Registration Dossier

Administrative data

Description of key information

Skin irritation/corrosion: Six studies are used to cover this endpoint in a 'weight-of-evidence' approach.
In a test (Krivograd, 2012), the pH value of the cerium ammonium trinitrate was determined to be 0.61 and the acidic reserve 26.29. The calculation for corrosivity was -1.58 (lower than -0.5). Therefore the substance was considered to be corrosive.
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). Cerium ammonium nitrate 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). Cerium ammonium nitrate was considered to be non-irritating to the skin.
Two non-GLP local tolerance studies were performed according to OECD guideline 404 (Guillot, 1984). In one, scored as Klimisch 3, definitive conclusion could not be drawn. In the second, scored K2, cerium ammonium nitrate was considered to be non-irritating to the skin.
Finally, a K1 acute dermal irritation study 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 (Bradshaw, 2013). Cerium ammonium nitrate was considered to be corrosive.
In conclusion, cerium ammonium nitrate is evaluated as corrosive to the skin (Cat 1C) according to CLP.
Eye irritation:
Guillot (1984) determined, in a K2 study, the eye irritation potential of cerium ammonium nitrate in New Zealand White rabbits according to OECD Guideline 405. Cerium ammonium nitrate was observed to be severely irritating to the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
from 5 March 2013 to 18 June 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Leicestershire, UK
- Age at study initiation: 12-20 weeks
- Weight at study initiation: 2.6 kg
- Housing: suspended cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23°C
- Humidity (%): 30-70%
- Air changes (per hr): at least 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12h light (6-18h); 12h dark (18-6h)

IN-LIFE DATES: From: 5 March 2013 To: 5 March 2013
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
water
Controls:
other: not needed
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g of the test item was used as supplied, moistened sufficiently with 0.5 mL of distilled water to achieve a paste.


Duration of treatment / exposure:
3 minutes
1 hour
4 hours
Observation period:
immediately after removal and approximately 1h later
Number of animals:
one male
Details on study design:
TEST SITE
- Area of exposure: 3 suitable sites on the back of the rabbit
- Type of wrap if used: 2.5 cm x 2.5 cm cotton gauze patch, secured with a strip of surgical adhesive tape. The trunk was wrapped in an elasticated corset for the duration of the exposure period.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): residual test item was removed by gentle swabbing with cotton wool soaked in distilled water
- Time after start of exposure: immediately

SCORING SYSTEM:
Erythema and eschar formation:
No erythema - 0
Very slight erythema (barely perceptible) - 1
Well-defined erythema - 2
Moderate to severe erythema - 3
Severe erythema (beff redness) to eschar formation preventing grading of erythema - 4

Edema formation:
No edema - 0
Very slight edema (barely perceptible) - 1
Slight edema (edges of area well defined by definite raising) - 2
Moderate edema (raised approximately 1 millimeter) - 3
Severe edema (raised more than 1 millimeter extending beyond the area of exposure) - 4
Irritation parameter:
overall irritation score
Remarks:
3 min exposure
Basis:
animal #1
Time point:
other: immediately and 1 hour
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
overall irritation score
Remarks:
1 hour exposure
Basis:
animal #1
Time point:
other: immediately and 1 hour
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
erythema score
Remarks:
4 hour exposure
Basis:
animal #1
Time point:
other: immediately
Score:
2
Max. score:
4
Reversibility:
not reversible
Remarks on result:
other: purple/green colored dermal necrosis, animal was killed for humane reasons after the observation
Irritation parameter:
edema score
Remarks:
4 hour exposure
Basis:
animal #1
Time point:
other: immediately
Score:
2
Max. score:
4
Reversibility:
not reversible
Remarks on result:
other: animal killed for humane reasons after the observation
Irritant / corrosive response data:
3-Minute and 1-Hour semi-occluded applications of the test item to the intact skin of one rabbit produced no evidence of skin irritation.

A single 4-Hour semi-occluded application of the test item to the intact skin of one rabbit produced: Well-defined erythema and slight oedema were noted at the test site immediately after patch removal. Purple/green colored dermal necrosis, approximately 2 cm x 2 cm in size and extending approximately 1 cm below and 1 cm to the rear of the test site, was also noted at the observation.

