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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 March 2015 to 20 April 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Read-across to structurally similar substance.
Cross-reference
Reason / purpose:
other: read-across target
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
Read-across to structurally similar substance.
Reason / purpose:
read-across source
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
20 %
No. with + reactions:
4
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
20 %
No. with + reactions:
2
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
20 %
No. with + reactions:
15
Total no. in group:
20
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
20 %
No. with + reactions:
7
Total no. in group:
20
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
5 %
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
5 %
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
5 %
No. with + reactions:
4
Total no. in group:
20
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
5 %
No. with + reactions:
4
Total no. in group:
20

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
It is known from previous experience with several water soluble Rare Earth compounds that their irritating potential may confound the results of LLNA tests. Additionally, insoluble inorganic forms are often not able to penetrate the skin. Therefore, the skin sensitisation potential test method according to OECD guideline 406 was preferred above the LLNA tst according to OECD guideline 429.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: female (nulliparous and non-pregnant) guinea pigs, Dunkin Hartley strain, supplied by Charles River Italia S.p.A., Calco (Lecco), Italy (breeder: Charles River France Laboratories, Domaine des Oncins B.P. 0109, F 69592 L’ARBRESLE CEDEX, France)
- Age at order: 4 - 5 weeks
- Weight at arrival: 258 - 269 grams
- Housing: up to 5 animals per cage, noryl cages measuring 74.3 x 54.3 x 25 cm, daily inspected and changed as necessary (at least 3 times/week)
- Diet (e.g. ad libitum): ad libitum, 8GP17 (Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI) Italy))
- Water (e.g. ad libitum): ad libitum, via a water bottle
- Acclimation period: at least 5 days, veterinary health check during acclimatisation period
- Components present in the drinking water or diet were not at a level likely to interfere with the purpose or conduct of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 55 ± 15%
- Air changes (per hr): 15 - 25
- Photoperiod (hrs dark / hrs light): 12/12, artificial fluorescent lighting

Study design: in vivo (non-LLNA)

Induction
Route:
other: intradermal and topical
Vehicle:
corn oil
Concentration / amount:
- Induction by intradermal injection: (tolerated by the test system) 0.1 mL of 0.5 % w/v in FCA or corn oil;
- Induction by topical application: 0.4 mL, concentration of 20 % w/v in corn oil
Day(s)/duration:
Intradermal: day 1 and Topical: day 7 (for 48 hours)
Adequacy of induction:
highest technically applicable concentration used
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
- Challenge phase: 0.2 mL, concentration of 20 % w/v in corn oil;
Day(s)/duration:
Day 21 for 24 hours
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
- Re-challenge phase: 0.2 mL, concentration of 5 % w/v in corn oil.
Day(s)/duration:
Day 28 for 24 hours
No. of animals per dose:
Range finding tests:
- intradermal injection tolerance test: 1 group of 2 animals
- topical application tolerance test: 1 group of 5 animals

Main study:
- test group: 20 animals
- control group 10 animals
Details on study design:
RANGE FINDING TESTS:
A. INTRADERMAL INJECTION TOLERANCE TEST
- 1 group of 2 animals was allocated to the intradermal injection tolerance test.
- No. of exposures: a single intradermal injection of 0.1 mL
- Site: the hair over the scapulae was removed in the allocated animals using an electric clipper with suitable blade. Six sites were selected on each animal over the shaved scapulae. Each site was injected with a single concentration of the test material.
- Concentrations: 20, 10, 5, 1, 0.5 and 0.25% w/v in corn oil; concentration of 20% w/v was found not administrable due to the viscosity of the test material formulation and concentration of 10% was administered with difficulty.
- Readings: approximately 24 hours after dosing and on day 6
- Evaluation: according to the Draize scoring scale

B. TOPICAL APPLICATION TOLERANCE TEST
- 1 group of 5 animals was allocated to the topical application tolerance test. The hair over the scapulae was removed using an electric clipper with suitable blade. Each animal was then injected intradermally at the prepared site with two injections, each of 0.1 mL, of emulsified Freund’s complete adjuvant.
- No. of exposures: 1 percutaneous exposure seven days after preparation of the test site
- Site: flank, clipped free of hair
- Concentrations: 20, 10, 5, 1, 0.5 and 0.25% w/v in corn oil
- Each animal was dosed with 2 concentrations of the test material, 1 on each flank such that a total of 5 concentrations of the test material were each dosed in duplicate. A gauze patch measuring at least 20 x 20 mm was soaked with 0.2 mL of the test material and placed onto the selected treatment site. When both sites of the animal had been treated, they were covered with a strip of synthetic film and the trunk wrapped with an elastic adhesive bandage to maintain the test material in contact with the skin (occlusive barrier).
- Exposure period: 24 hours, after removing of the adhesive dressings and gauze patches, the treated sites were gently cleaned by washing with lukewarm water.
- Readings: approximately 24 hours and 48 hours after removal of the dressings.
- Evaluation: scoring scale was according to the following: no visible change = 0; discrete or patchy erythema = 1; moderate and confluent erythema = 2; intense erythema and swelling = 3


