Registration Dossier

Administrative data

Description of key information

The skin sensitisation potential of cerium ammonium nitrate was evaluated using the LLNA method (OECD 429 and EU Method B42). The substance was tested at 25%, 10% and 5% (w/w) concentrations. Based on the results of this study (Henzell, 2013) the test result was considered ambiguous and then disregarded. Therefore, a Guinea pig maximisation test (GPMT) following OECD guideline 406, EU Method B.6 and US EPA OPPTS 870.2600 was performed. The result of this test (Török-Bathó, 2013) concluded that this substance is clearly a skin sensitiser. Therefore the substance is classified as a skin sensitiser (Category 1A).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
from 26 February 2013 to 02 May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Minor variations from the target relative humidity and temperature ranges were observed during the study. These deviations were considered to have no impact on the animal health and in the study.
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
An LLNA test was performed first. However, the results of the LLNA were considered ambiguous (expert judgement) and, on this basis, sensitization could not be confirmed (for more info see WoE_K2_Skin sensitisation study of Henzell, 2013). It was decided to perform an GPMT to be able to conclude on the skin sensitization potential.
Species:
guinea pig
Strain:
other: LAL/HA/BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: LAB-ÁLL Bt. Budapest, 1174 Hunyadi u. 7.
- Age at study initiation: Young adult, 5-6 weeks
- Weight at study initiation: 324 – 398 g
- Housing: macrolon cages size IV with laboratory bedding. 5 animals/cage to allow socialization.
- Diet (e.g. ad libitum): CuniFort Diet for Rabbits (Bonafarm-Bábolna Takarmány Ltd.) ad libitum. This diet is classified as being suitable for guinea pigs and used by the breeder/supplier, animals were fully adapted to this diet on arrival.
- Water (e.g. ad libitum): Animals received tap water from municipal supply containing 50 mg/100 mL ascorbic acid, ad libitum.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.0 – 23.5 °C
- Humidity (%): 22 – 52%
- Air changes (per hr): 15-20 air exchange/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily from 6 a.m. to 6 p.m. (artificial light)

IN-LIFE DATES: From: 27 February 2013 To: 16 April 2013
Route:
intradermal and epicutaneous
Vehicle:
physiological saline
Remarks:
(NaCl 0.9%)
Concentration / amount:
MAIN STUDY
- Intra-dermal induction exposure: 0.1% (w/v)
- Dermal induction exposure: 100% (w/v)
- Challenge exposure: 25% (w/v) as highest non-irritant concentration; 12.5% (w/v) as safeguard concentration
- Re-challenge exposure: 25% (w/v) as highest non-irritant concentration; 12.5% (w/v) as safeguard concentration
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Remarks:
(NaCl 0.9%)
Concentration / amount:
MAIN STUDY
- Intra-dermal induction exposure: 0.1% (w/v)
- Dermal induction exposure: 100% (w/v)
- Challenge exposure: 25% (w/v) as highest non-irritant concentration; 12.5% (w/v) as safeguard concentration
- Re-challenge exposure: 25% (w/v) as highest non-irritant concentration; 12.5% (w/v) as safeguard concentration
No. of animals per dose:
MAIN STUDY
- Intra-dermal induction exposure: 10 in control group and 20 in test group. The same animals were used for the dermal induction exposure and the challenge exposure.
- Re-challenge exposure: the same test animals with the same control animals and 10 naive control animals
Details on study design:
RANGE FINDING TESTS:
- Intra-dermal injection: 0.01, 0.1, 0.5, 1, 2.5 and 5% (w/v) concentrations were used for intra-dermal injection. 0.1 mL was injected per concentrationinto the hair free skin of the flanks. Each concentration was injected in duplicate. Local effects (for erythema and oedema, any other observations of changes to the skin) were examined and scored 1, 24, 48 and 72 hours after treatment.

- Dermal application: 25, 50, 75 and 100% (w/v) concentrations were used for dermal application. Two animals were used per concentration. 0.5 mL was applied onto the clipped and shaved skin of the animals per concentration (occlusive exposure). Time of exposure was 24 hours. Skin effects were scored for erythema and oedema, any other observations of changes to the skin were recorded. Local effects were examined and scored 1, 24, 48 and 72 hours after patch removal.

- Control animals were treated with saline.

MAIN STUDY
A. INDUCTION EXPOSURE

- No. of exposures:
Intra-dermal: A series of three injections was administered on each side of the scapular region of treatment group animals, as follows, resulting in six injections per animal:
2 injections with 0.1 mL of Freund's Complete Adjuvant mixed with physiological saline (1:1) (v/v),
2 injections with 0.10 mL of the test item in saline solution at 0.1% (w/v) concentration,
2 injections with 0.1 mL of test item in 0.1% (w/v), formulated in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

Dermal: the same inter-scapular region which received the intradermal injections, were used for dermal induction exposure. One dermal application was used.

- Exposure period: time of exposure is not applicable for intra-dermal injection; dermal induction exposure period was 48 hours.

- Test groups: test item in saline

- Control group: The control animals were treated similarly as the test group. However, the vehicle without the test item was used for injections.