The skin reactions noted were considered to be irreversible and indicative of full thickness tissue destruction, therefore the animal was killed for humane reasons immediately after the observation, in accordance with Company policy and current UK Home Office guidelines.
Interpretation of results:
Category 1C (corrosive) based on GHS criteria
Conclusions:
The test item was classified as corrosive to rabbit skin (based on one animal only). The test item was classified as Category 1C according to the CLP Regulation 1272/2008.
Endpoint:
skin irritation: in vitro / ex vivo
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Between 16 October 2012 and 19 October 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Source strain:
other: not applicable
Vehicle:
other: 0.9% w/v sodium chloride solution added for wetting of the test item
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 EPISKIN TM 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.

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, 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 EPISKIN TM 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 following procedure: 20 mg of the test item was added to 2.2 mL of a 0.3 mg/mL 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. The MTT solution containing the test item did not turn blue. This was taken to indicate the test item did not reduce MTT.
Control samples:
yes, concurrent negative control
yes, concurrent positive 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 added for wetting of the test item.
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 100 μL of 0.9% w/v sodium chloride solution
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of glacial acetic acid
Duration of treatment / exposure:
3, 60 and 240 minutes
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
Duplicate exposures of EPISKIN TM tissues to test item, positive control and negative control.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-minute application
Value:
102.3
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1-hour application
Value:
95.8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
240-minute application
Value:
65.2
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Colour interference with MTT: The MTT solution containing the test item did not turn blue. This was taken to indicate the test item did not reduce MTT.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD540 for the negative control treated tissues was 0.791. The negative control acceptance criterion was therefore satisfied.
- Acceptance criteria met for positive control: 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.
Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be a non-corrosive (>= 35; relative mean tissue viability percentage of negative control criteria).
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
from 20 November 2012 to 26 November 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Source strain:
other: not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
The EPISKIN model kit was purchased from SkinEthic Laboratories, Lyon, France, received on 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.

Pre-Incubation (Day 0: tissue arrival)
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 µL 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, 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.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL: 10 mg of ground-up test substance was applied to tissues moistened with 5 µL sterile distilled water
NEGATIVE CONTROL: 10 µL of DPBS
POSITIVE CONTROL: 10 µL of SDS 5% w/v
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
Triplicate exposures of EPISKIN TM tissues to test item, positive control and negative control.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15-minute application
Value:
74.4
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Colour interference with MTT: The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: 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.
- Acceptance criteria met for positive control: 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.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 9.7%. 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).
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:
from 31-05-2012 to 14-06-2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Principles of method if other than guideline:
Electrochemic determination of pH
Determination of the acidic reserve via titration
The determination of the pH-value was in compliance with TIAG standard operating procedures AANC Determination of the pH value (2012).
GLP compliance:
no
Remarks:
This study was performed not GLP compliant, but does meet the TIAG standard procedures for physicochemical testing.
Species:
other: not applicable
Strain:
other: Not applicable
Details on test animals 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:
Preparation of test item:
At first, a 10% solution of the test item was prepared by stirring 10 g sample material with 90g H2O dem. (Milli-Q water -
Type 1 water grade) for 30 minutes and then adjusting it to a temperature of 20°C.

Calibration of the pH-meter:
Prior to the measurement the pH meter was checked and calibrated with a buffer solution in the expected measurement
range. The pH meter was calibrated via a 3-point calibration. The slope of the electrode and the result of the control buffer
were either listed in the "Prüfmittelkontrollblatt" or "calibration report". The electrode was rinsed with H2O dem., wiped dry
and immersed for measurement into the solution.

Determination of the pH-value of the test item
The pH is determined 3 times from a 10% solution of the test item under continuous stirring. The measured pH was
protocolled.


Apparatus and auxiliary material:
- Analytical scales with an accuracy of 0.01g
- pH-meter with pH electrode or 809 Titrando v. Metrohm
- Buffer solutions 1.09, 4.01, 6.00
- Magnetic stirrer with magnetic stir bar
- Beakers
- Flask
- Water bath
- Pipettes
- Milli-Q water

Determination of the acid reserve by titration:
For the determination of the acid reserve the titration volume (in mL) of a 1 mol/L NaOH, which is required to achieve a pH of 4, was determined for each test item.

Calculation of the acid reserve:
Acid reserve = titration volume [mL] x 0.4

If the pH minus 1/12 acid reserve <= -0.5 then the test item is considered as corrosive.
Remarks on result:
other: The substance is considered to be corrosive
Irritant / corrosive response data:
Corrosive

A pH value of 0.61 was determined.

The calculated acid reserve (arithmetic mean of 3 replicate measurements) was 26.29. The pH minus 1/12 acid reserve was -1.58 (<= -0.5). Thus cerium ammonium nitrate is considered as corrosive.

