCEPTANCE CRITERIA:
The test was acceptable if
- the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
- the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

Irritation parameter:

in vitro irritation score

Remarks:

(mean)

Value:

2.97

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- with the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.48).
- distinct opacity of the corneae leading to a mean IVIS of 110.15 was caused after treatment with the positive control (10% (w/v) Benzalkonium chloride in saline) corresponding to the classification Irreversible effects on the eye / serious eye damage (CLP (Cat 1)).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: opacity and permeability of the negative control are less than the respective established upper limits for background opacity and permeability.
- Acceptance criteria met for positive control: the positive control falls within two standard deviations of the current historical mean.

Please refer to the field "Any other information on results incl. tables" below.

Table 1: Results after 240 minutes incubation time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS
		Mean		Mean
Negative Control	0	0.33	0.079	0.077	1.19	1.48
	1		0.076		2.14
	0		0.075		1.13
Positive Control	117.67*		0.003*		117.72	110.15
	104.67*		0.017*		104.93
	107.67*		0.009*		107.81
Magnesium diniobate	2.67*		0.005*		2.75	2.97
	2.67*		0.016*		2.91
	2.67*		0.039*		3.26

*corrected values

Table 2: Historical data

	Positive Control	Negative Control
Mean IVIS	122.01	1.32
Standard Deviation IVIS	15.51	0.33
Range of IVIS	98.49 – 167.85	0.73 – 2.04
Mean Opacity t240min	120.03	0.17
Standard Deviation Opacity t240min	18.45	0.25
Range of Opacity t240min	88.67 – 183.00	-.33 – 0.67
Mean Permeability	0.147	0.184
Standard Deviation Permeability	0.257	0.281
Range of Permeability	0.000 – 1.438	0.055 – 1.100

Values of 32 studies with solid test items performed from February 2015 (calendar week 7) until October 2016 (calendar week 40)

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, based on a valid OECD 437 test and under the experimental conditions reported, magnesium diniobate does not meet the requirements for classification as UN GHS Category 1 or 2 (2A or 2B) and is referred to as UN GHS No Category.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-02-23 to 2017-03-16

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:

2015-07-28

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model

Version / remarks:

2015-06-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-09-14

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

Details on test animals or tissues and environmental conditions:

JUSTIFICATION OF THE TEST METHOD
In a prevalidation study performed by Avon Products Inc. and MatTek Corporation, the in vitro eye irritation using the human cornea model EpiOcular™ and measurement of cell viability
by dehydrogenase conversion of MTT into a blue formazan salt has turned out to be a sufficiently promising predictor for eye irritancy potential.
The EpiOcular ™ Eye Irritation Test (EIT) has been validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe between 2008 and 2013.

HUMAN RECONSTRUCTED CORNEA MODEL
- Model used: human cornea model EpiOcular™ (source: MatTek Corporation, 82105 Bratislava, Slovakia)
The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes that have been cultured to form a stratified squamous epithelium morphologically similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a nonkeratinized surface with a cornea-like structure analogous to that found in vivo.
- Tissue lot number: 23771

FUNCTIONAL MODEL CONDITIONS
- Viability: tissues pass analysis for tissue viability
- Barrier function: tissues pass analysis for tissue functionality
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1- virus, Hepatitis B virus, and Hepatitis C virus
Please also refer to the field "Attached background material" below.

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): approx. 50 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 50 µL

Duration of treatment / exposure:

6 hours

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

duplicates

Details on study design:

ASSESSMENT OF DIRECT MTT REDUCTION
The test item was evaluated for its potential to directly reduce MTT. For this purpose approx. 50 mg of the test item were added to 1 mL MTT solution (1 mg/mL; in DMEM) in a glass tube, and the mixture was incubated in the dark at standard culture conditions for three hours. A control (50 μL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was run concurrently. If the MTT solution colour would turn blue/purple, the test item was presumably to have reduced the MTT.

ASSESSMENT OF COLOURED OR STAINING MATERIALS
The test item was checked for its colourant properties, which might interfere with the quantitative photometric MTT measurement.
Since the test item was non-coloured additional tests had to be performed to assess if it became coloured after contact with water or isopropanol. For this purpose, approx.
50 mg of the test item were added to 1.0 mL of water and to 2 mL isopropanol in a glass tube, respectively. The water mixture was incubated in the dark at standard culture conditions for 1 hour, whereas the isopropanol mixture was incubated for 3 hours at room temperature.

