Praseodymium trinitrate

Endpoint summary

Description of key information

Skin irritation/corrosion:
Four reliable studies are used in a ‘Weight-of-evidence’ approach.
Based on the determination of the pH (1.81) and the acidic reserve (0.424), the calculation for corrosivity was 1.775 (> -0.5). Therefore praseodymium trinitrate was considered not to be corrosive (Rhodia Operations, 2012). This test was scored K2.
A K1 in vitro skin corrosion test in an EPISKIN reconstructed human epidermis model was performed according to OECD Guideline 431 and EU Method B.40 (Warren, 2013). Praseodymium trinitrate was considered to be non-corrosive to the skin.
A K1 in vitro skin irritation test in an EPISKIN reconstructed human epidermis model was performed according to OECD Guideline 439 and EU Method B.46 (Warren, 2013). Praseodymium trinitrate was considered to be non-irritating to the skin.
Finally, a K1 in vivo skin irritation test in New Zealand White rabbits was performed according to OECD Guideline 404, Commission Regulation (EC) NO 440/2008, B.4 and OPPTS Guideline 870.2500 and in compliance with GLP (Matting, 2014). Praseodymium trinitrate was considered to be non-irritating to the skin.

Eye irritation:
No data were available for the eye irritation endpoint. However, on the basis of expert judgment and the available information on other Rare Earth nitrates (e.g. cerium trinitrate, lanthanum trinitrate, neodymium trinitrate), it is very probable that praseodymium trinitrate should be considered as a substance causing eye damage Cat 1 and needs a H318 hazard classification according to CLP. This classification is kept in order to protect workers.

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:

2012-10-16 - 2012-10-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

in vitro reconstructed human epidermis model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on test system:

The EPISKIN model kit was purchased from SkinEthic Laboratories, Lyon, France, received 16 October 2012.
The EPISKINTM model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
The procedure followed is based on the recommended EpiSkinTM Skin Corrosivity Test protocol INVITTOX No 118. The test item is applied topically to the stratum corneum surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. The test is based on the experience that corrosive chemicals are cytotoxic after a short term exposure to the EPISKINTM model. Corrosive chemicals are able to penetrate the stratum corneum and are sufficiently cytotoxic to cause cell death in the underlying cell layers. Toxicity is determined by the metabolic conversion of the vital dye MTT to formazan by viable cells in the test item treated cultures relative to the negative control.

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg of ground-up test substance was applied to tissues, with 100 µL 0.9% w/v sodium chloride solution was added for wetting of the test item.

Duration of treatment / exposure:

3, 60 and 240 minutes

Number of replicates:

Duplicate exposures of EPISKIN tissues to test item, positive control and negative control.

Details on study design:

Pre-Incubation (Day 0: tissue arrival)
2.2 ml of maintenance medium, warmed to approximately 37ºC, was pipetted into two wells of the first column of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (2 units per plate). A different 12-well plate was used for each test item, control and time point. The tissues were incubated at 37ºC, 5% CO2 in air overnight. After 24 hours the medium underneath the tissues was refreshed and the tissues were returned to the incubator for a further 24 hours.

Application of Test Item and Rinsing (Day 2)
2.2 mL of assay medium, warmed to approximately 37ºC, was pipetted into 2 wells of the second and third columns of the 12-well plate. The tissues were transferred into the second column. Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes. Duplicate tissues were treated with the positive and negative control items for an exposure period of 240 minutes. 20 mg of the solid test item was applied topically to the corresponding tissues ensuring uniform coverage of the tissues. 100 μL of 0.9% w/v sodium chloride solution was added for wetting of the test item. Duplicate tissues, treated with 50 μL of 0.9% w/v sodium chloride solution served as negative controls. Duplicate tissues, treated with 50 μL of glacial acetic acid served as positive controls. The treated tissues were kept in a biological safety cabinet at room temperature for the appropriate exposure period.
At the end of each exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Phosphate Buffered Saline Dulbeccos (PBS) with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test item. Each rinsed tissue was placed into the third column of the 12-well plate until all tissues were rinsed. 2.0 mL of a 0.3 mg/mL MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide solution), freshly prepared in assay medium, was pipetted into 2 wells of the fourth column of each 12 well plate. The tissues were transferred into the MTT filled wells. The tissues were incubated for 3 hours ±5 minutes at 37ºC, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was taken using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into labelled 1.5 mL micro tubes containing 500 μL of acidified isopropanol. Each tube was plugged, mixed thoroughly and stored overnight at room temperature, protected from light, to extract formazan crystals out of the MTT-loaded tissues.