Interpretation of results:
Category 1 (corrosive) based on GHS criteria
Conclusions:
The pH value was determined to be 0.61 and the acidic reserve 26.29. The calculation for corrosivity was -1.58 (lower than -0.5). Therefore the substance was considered to be corrosive.
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 16 May 1984 to 19 May 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: several accredited breeders supply animals to the Hazleton laboratories: Charles River, E.G.A.V. and Elevage Scientifique des Dombes
- Age at study initiation: no data
- Weight at study initiation: 2.5 kg ± 200g
- Housing: Individual polystyrene cages, measuring 540 x 360 x 315 mm with a perforated polystyrene floor in a ventilated and air conditioned room.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C ± 3 °C throughout the year
- Humidity (%): 30 - 70%
- Air changes (per hr): 12 to 14 times per hour (pre-filtered air: 5 - 10 µ)
- Photoperiod (hrs dark / hrs light): lighting is artificial for 12 hours per day
Type of coverage:
semiocclusive
Preparation of test site:
shaved
Vehicle:
water
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g. Test material was mixed with 0.05 ml water before exposure
Duration of treatment / exposure:
4 hours
Observation period:
The cutaneous primary irritation is evaluated 1 hour after removal of the semi-occlusive patch and of hydrophilic gauze pads, and again 24, 48 and 72 hours later.

Number of animals:
3 rabbits

Details on study design:
TEST SITE
- Area of exposure: the flanks of the animal: ± 6 cm², recovered with a Codex hydrophilic eight layer gauze pad of about 2.5 cm².
- Type of wrap if used: The test substance and gauze pad are held in contact with the skin with a semi-occlusive patch: 10 cm wide adhesive perforated tape applied on a crimped gauze bandage covering more particularly the whole clipped surface (to avoid possible orthoergic reactions) and wrapped around the animal (without blocking the respiratory and abdominal movements).

REMOVAL OF TEST SUBSTANCE
- After 4 hours of exposure, the bandagings are removed, and if necessary the excess of the test substance which has not penetrated is wiped with a gauze pad moistened with deionized water (or a non-irritant solvent).

SCORING SYSTEM:
Draize scoring
Irritation parameter:
erythema score
Basis:
mean
Time point:
other: 24, 48, 72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible within: 24h
Irritation parameter:
edema score
Basis:
mean
Time point:
other: 24, 48, 72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritant / corrosive response data:
After 1h, two animals showed slight erythema which was not present anymore after 24h. No other effects were noted.

The red colour of the substance made the erythema observation imprecise. The surplus substance that didn't penetrate into the skin was removed with a gauze and water. The reading was done one hour later.

Interpretation of results:
GHS criteria not met
Conclusions:
After 4 hours exposure, cerium ammonium nitrate was considered to be non irritating to the skin. No classification is warranted.
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
18 April 1984
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Well documented non GLP study performed according to OECD Guideline 404.
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: several accredited breeders supply animals to the Hazleton laboratories: Charles River, E.G.A.V. and Elevage Scientifique des Dombes
- Age at study initiation: no data
- Weight at study initiation: 2.5 kg ± 200 g
- Housing: individual polystyrene cages, measuring 540 x 360 x 315 mm with a perforated polystyrene floor in a ventilated and air conditioned room.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C ± 3 °C throughout the year
- Humidity (%): 30 - 70%
- Air changes (per hr): 12 to 14 times per hour (pre-filtered air: 5 - 10 µ)
- Photoperiod (hrs dark / hrs light): lighting is artificial for 12 hours per day


Type of coverage:
semiocclusive
Preparation of test site:
shaved
Vehicle:
water
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g. Test material was mixed with 0.05 ml water before exposure.


Duration of treatment / exposure:
4 hours

Observation period:
The cutaneous primary irritation is evaluated 1 hour after the removal of the semi-occlusive patch and of hydrophilic gauze pads (i.e. 5 hours after the application of the test substance), and again 24, 48 and 72 hours later. The observation period was extended until 14 days in order to show an eventual corrosive action of the test substance, characterized by the irreversibility of the lesions observed.
Number of animals:
6 rabbits

Details on study design:
TEST SITE
- Area of exposure: the flanks of the animal: ± 6 cm2, recovered with a Codex hydrophilic eight layer gauze pad of about 2.5 cm square.
- Type of wrap if used: The test substance and gauze pad are held in contact with the skin with a semi-occlusive patch: 10 cm wide adhesive perforated tape applied on a crimped gauze bandage covering more particularly the whole clipped surface (to avoid possible orthoergic reactions) and wrapped around the animal (without blocking the respiratory and abdominal movements).

