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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:
27 February 2013 - 30 April 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well performed study following the OECD guideline 406, EU Method B.6 and US EPA OPPTS 870.2600 with no major deviations. 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
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
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 weeks
- Weight at study initiation: 274 – 379 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 – 23.2 °C
- Humidity (%): 22 – 47%
- 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: 07 April 2013
Route:
intradermal and epicutaneous
Vehicle:
physiological saline
Remarks:
NaCl 0.9%
Concentration / amount:
MAIN STUDY
- Intra-dermal induction exposure: 0.01% (w/v)
- Dermal induction exposure: 100% (w/v)
- Challenge exposure: 100% (w/v) as highest non-irritant concentration; 50% (w/v) as safeguard concentration
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Remarks:
NaCl 0.9%
Concentration / amount:
MAIN STUDY
- Intra-dermal induction exposure: 0.01% (w/v)
- Dermal induction exposure: 100% (w/v)
- Challenge exposure: 100% (w/v) as highest non-irritant concentration; 50% (w/v) as safeguard concentration
No. of animals per dose:
MAIN STUDY
- Intra-dermal and dermal induction exposure: 10 in control group and 20 in test group.
- Challenge exposure:
Control group: 5 (Left flank) - 5 (Right flank)
Test group: 10 (Left flank: 100 % test substance) - 10 (Right flank: 50 % test substance)
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 concentration into 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 0.1 mL of the test item in saline solution at 0.01% (w/v) concentration,
2 injections with 0.1 mL of test item in 0.01% (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 throughout

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 100 % (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 (50 % (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 100 % (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 (50 % (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: 100 % (w/v) as highest non-irritant concentration; 50 % (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.
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 chemical
Dose level:
100 % (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: 100 % (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 chemical
Dose level:
100 % (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: 100 % (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:
100 % (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: 100 % (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:
100 % (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: 100 % (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 chemical
Dose level:
50% (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: 50% (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 chemical
Dose level:
50% (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: 50% (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:
50% (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: 50% (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:
50% (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: 50% (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.

Local / Clinical observations: During the induction period very slight erythema was observed in all test item treated animals, 1 hour after the patch removal. No further local skin effects were observed in either the test group or in the control group. No clinical signs were noted during the study.

Range finding test

- Intra-dermal application: the test item at concentrations of 0.1, 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 48 hours after the treatment at the treatment site away from the necrotic foci at concentrations of 0.5, 1, 2.5 and 5% (w/v). The concentration of 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 very slight erythema (score 1) at 1 hour after patch removal. At concentration of 50 % (w/v) and 24 hours after the patch removal, barely perceptible erythema (score 1) was observed in one of two animal but 0/2 animals at the higher 100% application; this observation was considered to be an individual response and not a clear treatment related response. Concentration of 25% (w/v) produced no reaction (scores 0-0) on the skin of guinea pigs.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the present assay the test item Cerium trichloride was shown to have no sensitisation potential and classified as a non-sensitizer, according to current EU-regulations.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Skin sensitisation:

The potential for skin sensitisation of cerium trichloride was evaluated using the local lymph node assay in the mouse (Henzell G, 2013) 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 the test item to dorsal ear surface as a suspension in ethanol/water 7:3 at concentrations of 10, 25 and 50% 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). At concentrations of 50% and 25%, the stimulation indexes were only slightly higher than the three-fold increase in 3HTdR incorporation compared to the control values (4.29 and 4.44 respectively). It was also observed that the test item had the potential to cause irritation as shown in the main test animals (50% and 25%). On this basis the test item could not be clearly classified as a sensitiser. 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 ammonium nitrate: ambiguous in LLNA but clearly 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 trichloride, 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. The test item, formulated in saline solution, was applied to twenty animals in the induction exposure phase (intra-dermal and dermal exposure). The dose for the intra-dermal injection was 0.01 % (w/v) and 100 % (w/v) for the dermal occlusive exposure. Ten 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 100 % (w/v). Remaining ten tested animals and five control animals were treated with 0.5 mL of the test item at a concentration of 50 % (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. After the challenge with the test item at concentrations of 100 or 50% (w/v) in saline, no positive response was observed in the treated animals. The mean of the scores of dermal reactions was 0.00 according to the 24 and 48-hours results. The right shaved flank area of all animals was treated with saline and no reaction was noted. After the challenge with the test item at concentrations of 100 or 50% (w/v) in saline no visible changes were found at the 24 and 48 hours examinations. The right shaved flank area of control animals was treated with saline and no reaction was noted. On the basis of the results of this study, the substance should be considered as not sensitising to the skin. This study is scored as Klimisch 1.


Migrated from Short description of key information:
Skin sensitisation:
Cerium trinitrate was tested using the LLNA method (OECD 429 and EU Method B42 and in compliance with GLP). The substance was tested at 50%, 25% and 10% (w/w) concentrations. Based on the results of this K2 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, and in compliance with GLP, was performed. The result of this K1 test (Török-Bathó, 2013) concluded that this substance is clearly not a skin sensitiser.

Justification for selection of skin sensitisation endpoint:
Although this endpoint is covered by 'weight-of-evidence' approach, only the results of one of the two studies available are considered reliable.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

On the basis of the available data and according to CLP and DSD criteria, the substance should not be considered as a skin sensitiser.

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