- Site: scapular region

- Frequency of applications: not applicable

- Duration: 0-8 d

- Concentrations: same throughtout

B. CHALLENGE EXPOSURE

- No. of exposures: one

- Day(s) of challenge: two weeks after the topical induction application, the animals were exposed to a dermal challenge dose (day 22 of treatment)

- Exposure period: 24 hours

- Test groups: 0.5 mL of the test item at 25% (w/v) concentration in saline (highest non-irritant dose) was applied to the left flank of 10 test animals. . The remaining 10 animals were treated with approximately 0.5 mL of the 50 % dilution of the maximum dermal challenge dose (12.5% (w/v) in saline as a safeguard dose) to the left flank. The right shaved flank area of all animals was treated with saline.

- Control group: 0.5 mL of the test item at 25% (w/v) concentration in saline (highest non-irritant dose) was applied to the left flank of 5 control animals. . The remaining 5 animals were treated with approximately 0.5 mL of the 50 % dilution of the maximum dermal challenge dose (12.5% (w/v) in saline as a safeguard dose) to the left flank. The right shaved flank area of all animals was treated with saline.

- Site: left and right flank

- Concentrations: 25% (w/v) as highest non-irritant concentration; 12.5% (w/v) as safeguard concentration

- Evaluation (hr after challenge): 24 and 48 hours after the patch removal.

OTHER:
Time of observations after intra-dermal induction: 24 hours after treatment
Time of observations after dermal induction: 1, 24, 48 and 72 hours after the patch removal
Challenge controls:
No naive control group was used for the challenge exposure but for the re-challenge exposure.
Positive control substance(s):
no
Remarks:
The sensitivity and reliability of the experimental procedure is assessed twice a year by use of items which are known to have moderate skin sensitisation properties such as 2-Mercaptobenzothiazole.
Positive control results:
The sensitivity and reliability of the experimental procedure is assessed twice a year by use of items which are known to have moderate skin sensitisation properties such as 2-Mercaptobenzothiazole. On the basis of the results of the most recent reliability check study (start of experiment: 28 November 2012, date of report: 21 January 2013), the reference item 2-Mercaptobenzothiazole was classified as a skin sensitizer. This demonstrated that the experimental procedure and the test system were appropriate.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25 % (w/v)
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
No
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25 % (w/v). No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: No.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25 % (w/v)
No. with + reactions:
7
Total no. in group:
10
Clinical observations:
No
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25 % (w/v). No with. + reactions: 7.0. Total no. in groups: 10.0. Clinical observations: No.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25 % (w/v)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 25 % (w/v). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25 % (w/v)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25 % (w/v). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
12.5 % (w/v)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 12.5 % (w/v). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
12.5 % (w/v)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 12.5 % (w/v). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
12.5 % (w/v)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 12.5 % (w/v). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
12.5 % (w/v)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 12.5 % (w/v). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No.

Body weight : there were no notable differences between the test animal group and the control group.

Mortality: there were no moribund or dead animals during the study.

Clinical observations: no clinical response to treatment were observed in either the test group or in the control group during the course of the study.

Range finding test

- Intra-dermal application: the test item at concentrations of 0.5, 1, 2.5 and 5% (w/v) produced focal necrosis from 24 hours after the treatment; therefore these concentrations were not acceptable for the main study. Very slight erythema (score 1) was also observed at 1, 24 and 48 hours after the treatment at the treatment site away from the necrotic foci at concentrations of 1, 2.5 and 5% (w/v). Concentrations of 0.1 and 0.01% (w/v) caused no reaction (scores 0-0 for erythema and oedema) in the skin of guinea pigs.

- Dermal application: It was found that 0.5 ml of the test item formulations at concentrations of 100, 75 and 50% (w/v) caused erythema (score 1 or 2) at 1, 24, 48 or 72 hours after the patch removal. Dry and scaly skin was seen 48 and 72 hours after the patch removal on the treated area at concentrations of 100, 75 and 50% (w/v). Concentration of 25% (w/v) caused barely perceptible erythema (score 1) only 1 hour after the treatment in one animal.

- Re-challenge test group results:

Re-challenge with the test item at a concentration of 25% (w/v) in saline elicited discrete and/or moderate erythema (scores 1 or 2) on the skin surface of previously induced guinea pigs. Dry skin was observed in 6/10 animals at the 48 hour observation. The net response value represented an incidence rate of 100% and 60% and the net score value of 1.30 and 0.60 according to the 24 and 48 hours observations.

Following re-challenge with 12.5% (w/v), discrete erythema (score 1) was observed in 2 animals at 24 hours. No further observation was detected. The net response value represented an incidence rate of 20% and 0% and the net score value of 0.20 and 0.00 according to the 24 and 48 hours observations.

- Re-challenge control group results:

Re-challenge with the test item at a concentration of 25 and 12.5% (w/v), the mean of the scores was 0.00 according to the 24 and 48-hours results in the control animals. Dry and scaly skin was seen in one originally appointed control animal skin on the treated site at both doses.