MAIN STUDY
A. 1) INDUCTION EXPOSURE (intradermal injection)
- No. of exposures: three pairs of intradermal injections (day 1)
- Site: the hair over the scapulae was removed over an area of approximately 20 x 40 mm using an electric clipper with a suitable blade.
- All injections were made at the edge of the prepared site and the anterior and median injections were positioned close together and distant from the posterior injections. A volume of 0.1 mL was injected at each site.
- Test groups: anterior: emusified Freund's complete adjuvant; median: 0.5% w/v test material in the vehicle (corn oil); posterior: 0.5% w/v test material in emulsified Freund's complete adjuvant
- Control group: anterior: emulsified Freund's complete adjuvant; median: vehicle (corn oil); posterior: vehicle mixed with emulsified Freund's complete adjuvant
- Frequency of applications: single intradermal injection (day 1) with a graduated syringe
- Readings: approximately 24 hours after injection
- Evaluation: according to the Draize scoring scale

A. 2) INDUCTION EXPOSURE (topical application)
- No. of exposures: one toppical application six days after injection (day 7)
- Exposure period: 48 hours
- Site: the hair was removed on the area surrounding the injection sites by means of an electric clipper
- A gauze patch measuring 20x40 mm was soaked with 0.4 mL of the test material at concentration of 20% w/v in corn oil or vehicle (corn oil) according to the group and then placed over the injection sites. The treatment site was covered with a strip of synthetic film and the trunk wrapped with an elastic adhesive bandage to maintain the test item in contact with the skin (occlusive barrier).
- After removal of the dressings, the treated sites were gently cleaned by washing with lukewarm water
- Readings: approximately 24 hours after removal of the dressings
- Evaluation: according to the Draize scoring scale

B. CHALLENGE EXPOSURE
- No. of exposures: single percutaneous exposure
- Day(s) of challenge: 21 days after the first induction phase
- Exposure period: 24 hours
- Site: flanks, clipped free of hair to expose areas of approximately 50 mm x 50 mm on each flank
- A gauze patch measuring 20x20 mm was soaked with 0.2 mL of the test material at concentration of 20% w/v in corn oil (or vehicle, corn oil) and then placed over the centre of the right (or left) flank. When both sites of the animal had been treated, they were covered with a strip of synthetic film and the trunk wrapped with an elastic adhesive bandage to maintain the test material and vehicle in contact with the skin (occlusive barrier).
- After removal of the dressings and patches, the treated sites were washed with lukewarm water. The treated sites were closely clipped to remove any hair that may have grown approximately 21 hours after the removal of the dressings.
- Evaluation (hr after challenge): approximately 24 hours and 48 hours after removal of the dressings
- Evaluation: scoring scale was according to the following: no visible change = 0; discrete or patchy erythema = 1; moderate and confluent erythema = 2; intense erythema and swelling = 3

C. RECHALLENGE
- No. of exposures: single percutaneous exposure
- Day(s) of challenge: 28 days after the first induction phase
- Exposure period: 24 hours
- Site: flanks, clipped free of hair to expose areas of approximately 50 mm x 50 mm on each flank
- A gauze patch measuring 20x20 mm was soaked with 0.2 mL of the test material at concentration of 5% w/v (or vehicle, corn oil) and then placed over the centre of the right (or left) flank. When both sites of the animal had been treated, they were covered with a strip of synthetic film and the trunk wrapped with an elastic adhesive bandage to maintain the test material and vehicle in contact with the skin (occlusive barrier).
- After removal of the dressings and patches, the treated sites were washed with lukewarm water. The treated sites were closely clipped to remove any hair that may have grown approximately 21 hours after the removal of the dressings.
- Evaluation (hr after challenge): approximately 24 hours and 48 hours after removal of the dressings
- Evaluation: scoring scale was according to the following: no visible change = 0; discrete or patchy erythema = 1; moderate and confluent erythema = 2; intense erythema and swelling = 3
Positive control substance(s):
yes
Remarks:
alpha-hexylcinnamaldehyde