REMOVAL OF TEST SUBSTANCE
- After 4 hours of exposure, the bandagings are removed, and if necessary the excess of the test substance which has not penetrated is wiped with a gauze pad moistened with deionized water (or a non-irritant solvent). The animals are then returned to their individual cage.

SCORING SYSTEM:
Draize scoring
Irritation parameter:
erythema score
Basis:
other: mean (of 6 animals)
Time point:
other: 24, 48, 72 hours
Score:
1
Max. score:
4
Reversibility:
not fully reversible within: 14 days
Irritation parameter:
edema score
Basis:
mean
Time point:
other: 24, 48, 72 hours
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritant / corrosive response data:
One animal showed significant effects: already after 1h half of the test surface showed signs of burns, which recovered after 7 days and 14 days, although severe erythema was still observed. Four animals showed slight erythema which was fully reversible within 72h. One animal did not show any adverse skin effects.

The red colour of the substance made the erythema observation imprecise. The surplus substance that didn't penetrate into the skin was removed with a gauze and water. The reading was done one hour later.

Interpretation of results:
study cannot be used for classification
Conclusions:
Under the conditions of the test, the substance was found to be slightly irritating in 5 of the 6 animals. One animal showed significant skin lesions though. No definite conclusion can be drawn on the classification and labeling for skin irritation/corrosion. A second test has been performed.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 April 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: several accredited breeders supply animals to the Hazleton laboratories: Charles River, E.G.A.V. and Elevage Scientifique des Dombes
- Age at study initiation: no data
- Weight at study initiation: 2.5 kg ± 200 g
- Housing: individual polystyrene cages, measuring 540 x 360 x 315 mm with a perforated polystyrene floor in a ventilated and air conditioned room.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 8 days to allow time for acclimatization

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C ± 3 °C throughout the year
- Humidity (%): 30 - 70%
- Air changes (per hr): 12 to 14 times per hour (pre-filtered air: 5 - 10 µ)
- Photoperiod (hrs dark / hrs light): lighting is artificial for 12 hours per day
Vehicle:
unchanged (no vehicle)
Controls:
other: The left eye served as control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg
Duration of treatment / exposure:
The upper and lower eyelids are held together for ten seconds after administration to avoid any loss of test substance.
3 rabbits: wash-out after 4 seconds
3 other rabbits: wash-out after 30 seconds
Observation period (in vivo):
The examinations are always carried out under the same conditions in particular for the lighting. They are made by comparison with the control eye, 1hour after the instillation, and then 24, 48 and 72 hours later. For evaluating the reversibility or irreversibility of the lesions the observation period was prolonged for 7 days.
Number of animals or in vitro replicates:
6 rabbits
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): The eyes of 3 rabbits are washed out 4 seconds after the instillation, care being taken not to cause injury, and on the 3 other animals 30 seconds after the pplication of the test substance. About 50 mL of the rinsing solution at about 20°C is administered using a plastic jet at moderate pressure. The control eye is washed out under the same conditions as the treated one. The excess of liquid is immediately wiped away with a Codex hydrophilic gauze pad.

SCORING SYSTEM: Draize

TOOL USED TO ASSESS SCORE: hand-slit lamp / Heine's ophtalmoscope
Irritation parameter:
iris score
Basis:
mean
Time point:
other: 24, 48, 72 hours
Score:
2
Max. score:
2
Reversibility:
not fully reversible within: 7 days
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
other: 24, 48, 72 hours
Score:
4
Max. score:
4
Reversibility:
not fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
other: 24,48, 72 hours
Score:
3.94
Max. score:
4
Reversibility:
not fully reversible within: 7 days
Remarks on result:
other: chemosis
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
other: 24, 48, 72 hours
Score:
1.89
Max. score:
3
Reversibility:
not fully reversible within: 7 days
Remarks on result:
other: redness
Irritant / corrosive response data:
On day 7 total necrosis of the eye was observed with loss of the vitreous humor of the eye in 3 of 6 rabbits. In the other 3 rabbits, the observed lesions were not reversible.
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Under the conditions of the test the substance was observed to be severely irritating. Based on the criteria of the CLP Regulation, the substance is classified as Irreversible effects on the eye category 1.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion:

Six studies are used in a 'Weight-of-Evidence' approach: one K2 test (Krivograd, 2012), two K1 in vitro studies (Warren, 2013), two in vivo non-GLP studies (K3 and K2) by Guillot (1984) and one K1 acute dermal irritation study (Bradshaw, 2013).