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
Under the conditions of the present assay the test item cerium ammonium nitrate was shown to have a sensitisation potential and is classified as a sensitizer, according to current EU regulations (Commission Regulation (EC) No 440/2008 of 30 May 2008 method B.6). According to the to current Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, amending Regulation (EC) No 1907/2006 and UN Globally Harmonised System of Classification and Labelling of Chemicals, cerium ammonium nitrate requires classification as a skin sensitizer (Category 1A).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Skin sensitisation:

The potential for skin sensitisation of cerium ammonium nitrate was evaluated using the local lymph node assay in the mouse (Henzell, 2013) evaluated the potential for skin sensitisation of cerium ammonium nitrate using the local lymph node assay (LLNA) in the mouse according to OECD 429 guideline and EU method B.42. Following the results of a preliminary study, three groups, each of four animals, were treated with 25 µL of the test item to dorsal ear surface as a suspension in ethanol/water 7:3 at concentrations of 5, 10 and 25% w/w. An additional group of four animals was treated only with the vehicle (negative control). A concurrent positive control group, using four animals, was also tested with hexyl cinnamic aldehyde (15% in vehicle). A stimulation index of 6.13 was obtained at a test concentration of 25% suggesting a sensitisation response. However irritation potential was noted in pre-test and main test animals. Concentrations of 5% and 10% gave an stimulation index of < 3. The results of this study were considered therefore ambiguous (expert judgment) and, on this basis, sensitization could not be confirmed.

This study has been scored Klimish 2 to reflect the ambiguity of the results (expert judgment) and the use of this study in a 'weight-of-evidence' approach according to the ECHA Practical guidance 2 'How to report weight of evidence'. Thus, the guideline indicates that a 'weight-of-evidence' approach should be used when several studies are available which give conflicting results.

A similar ambiguity has been observed in reliable experimental data from other 'heavy' rare earth metal compounds:

- Cerium trichloride: ambiguous in LLNA but clearly no sensitiser to the skin in a GPMT test.

- Lanthanum trinitrate: ambiguous in LLNA but clearly no sensitiser to the skin in a GPMT test.

In order to clarify the skin sensitisation potential of cerium ammonium nitrate, a Guinea Pig Maximisation test (Török-Bathó M, 2013) was performed according to the OECD guideline 406, EU Method B.6 and EPA OPPTS 870.2600. Cerium ammonium trinitrate, formulated in saline solution, was applied to 20 animals in the induction exposure phase (intra-dermal and dermal exposure). The dose for the intra-dermal injection was 0.1 % (w/v) and 100 % (w/v) for the dermal occlusive exposure. 10 animals were dosed with the saline only (control group). Fourteen days later, ten treated animals and five control animals were treated with 0.5 mL of the test item at a concentration of 25% (w/v). Remaining ten tested animals and five control animals were treated with 0.5 mL of the test item at a concentration of 12.5% (w/v) as safeguard dose. There were no dead animals or adverse clinical responses, related to treatment, observed in either the test group or in the control group. There were no notable differences in bodyweights between the test animal group and the control group.

The net response value after the challenge exposure of the test group animals at 25% (w/v) represented an incidence rate of 100% and 70% at 24 and 48 hours observation time, respectively. The mean of the dermal scores were 1.40 and 0.70 at 24 and 48 hour, respectively. Dry, cracked skin was detected in all animals. Following challenge exposure with 12.5% (w/v) of the treated animals (test group), there were no skin reactions in the test animals. The net response value represented an incidence rate of 0% and the net score value of 0.00 and 0.00 at 24 and 48 hours, respectivelly. Following challenge exposure with the test item of control animals at a concentration of 25 and 12.5% (w/v), the net response value represented an incidence rate of 0% and the net score value of 0.00 and 0.00 at 24 and 48 hours, respectivelly. To confirm the results obtained in the first challenge, a second challenge (re-challenge) was performed 7 days after the last scoring of the first challenge. The same test animals with the same control animals and 10 naive control animals were treated on the right flank with the test item with the challenge or the safeguard dose and with vehicle on the left flank, in the same manner as that described in the first challenge. Dry skin was observed in 6/10 animals at the 48 hour observation after re-challenge with the test item at a concentration of 25% (w/v) in saline. The net response value represented an incidence rate of 100% and 60% and the net score value of 1.30 and 0.60 at the 24 and 48 hours observation time-point, respectivelly.No further observation was detected following re-challenge with 12.5% (w/v) of test group animals. The net response value represented an incidence rate of 20% and 0% and the net score value of 0.20 and 0.00 at 24 and 48 hours observation time-point, respectivelly.The mean of the scores after re-challenge with the test item at a concentration of 25 and 12.5% (w/v) of control animals was

0.00 at 24 and 48-hours. Dry and scaly skin was seen in one originally appointed control animal skin on the treated site at both doses.

The re-challenge confirmed the results obtained in the challenge exposure.

On the basis of the results of this study, cerium ammonium trinitrate is a skin sensitiser and should be classified according to CLP regulations.


Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results of the in vivo skin sensititsation test (Guinea pig maximisation test) and according to the criteria of the CLP Regulation, cerium ammonium nitrate should be classified as a skin sensitiser (Category 1A).