Absorbance/Optical Density Measurements (Day 3)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution. For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader

Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT according to the procedure below: 20 mg of the test item was added to 2.2 mL of a 0.3 mg/m\L MTT solution freshly prepared
in assay medium. The solution was incubated in the dark at room temperature for 3 hours. Untreated MTT solution was used as a control. If the MTT solution containing the test item turns blue relative to the control, the test item was presumed to have reduced the MTT. No color change was observed.

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

test item after 3-minute exposure

Value:

101.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not corrosive

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

test item after 60-min exposure

Value:

111.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not corrosive

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

test item after 240-min exposure

Value:

98.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not corrosive

Quality criteria:

The relative mean tissue viability for the positive control treated tissues was 5.8% relative to the negative control treated tissues following the 240-minute exposure period. The positive control acceptance criterion was therefore satisfied. The mean OD540 for the negative control treated tissues was 0.791. The negative control acceptance criterion was therefore satisfied.

Interpretation of results:

GHS criteria not met

Conclusions:

The relative mean viability of the test item treated tissues after the 240 minutes exposure was 98.6% (>= 35; relative mean tissue viability percentage of negative control criteria). The test item was therefore considered to be non-corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013-12-03 - 2013-12-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

relative humidity was out (min 23%) of target range (30-70%) during the study; this deviation is not considered to have impact on the outcome of the study and the interpretation of the results

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

relative humidity was out (min 23%) of target range (30-70%) during the study; this deviation is not considered to have impact on the outcome of the study and the interpretation of the results

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Deviations:

yes

Remarks:

relative humidity was out (min 23%) of target range (30-70%) during the study; this deviation is not considered to have impact on the outcome of the study and the interpretation of the results

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: S&K-LAP Kft, 2173 Kartal, Csaszar ut 135, Hungary
- Age at study initiation: 16 weeks old
- Weight at study initiation: 3860-3920 g
- Housing: Rabbits were individually housed in AAALAC approved metal wire rabbit cages. Cages were of an open wire structure and cages were placed together to allow some social interaction with rabbit(s) in adjoining cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 42 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.3 - 21.1 °C
- Humidity (%): 23 - 70%
- Air changes (per hr): 15 - 20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily light, from 6.00 a.m. to 6.00 p.m.

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

other: The untreated skin of each animal served as a control.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The powdered test item was used as a single dose of 0.5 g applied to the test area.

Duration of treatment / exposure:

4 hours

Observation period:

Animals were examined for signs of erythema and oedema, and the responses scored at 60 minutes and then at 24, 48 and 72 hours after patch removal. The duration of the study was sufficient to evaluate the reversibility or irreversibility of the effects observed.

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: 6 cm² of intact skin, back and flanks of animals
- On the day of treatment, 0.5 g of the test item was placed on a surgical gauze pad (approximately 5 cm x 5 cm) and sufficient water was added to dampen the material to ensure good contact with the skin. This gauze pad was applied to the intact skin of the clipped area and water was kept in contact with the skin by a patch with a surrounding adhesive hypoallergenic plaster. The entire trunk of the animals was then wrapped with plastic wrap held in place with an elastic stocking.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The semi-occlusive dressing was removed and the skin was flushed with lukewarm tap water to clean the application site.

SCORING SYSTEM: The dermal irritation scores were evaluated according to the scoring system by Draize (1959)

Irritation parameter:

primary dermal irritation index (PDII)

Remarks:

4 hours exposure

Basis:

mean

Remarks:

(three animals)

Time point:

24/48/72 h

Score:

0

Max. score:

8

Reversibility:

fully reversible

Irritation parameter:

erythema score

Remarks:

4 hours exposure

Basis:

mean

Remarks:

(three animals)

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Remarks:

4 hours exposure

Basis:

mean

Remarks:

(three animals)

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritant / corrosive response data:

At observation 1, 24, 48 and 72 hours after patch removal, there were no observed clinical signs noted on the skin of the treated animals. As no clinical signs were observed at 72 hours after patch removal, the study was terminated after the 72 hours observation.

Other effects:

No mortality was observed during the study. There was no test item related effect on body weight. There were no test item related general clinical signs noted.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of this study, praseodymium trinitrate does not require classification as skin irritant.

Reference 3

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012-11-20 - 2012-11-26

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)

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)

Test system:

human skin model

Remarks:

Human-derived epidermal keratinocytes

Source species:

human

Cell type:

non-transformed keratinocytes

Details on test system:

The EPISKIN model kit was purchased from SkinEthic Laboratories, Lyon, France, received 20 November 2012.
The EPISKIN model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg of ground-up test substance was applied to tissues moistened with 5 µL sterile distilled water

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Triplicate exposures of EPISKIN tissues to test item, positive control and negative control.

Details on study design:

Pre-incubation (day 0: tissue arrival)
On arrival of the tissue, 2 ml of maintenance medium, warmed to approximately 37°C, was pipetted into the first column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37°C, 5% C02 in air overnight.