PRE-EXPERIMENT CONDITIONS
- on day of receipt of the EpiOcular™ tissues, a equilibration step was started (15 minutes at room temperature in the shipping container).
- after the equilibration step, each tissue was inspected for air bubbles between the agarose gel and insert.
- insert was transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in Assay Medium.
- after one hour, the Assay Medium was replaced by fresh Assay Medium at 37 °C
- EpiOcular™ tissues were incubated at standard culture conditions overnight (about 18 hours).

EXPERIMENTAL PERFORMANCE
- after the overnight incubation, the tissues were pre-wetted with 20 μL of Ca++& Mg++free-DPBS and were incubated at standard culture conditions for 30 minutes.
- after the 30 minute period, the test and control item were applied topically onto the EpiOcular™ tissues.
- tissues were incubated at standard culture for 6 hours.
- after the 6 hours, the test item was removed by rinsing the tissues several times with Ca++& Mg++-free DPBS (brought to room temperature).
- it was not possible to remove the visible test item completely, but no further rinsing was done.
- after rinsing, the tissues were transferred to and immersed in assay medium (room temperature) in a 12-well plate for 25 minutes immersion incubation at room temperature (to remove any test item adsorbed by the tissue).
- next, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert was blotted and transferred into the appropriate well of a 6-well plate containing warm assay medium.
- tissues were incubated for about 18 hours at standard culture conditions (post-treatment incubation).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- at the end of the post-treatment incubation, each insert was removed from the plate and blotted.
- tissues were placed into a 24-well plate containing 0.3 mL of MTT solution.
- plate was incubated for 180 minutes at standard culture conditions.
- next, inserts were removed from the 24-well plate, bottom of the insert was blotted, and then transferred into 6-well plate containing 2 mL isopropanol in each well.
- plates were sealed with parafilm or a standard plate sealer, and were extracted (shaken for 2.5 hours at room temperature).
- tissues were not pierced.
- corresponding negative, positive, and additional viable tissues (without MTT addition) were treated identically without piercing.
- extract solution was mixed and two 200 μL aliquots were transferred into the appropriate wells of a 96-well plate(s).
- absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices). No reference wavelength measurement was used.

DATA EVALUTION
1) mean optical density (OD) value of the blank control wells (ODBlk) for each experiment was calculated.
2) mean ODBlk from each mean OD value of the same experiment was subtracted (blank corrected values).
3) mean value of the two replicates for each tissue was calculated.
4) mean value of the two relating tissues for each control (negative control (NC) and positive control (PC) and test item (TI) was calculated (ODTI, ODNC, ODPC).
5) mean OD value of the negative control corresponds to 100% viability.
Corrected negative control OD = Negative Control OD - ODBlk = 100% Viability

6) percent viability of each of the two relating tissues for each control and test item relative to the negative control (= 100%) was calculated.
Viability (%) = 100 x ((ODTi/ODPC/ODNC) / mean ODNC)
7) difference of the viability between duplicate tissues was calculated (difference >20% the test is considered as non-valid).
8) mean viability of the test item (TI viability) was calculated and the test item was classified according to the prediction model.

PREDICTION MODEL
- test item-treated tissue viability is > 60% relative to the negative control treated tissue viability. Test item is identified as not requiring classification and labelling according to
GHS (No Category), i.e. is not an eye irritant.
- test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability. Test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1), is an eye irritant.

ACCEPTABILITY CRITERIA
The results are acceptable, if:
1) negative control optical density is > 0.8 and < 2.5,
2) mean relative viability of the positive control is below 50% of the negative control viability.
3) difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This
applies also to the viabilities of freeze-killed tissues (items and negative control) and the additional viable tissues (without MTT addition) which are calculated relative to the negative control.
4) results of the positive and negative controls of the test method demonstrate reproducibility over time.

Irritation parameter:

other: % tissue viability

Value:

93.1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The laboratory had demonstrated technical proficiency by correctly predicting the irritation potential of fifteen proficiency chemicals listed in Table 1 of OECD TG 492.
Please also refer to the field "Attached background material" below.