The pH-value of a 10% solution of cerium ammonium trinitrate was determined electrochemically (Krivograd, 2012) in a K2 test. The solution

was prepared by stirring 10g sample material with 90g H2O demineralised (Milli-Q water - Type 1 water grade) during 30 minutes and then adjusting it to a temperature of 20°C. Prior to the measurement, the pH-meter was checked and calibrated with a buffer solution in the expected measurement range. The pH-meter was calibrated via a 3-point calibration. The pH was determined 3 times from the 10% solution of the test item under continuous stirring

The measured pH-value for cerium ammonium nitrate was 0.61. Therefore, the determination of the acid reserve was necessary. The calculated acid reserve (arithmetic mean of 3 replicate measurements) was 26.29. The pH minus 1/12 acid reserve was -1.58 (<= -0.5). Thus cerium ammonium nitrate was considered as corrosive.

In an in vitro skin corrosion test using the EPISKIN TM reconstructed human epidermis model (K1 study), cerium ammonium nitrate was tested according to the OECD 431 test guideline and EU Method B40 (Warren, 2013a). In this test, 20 mg of ground-up test substance was applied to tissues for 3, 60 and 240 minutes, with 100 µL 0.9% w/v sodium chloride solution added for wetting of the test item. The relative mean viability of the test item treated tissues after the 240 minutes exposure was 65.2%. The test item was considered to be non-corrosive.

In an in vitro skin irritation test using the EPISKIN™ reconstructed human epidermis model (K1 study), cerium ammonium nitrate was tested according to the OECD 439 test guideline and the EU method B.46 (Warren, 2013b). In this test, 10 mg of ground-up test substance was applied to tissues, moistened with 5 µL sterile distilled water. 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. The relative mean viability of the test item treated tissues was 74.7% after a 15-minute exposure period. The test item was considered to be a non-irritant.

Guillot (1984) performed two skin irritation tests in New Zealand White rabbits according to OECD Guideline 404. In the first test (K3) cerium ammonium nitrate was observed to be slightly irritating, however no definite conclusion could be drawn on the classification and labeling for skin irritation/corrosion. In the second test (K2), cerium ammonium nitrate was considered to be non-irritating to the skin. Although both studies were well performed, the results were contradictory.

More recently, an in vivo acute dermal irritation study (K1) in rabbits was performed (Bradshaw, 2013) according to OECD guideline 404 and EU method B.4 and in compliance of GLP regulations. In this test, the substance was applied in a semi-occlusive dressing at three locations on the back of a single rabbit for respectively 3 min, 1 hour and 4 hours of exposure. Both the 3 min and 1 hour exposure did not produce evidence of skin irritation. However, a single 4 hour application produced well-defined erythema, slight edema and purple/green colored dermal necrosis. The skin reactions noted were considered to be irreversible and indicative of full thickness tissue destruction. The test item was classified as skin corrosive category 1C according to the criteria of the CLP Regulation EC No 1272/2008.

In conclusion, cerium ammonium nitrate is considered corrosive.

Eye irritation:

In the study by Guillot (1984) 6 rabbits were instilled 0.1 g of test substance in one eye, while the other eye served as control. The examinations have been carried out under the same conditions in particular for the lighting. They are made by comparison with the control eye, 1 hour after the instillation, and then 24, 48 and 72 hours later. For evaluating the reversibility or irreversibility of the lesions the observation period was prolonged for 7 days. On day 7 total necrosis of the eye was observed with loss of the vitreous humor of the eye in 3 of 6 rabbits. In the other 3 rabbits, the observed lesions were not reversible.

Justification for classification or non-classification

In an in vivo acute dermal irritation study in rabbits, a single 4-hour application produced well-defined erythema, slight oedema and grey/green colored dermal necrosis. The skin reactions noted were considered to be irreversible and indicative of full thickness tissue destruction. The test item was classified as corrosive category 1C (H314: causes severe skin burns and eye damage) according to the criteria of the CLP Regulation EC No 1272/2008.

Guillot (1984) determined in a K2 study the eye irritation potential of cerium ammonium nitrate in New Zealand White rabbits according to OECD Guideline 405. Cerium ammonium nitrate was observed to be severely irritating to the eyes. Therefore, the substance is classified for eye damage category 1 according to CLP regulation.