Application of test item and rinsing (day 1)
2 ml of maintenance medium, warmed to approximately 37°C, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. Approximately 10 mg of the test item was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 µl of sterile distilled water to improve contact between the solid test item and the epidermis. Triplicate tissues treated with 10 µl of DPBS served as the negative controls and triplicate tissues treated with 10 µI of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After 7 minutes contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period. The plate(s) were kept in the biological safety cabinet at room temperature for 15 minutes. At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37°C, 5% C02 in air for 42 hours.

MTT Loading/Formazan Extraction (day 3)
Following the 42-hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenise the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30°C for possible inflammatory mediator determination. 2 mL of a 0.3 mg/ml MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide solution), freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12 well plate(s). The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% C02 in air. At the end of the 3-hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN™ biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT -loaded tissues.

Absorbance/optical density measurements (day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution. For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as 'blanks'. The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

MTT reducing capacity:
10 mg of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37°C, 5% C02 in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test item turns blue, the test item is presumed to have reduced the MTT. No color change was observed.

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

test item after 15-min exposure

Value:

94.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

The relative mean tissue viability for the positive control treated tissues was 5.5% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.2%. The positive control acceptance criterion was therefore satisfied. The mean OD540 for the negative control treated tissues was 0.745 and the standard deviation value of the percentage viability was 1.0%. The negative control acceptance criterion was therefore satisfied. The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 6.0 %. The test item acceptance criterion was therefore satisfied.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be a non-irritant (>50% relative mean tissue viability percentage of negative control).

Reference 4

Endpoint:

skin irritation / corrosion, other

Remarks:

other: Determination of pH-value and acidic reserve

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012-06-12

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

Well documented determination of pH value and acidic reserve according to accepted scientific methods.

Principles of method if other than guideline:

Electrochemic determination of pH
Determination of the acidic reserve via titration

GLP compliance:

no

Test system:

other: Not applicable

Species:

other: not applicable

Strain:

other: Not applicable

Details on test animals or test system and environmental conditions:

Not applicable

Type of coverage:

other: Not applicable

Preparation of test site:

other: Not applicable

Vehicle:

not specified

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 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
Acid reserve = titration volume [mL] x 0.4

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

Remarks on result:

other: The substance is considered not to be corrosive

Remarks on result:

other: The substance is considered not to be corrosive

Irritant / corrosive response data:

Not corrosive (based on an acidic reserve of 0.424 and pH of 1.81, the calculation for corrosivity was 1.775 which is higher than -0.5.

A pH value of 1.81 was determined.

Interpretation of results:

other: not corrosive

Conclusions:

The pH value of a 10% solution of praseodymium trinitrate was determined to be 1.81 and the acidic reserve 0.424. Based on these data, the calculation for corrosivity was 1.775 (> -0.5). Therefore the substance was considered not to be corrosive.

Not corrosive (based on a pH of 1.81, which is higher than -0.5)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion:

Four studies are used in a 'Weight-of-Evidence' approach: one K2 test (Rhodia operations, 2012), two K1 in vitro studies (Warren, 2013) and one K1 acute dermal irritation study (Matting 2014).

The pH-value of a 10% praseodymium trinitrate solution was determined electrochemically at 20°C with a calibrated pH meter: a pH value of 1.81 was determined. In addition, the acidic reserve of the solution was determined via titration of a 1 mol/L NaOH solution at 20°C. The acidic reserve was determined to be 0.424. The calculation for corrosivity was 1.775 (> -0.5). Therefore the substance was considered not to be corrosive (Rhodia Operations, 2012).

An in vitro skin corrosion test in an EPISKIN reconstructed human epidermis model was performed according to OECD Guideline 431 and EU Method B.40 (Warren, 2013). Duplicate tissues were treated with 20 mg of the solid test item for exposure periods of 3, 60 and 240 minutes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for loading in a MTT solution ( (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide solution). After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 540 nm (OD540). Data were presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues after the 240 minutes exposure was 98.6% (>= 35, relative mean tissue viability percentage of the negative control criteria). Praseodymium trinitrate was considered to be non-corrosive to the skin.

An in vitro skin irritation test in an EPISKIN reconstructed human epidermis model was performed according to OECD Guideline 439 and EU Method B.46 (Warren, 2013). Ten mg of ground-up test substance was applied to tissues moistened with 5 µL sterile distilled water for 15 minutes. EPISKIN tissues were exposed in triplicate to test item, positive control and negative control. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96 -well plate. The optical density was measured at 540 nm. Data were presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues was 94.1% after the 15 -minute exposure period. Praseodymium trinitrate was considered to be non-irritating to the skin (>50% relative mean tissue viability percentage of negative control.