OTHER EFFECTS:
- Colour interference with MTT: the colour interference pre-experiment to investigate the colour change potential of the test item in water or isopropanol did not result in a change in colour. Therefore, an additional test with viable tissues without MTT addition was not necessary.
- Direct-MTT reduction: optical evaluation of the MTT-reducing capacity of the test item with the MTT-reagent did not recognise a blue colour. Therefore, an additional test with freeze-killed tissues was not necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, negative control OD is > 0.8 and < 2.5 (values between 1.038 and 1.117).
- Acceptance criteria met for positive control: yes, mean relative viability of the positive control is below 50% of the negative control viability (37.0%).
- Acceptance criteria met for variability: yes, difference of viability between the two relating tissues of a single item is < 20% (values between 2.0% and 4.9%) in the same run (for positive and negative control tissues and tissues of single test items).
- Reproducibility of negative and positive control: results of the positive and negative controls of the test method demonstrate reproducibility over time. Even though the negative control absorbance is slightly below the historical range, it is in the acceptance range of OECD TG 492.

Table 1: Absorbance of tissues after treatment for 6 hours with the test item and the controls

Dose Group	Absorbance Well 1 (Tissue 1/2)	Absorbance Well 2 (Tissue 1/2)	Mean Absorbance* (Tissue 1/2)	Mean Absorbance* Tissue 1 and 2 minus Mean Blank	Mean Absorbance of 2 Tissues*	Rel. Absorbance [%] Tissue 1 and 2**	Absolute Value of the Difference of the Rel. Absorbances [%] Tissue 1 and 2	Mean Rel. Absorbance [% of Negative Control]**
Blank	0.039	0.039	0.039	0.000
Negative Control	1.079	1.038	1.059	1.020	1.045	97.5	4.9	100.0
	1.103	1.117	1.110	1.071		102.5
Positive Control	0.440	0.446	0.443	0.404	0.387	38.6	3.3	37.0
	0.404	0.412	0.408	0.369		35.3
Test Item	0.974	1.072	1.023	0.984	0.974	94.1	2.0	93.1
	1.001	1.003	1.002	0.963		92.1

* Mean of two replicate wells after blank correction

** Relative absorbance [rounded values]: (100 X (absorbance test item/positive control))/ (absorbance negative control)

Table 2: Historical control data

Positive Control		Negative Control [OD570]
Mean Viability	32.47%	Mean Absorption	1.49
Rel. Standard Deviation	10.29%	Rel. Standard Deviation	0.24
Range of Viabilities	15.90% - 42.30%	Range of Absorbance	1.24 – 2.05
Mean Absorption	0.48
Rel. Standard Deviation	0.14
Range of Absorbance	0.22- 0.64

Data of 15 studies performed from July 2015 until end of December 2016

Interpretation of results:

GHS criteria not met

Conclusions:

It can be stated that in this study and under the experimental conditions reported, magnesium diniobate does not possess any eye irritating potential. Magnesium diniobate is identified as not requiring classification and labelling for eye irritation according to UN GHS (No Catogory).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Magnesium diniobate can safely be assumed to have a low potential for human inhalation hazard during handling or application. MPPD modelling indicates that magnesium diniobate does not penetrate to deep lung tissue (tracheobronchial: 0.11%; pulmonary: 0.03%), whereas the majority of the inhaled material (Head: 55.79%) would be cleared to the GI tract (by swallowing), where oral bioavailability will determine its uptake. Nearly all of the inhaled particles are swallowed and do not reach the pulmonary fraction of the lung. Any lung overload associated with inert particles can be excluded. In addition, magnesium diniobate is poorly water soluble (< 1 mg/L at 20°C, pH 7.3) indicating inertness and a corresponding lack of potential to become bioavailable. Furthermore, pH-related effects do not need to be assumed upon contact with respiratory tract epithelia. Based on extremely poor solubility and complete lack of irritant potential, it is considered justified to assume an absence of local effects. In sum, it is assumed that magnesium diniobate does not cause respiratory tract irritation.

Justification for classification or non-classification

Magnesium diniobate does not possess an eye or skin irritating potential based on three in vitro tests (OECD 437, OECD 492 and OECD 439) and does not require classification according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Based on available data, including dustiness, solubility, irritation potential, it is assumed that magnesium diniobate does not cause respiratory tract irritation.