Results and discussion

Positive control results:
Sensitisation reactions were observed in 60% of the animals. These results are considered to be acceptable for a weak sensitiser like the alpha-Hexylcinnamaldehyde.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
20 %
No. with + reactions:
4
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
20 %
No. with + reactions:
2
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
20 %
No. with + reactions:
15
Total no. in group:
20
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
20 %
No. with + reactions:
7
Total no. in group:
20
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
5 %
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
5 %
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
5 %
No. with + reactions:
4
Total no. in group:
20
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
5 %
No. with + reactions:
4
Total no. in group:
20

Any other information on results incl. tables

Preliminary tolerance test

The preliminary tolerance test to establish a suitable concentration for injection in the main sensitisation test indicated that the test material at a concentration of 0.5% w/v in corn oil was reasonably tolerated. No reaction was observed in the animals of the preliminary phase dosed topically at concentrations of 20, 10, 5, 1 and 0.5% w/v of the test material in corn oil. Therefore, the concentration of 20% w/v was judged to be non-irritant and was selected for use at topical induction of the main phase.

Induction

Well defined to moderate/severe erythema (scores of 2 and 3) was apparent at the intradermal injection sites following administration of Freund’s complete adjuvant (test and control group, anterior sites) and at sites treated with the Freund’s complete adjuvant mixed with the test material at concentration of 0.5% w/v in corn oil or vehicle alone (test and control group, posterior sites). Well defined to moderate/severe erythema (scores of 2 and 3) was also observed at sites treated with the test material at a concentration of 0.5% w/v in corn oil (test group, median sites). Very slight erythema (score of 1) was noted at median sites treated with the vehicle alone (control group). No erythema (score of 0) was observed following 48 hours of topical exposure with the test material at concentration of 20% w/v (test group) or the vehicle alone, corn oil (control group). Based on the above results and those seen in the preliminary phase, the test material at concentration of 20% w/v was selected to be used in the challenge phase.

Challenge

Discrete or patchy erythema to moderate and confluent erythema (scores of 1 and 2) was observed in 15 out of 20 animals of the test group at 24 hours following 24 hour topical exposure period to the test material at 20% w/v concentration in corn oil. At the observation performed 48 hours after the 24 hour topical exposure period, discrete or patchy erythema to moderate and confluent erythema (scores of 1 and 2) was still apparent in 7 out of 20 animals of the test group. Discrete or patchy erythema (score of 1) was observed in 4 out of 10 animals of the control group at 24 hours following 24 hour topical exposure period to the test material at 20% w/v concentration in corn oil. Reaction of the same entity was still noted in 2 out of 10 control animals at the observation performed at 48 hours after the 24 hour topical exposure period. No reaction to the vehicle alone was observed.

On the basis of these results, the test material at the concentration of 20% w/v appeared to be slightly irritant. Due to the incidence of control animals with an irritant reaction at the 24 hour observation, and since animals did not completely recover at the observation performed 48 hours after the 24 hour topical exposure, the concentration of 5% w/v of the test material in corn oil was selected to be used in the re-challenge phase.

Re-challenge

Discrete or patchy erythema (score of 1) was observed in 4 out of 20 animals of the test group at 24 and 48 hours following 24 hour topical exposure period to the test item at 5% w/v concentration in corn oil. No reaction was noted in animals of the control group, 24 and 48 hours following the 24 hour topical exposure period. No reaction to the vehicle alone was observed.

Body weight

Body weight changes were not observed.

Applicant's summary and conclusion

Interpretation of results:
other: Not classified in accordance with EU criteria
Conclusions:
Under the conditions of this study the test material is not sensitising to the skin. 
Executive summary:

The potential of the test material, Yttrium trinitrate, to induce and elicit delayed dermal sensitisation was assessed in the guinea pig using the maximisation test of Magnusson and Kligman. The study was performed in accordance with the standardised guidelines OECD 406 and EU Method B.6, under GLP conditions.

At challenge with the test material at 20% w/v concentration, reaction was observed in 75% of the animals of the test group and in 40% of control animals, indicating that irritation has occurred following dosing with the test material at 20% w/v in corn oil. Therefore, a re-challenge was performed one week later in the same animals with the test material at a concentration of 5% w/v in corn oil. Reaction was still observed in 20% of the animals of the test group, while no response was observed in animals of the control group.

These results indicate that the test material, Yttrium trinitrate, induced a slight skin sensitisation response in the guinea pig, since a slight positive reaction was observed in 20% of the test animals. However, since the incidence of the animals showing a positive reaction was lower than 30%, classification is not required.

Under the conditions of this study the test material is not sensitising to the skin.