Ytterbium trinitrate

Endpoint summary

Description of key information

Skin irritation:

Two reliable studies are used in a ‘Weight-of-evidence’ approach to assess the skin irritation/corrosion potential of the substance. First, in a study scored as K2 (Solvay, 2016), the pH-value of a 10% Ytterbium trinitrate solution was determined to be = 2.74 and its acidic reserve, determined via titration, was found to be 0.12. Thus, the calculation for corrosivity of the Ytterbium trinitrate was 2.73 that is well above the threshold of -0.5 to consider a substance as corrosive. Therefore, Ytterbium trinitrate was considered not to be corrosive to the skin.

In a second study performed according to OECD Guideline 439 (Orovezc, 2016) and in compliance with GLP, and thus scored as K1, Ytterbium trinitrate was found to be non-irritating to the skin in the in vitro EPISKIN reconstructed human epidermis model used for this study.

Furthermore, reliable studies available on other Rare Earth nitrates (which have been performed either by using in vivo skin irritation studies done according to OECD Guideline n° 404 or comparable guidelines (Cerium trinitrate, Lanthanum trinitrate, Neodymium trinitrate, Praseodymium trinitrate, Yttrium trinitrate and Terbium trinitrate) or by using in vitro skin irritation/corrosion studies done according to OECD Guideline n°431 and/or OECD guideline n° 439 (Gadolinium trinitrate, Dysprosium trinitrate, Lanthanum trinitrate, Neodymium trinitrate, Preseodymium trinitrate and Lutetium trinitrate)) have shown that none of these Rare-Earth nitrates need to be classified as a skin irritant according to CLP criteria.

Based on all data reported above, it is expected that Ytterbium trinitrate will not cause corrosion and irritation of the skin. Thus, Ytterbium trinitrate needs not to be classified according to CLP criteria. No further testing to cover this endpoint is deemed to be required.

Eye irritation:

No data is available on the substance for the eye irritation end-point. However, reliable available studies performed with other Rare Earth nitrates (done using, either in vivo eye irritation studies performed according to OECD Guideline n° 405 or comparable guidelines (Cerium trinitrate, Lanthanum trinitrate, Neodymium trinitrate, Yttrium trinitrate and Terbium trinitrate), or using in vitro eye irritation/corrosion study performed according to OECD Guideline n°437 (Gadolinium trinitrate), have shown that all these Rare-Earth nitrates have induced serious eye damage and thus, have been classified in the Category 1 with the hazard statement H318 “ Cause serious eye damage” according to CLP.

Based on these results, it is expected that Samarium trinitrate will cause serious eye damages and thus, needs to be classified in the Category 1 with the hazard statement H318 “ Cause serious eye damage” according to CLP. No further testing to cover this endpoint is deemed to be required.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From August 26th, 2016 to December 16th, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Performed under GLP compliance

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

September 22th, 2015

Test system:

human skin model

Source species:

human

Cell type:

other: Adult human-derived epidermal keratinocytes

Cell source:

other: three-dimensional human epidermis model

Justification for test system used:

The EPISKINTM (SM) model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: three-dimensional human epidermis model
- Tissue batch number(s): 16-EKIN-035
- Expiration date: 05 September 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature of pre-incubation: 37°C
- Temperature used during treatment / exposure: 15 minutes at room temperature (26.3 - 27.5°C)
- Temperature of post-treatment incubation: 37°C

APPLICATION
- Test item:
As the test item was solid, first an appropriate amount distilled water was applied to the epidermal surface in order to improve further contact between test item and epidermis and then the test item was applied evenly to the epidermal surface. If necessary, the test item was spread gently on the skin surface with a pipette tip (or other appropriate tool) without damaging the epidermis. The amount was sufficient to cover the epidermal surface and was calculated from the residual test item quantity after treatment.
- Negative and positive controls:
Negative control (PBS) or positive control (5% (w/v) SDS solution were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).
- Additional control for test item having colouring potential:
For potentially colouring test item, in addition to the normal procedure, two additional viable chemical-treated tissues were used for the non specific OD evaluation. These tissues followed the same treatment steps as the other tissues except for the MTT step: MTT incubation step was replaced by incubation with fresh Assay Medium. OD reading were made following the same conditions as the other tissues.

The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min).

REMOVAL OF TEST MATERIAL
After the 15 minutes incubation time, the EPISKINTM(SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
After rinsing the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5% CO2.

MTT TEST
After the 42 hours incubation, all EPISKINTM(SM) units (except of two colour control units) were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well). Then, all transferred EPISKINTM(SM) units were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 protected from light.

FORMAZAN EXTRACTION
After the incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.
A blank sample containing 2 mL of acidified isopropanol was processed in parallel.

CELL VIABILITY MEASUREMENTS
Following the formazan extraction, 2×200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
In the present study, the irritancy potential of test substances is predicted by the mean tissue viability of tissues exposed to the test item. The test item considered to be irritant to skin (Category 2), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.
Mean tissue viability % is ≤ 50%: Category 2 or Category 1
Mean tissue viability % is > 50%: Non-Irritant

VALIDITY OF THE TEST
The mean OD value of the three negative control tissues should be between 0.6 and 1.5, and the standard deviation value (SD) of the % viability values should be ≤ 18.
The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability values should be ≤ 18.
The SD calculated from individual % tissue viability values of the three test item treated replicates should be <18.
The mean OD value of the blank samples (acidified isopropanol) should be <0.1.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST ITEM
Around 85.2 mg of the test item

NEGATIVE AND POSITIVE CONTROLS
50 μL of negative control (PBS) or positive control (5% (w/v) SDS solution)

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 tissues

Irritation / corrosion parameter:

% tissue viability

Value:

85.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ADDITIONAL CONTROLS
As no colour change (yellow colour) was observed after three hours of incubation of the test item in MTT working solution, the test materials did not interact with MTT and additional controls and data calculations were not necessary. The false estimation of viability can be excluded.