Following a sequential test strategy for the assessment of the irritant/corrosive properties of praseodymium trinitrate, an acute dermal irritation study was performed (Matting, 2014) with praseodymium trinitrate (hydrated form) in New Zealand White rabbits. The study was performed according to the OECD Guidelines for Testing of Chemicals No. 404, Commission Regulation (EC) No 440/2008, B.4, OPPTS Guideline 870.2500 and Good Laboratory Practice. Parameters monitored during this study included mortality, body weight measurements and clinical observations. The irritancy of the test item was evaluated according to the Draize scoring method. The animals were treated by topical semi-occlusive application of 0.5 g to the intact shaved flank of 3 young adult New Zealand White rabbits. Sufficient water to damp the material was used to ensure good contact with the skin. The untreated skin of each animal served as control. The duration of treatment was 4 hours. The scoring of skin reactions for erythema and oedema was performed 1, 24, 48 and 72 hours after removal of the dressing. The primary irritation index was calculated by totalling the mean cumulative scores at 24, 48 and 72 hours and then dividing by the number of data points. Additional general examinations were performed daily. There was no mortality during the observation period. There was no test item related effect on body weight. At observation 1, 24, 48 and 72 hours after patch removal, there were no observed clinical signs noted on the skin of the treated animals. As no signs of irritation were observed at 72 hours after patch removal, the study was terminated after 72 hours observation. The animals' individual mean scores (considering readings at 24, 48 and 72 hours after patch removal) for erythema and oedema were 0.00, 0.00 and 0.00, respectively. The primary irritation index (considering readings at 24, 48 and 72 hours after patch removal) was calculated as 0.00.

In conclusion, the evaluation of the results of the abovementioned studies in a 'weight-of-evidence' approach demonstrates that praseodymium trinitrate is not a dermal irritant/corrosive substance.

In addition two studies have been disregarded:

Dunn (1980) investigated the in vivo skin corrosion potential of a substance containing 70% praseodymium nitrate and 30% neodymium nitrate in a study performed according to a method equivalent to a procedure specified in Title 49, Code of Federal Regulation articles 173.240, Appendix A in New Zealand White rabbits. Patches were removed after 4 hours and scored for destruction or irreversible alteration of the skin. The test sites were again observed at 24 and 48 hours. The study considered the mixture to be non-corrosive. However, due to the lack of a scoring system, no conclusion can be drawn on the skin irritation/corrosion potential of the mixture or its constituents.

(Bradshaw, 2013) performed an in vivo acute dermal irritation study in rabbits with praseodymium trinitrate (hydrated form). In this test, the substance was applied in a semi-occlusive dressing at three locations on the back of one rabbit for respectively 3 min, 1 hour and 4 hours of exposure and for 4 hours in one site of a second animal and in one site of a third animal.

Although no deviations were reported in the study, differences in the reversibility/irreversibility of the skin reactions were observed between animals after the same exposure period, with no apparent reason for these differences. This study was considered unconclusive.

However, other reliable experimental data available on this substance showed that praseodymium trinitrate is neither irritant nor corrosive:

- Measurement of the pH of a 10% w/w aqueous preparation (Bradshaw, 2013): pH value of 4.0 immediately and 3.9 after 10 minutes.

- Other information: Episkin study according to OECD 431(Warren, 2013) showed that praseodymium trinitrate is non-corrosive; Episkin study according to OECD 439 (Warren, 2013) showed that praseodymium trinitrate is non-irritant and an in vivo skin irritation study according to OECD 404 (Matting, 2014) showed no skin irritation.

In addition, reliable experimental data from other 'heavy' Rare Earth nitrates show that these nitrate salts are not irritant/corrosive for the skin:

- Cerium trinitrate: not irritant to skin in rabbits (Shapiro, 1990 and Gonnet, 1980)

- Lanthanum trinitrate: not irritant to skin in rabbits (Bradshaw, 2013)

Based on all the abovementioned information ('weight-of-evidence' approach), it can be concluded that the results of the study of Bradshaw (2013) are not conclusive but equivocal and that there is sufficient evidence to disregarded the study.

Eye irritation:

On the basis of expert judgment and the available information on other Rare Earth nitrates (e.g. cerium trinitrate, lanthanum trinitrate, neodymium trinitrate), it is very probable that praseodymium trinitrate should be considered as a substance causing eye damage Cat 1 and needs a H318 hazard classification according to CLP. Considering animal welfare, no further testing to cover this endpoint is required.

Justification for classification or non-classification

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

On the basis of expert judgment and the available information on other Rare Earth nitrates (e.g. cerium trinitrate, lanthanum trinitrate, neodymium trinitrate), it is very probable that praseodymium trinitrate should be considered as a substance causing eye damage Cat 1 and needs a H318 hazard classification according to CLP.