As the test item was coloured, two additional test item-treated tissues were used for the non-specific OD evaluation. The optical density (measured at 570 nm) of tissues were 0.039, Non-specific Colour % was calculated as 5.1%. This value was above 5%, therefore additional data calculation was necessary.

VIABILITY RESULTS
The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 1 below (Any other information on results incl. tables). The OD values for the test item treated skin samples showed 85.3% relative viability (after adjustment for non-specific colour).

VALIDITY OF THE TEST
After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.

The mean OD value of the three negative control tissues was in the recommended range (0.756). Standard deviation of the viability results for negative control samples was 4.5.
The positive control treated tissues showed 4.0% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.0.
The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 1.3.
The mean OD value of the blank samples (acidified isopropanol) was 0.046.

All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Table 1: Optical Density (OD) and the calculated relative viability % of the samples

 

Substance	Optical Density (OD)			TODTT	Viability (% RV)
		Measured	Blank corrected
Negative Control:	1	0.842	0.795	-	105.2
Phosphate buffered saline	2	0.779	0.732	-	96.9
	3	0.787	0.740	-	97.9
	mean	-	0.756	-	100.0
Positive Control:	1	0.075	0.029	-	3.8
5% (w/v) SDS* solution	2	0.084	0.038	-	5.0
	3	0.070	0.023	-	3.1
	mean	-	0.030	-	4.0
Test Item:	1	0.740	0.694	0.655	86.6
Ytterbium trinitrate	2	0.722	0.675	0.637	84.2
	3	0.727	0.681	0.642	84.9
	mean	-	0.683	0.645	85.3

* SDS: Sodium Dodecyl Sulphate

Notes:

1. Mean blank value was 0.046.

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

3. TOD_TT: The measured values were corrected for non-specific colour reduction (by subtracting the NSC_livingof 0.039) was applied for test item treated samples in each case.

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with Ytterbium trinitrate, the mean cell viability was 85.3% compared to the negative control (after adjustment for non-specific colour). This is above the threshold of 50%, therefore the test item was considered to be non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN model test with Ytterbium trinitrate (Batch number: 1612999), the results indicate that the test item is non-irritant to skin.

Executive summary:

An in vitro skin irritation test with Ytterbium trinitrate test item was performed in a reconstructed human epidermis model. EPISKINTM(SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The irritation potential of the test item was evaluated according to the OECD guideline No. 439.

 

Disks of EPISKINTM(SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2, in a > 95% humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at in an incubator with 5% CO2 protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

 

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. For each treated tissue, the viability was expressed as a % relative compared to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

 

Following exposure with Ytterbium trinitrate, the mean cell viability was 85.3% compared to the negative control (after adjustment for non-specific colour). This is above the threshold of 50%, therefore the test item was considered to be non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

 

In conclusion, in this in vitro EPISKIN model test with Ytterbium trinitrate (Batch number: 1612999), the results indicate that the test item is non-irritant to skin.

Reference 2

Endpoint:

skin irritation / corrosion, other

Remarks:

Determination of pH-value and acidic reserve

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Report date: August 20th, 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Electrochemic determination of pH
Determination of the acidic reserve via titration

GLP compliance:

no

Details on study design:

Purpose:
The higher the buffer capacity of a mixture (solution or slurry) is, the stronger the irritating and corrosive potential is, respectively. The physiological effects of acidic or alkaline solutions are not only determined by the pH value but also by the buffering capacity. That is why it is necessary to determine the alkaline or acidic reserve of identified products.

Method:
First, the pH-value of a 10% solution (or slurry) is determined electrochemically at 20°C with a calibrated pH meter (MA 235 Mettler Toledo). This value is recorded.

Determination of the acidic reserve titration:
- For the determination of the alkaline reserve: the titration volume (in mL) of a 0.5 mol/L H2SO4 solution, which is required to achieve a pH of 10 of a 10% solution (or slurry) at 20°C, is recorded.
- For the determination of the acid reserve: the titration volume (in mL) of a 1 mol/L NaOH solution, which is required to achieve a pH of 4 of a 10% solution (or slurry) at 20°C, is recorded.

Calculations:
Titration of the 10% solution/slurry
Alkaline or acid reserve = titration volume [mL] x 0.4

A product is classified as corrosive if:
pH + 1/12 alkaline reserve >= 14.5
pH - 1/12 acid reserve <= -0.5

Irritation parameter:

other: acid/alcaline reserve

Basis:

other: acid reserve

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Not corrosive (based on an acidic reserve of 0.12 and pH of 2.74, the calculation for corrosivity was 2.73 which is higher than -0.5).

Interpretation of results:

other: not corrosive

Remarks:

Based on pH and acidic reserve calculation

Conclusions:

The pH value of a 10% solution of ytterbium trinitrate was determined to be 2.74 and the acidic reserve 0.12. Based on these data, the calculation for corrosivity was 2.73 (> -0.5). Therefore the substance was considered not to be corrosive.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

Based on the results of the available data and the criteria of the EU CLP Regulation, Ytterbium trinitrate should not be classified for skin irritation.

Using the Weight of Evidence approach, Ytterbium trinitrate is considered as a substance causing eye damage Category 1 and needs a H318 hazard statement according to CLP.