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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:
From 19 August 2016 to 23 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Performed under GLP compliance

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

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
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
22 September 2015
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The skin sentisation potential test method (OECD Guideline 406) was preferred above LLNA (OECD Guideline 429) since previous experiences with several water soluble rare earth compounds learned that their irritating potential may confound the results of LLNA tests.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Remarks:
White crystals, deliquescent
Details on test material:
- Name of test material: Terbium trinitrate

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: CRL:HA Guinea pigs
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld.
- Females nulliparous and non-pregnant: yes.
- Age at study initiation: Young adult.
- Weight at study initiation: 356 – 396 g.
- Housing: Animals were housed in macrolon cages size IV, with 5 animals/cage to allow socialization.
- Diet (e.g. ad libitum): Animals received Cunigra Diet for Rabbits (produced by Bonafarm-Bábolna Takarmány Ltd., Hungary), ad libitum. This diet is classified as being suitable for Guinea pigs as the vitamin D level is high enough to meet the needs of this species.
- Water (e.g. ad libitum): Animals received tap water from municipal supply as for human consumption, containing at least 50 mg/100 mL ascorbic acid, ad libitum. The drinking water is routinely analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Acclimation period: 26 days.
- Healt status of animals: Only animals in acceptable health conditions were used for the test. Health status was certified by the Veterinarian.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.2 - 22.5 °C
- Humidity (%): 32 - 80%
- Air changes (per hr): 15-20 air exchange/hour.
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
- IN-LIFE DATES: From 12 September 2016 (randomisation) to 07 October 2016 (2nd and last examination at challenge).

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
0.1% (w/v) test item formulated in saline
Day(s)/duration:
Not applicable
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
100% (w/v) test item formulated in saline
Day(s)/duration:
48-hour exposure
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
25% (w/v) test item formulated in saline
Day(s)/duration:
Dermal application performed on Day 22, 24-hour exposure
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Preliminary test: 8 animals.
Main test: 5 animals for the control group, 10 animals for the test group.
Details on study design:
RANGE FINDING TESTS:
- Intradermal injection:
* Tested concentrations: A series of test item concentrations formulated in saline was tested to identify the primary irritation following intradermal injection: 0.1, 0.5, 1, 2.5 and 5% (w/v). 0.1 mL per concentration was injected intradermally into the hair free skin of the animals. Two concentrations were injected on the right side and another two concentrations on the left side of the animals. The highest concentration (5%) was also tested in a 1:1 mixture (v/v) of Freund's Complete Adjuvant and physiological saline solution. Each concentration was injected in duplicate. Two animals were used per concentration.
* Obtained results: Local effects were examined and scored 1, 24, 48 and 72 hours after the treatment. Skin effects were scored for erythema and oedema, any other observations of changes to the skin was recorded. 0.5, 1, 2.5 and 5% (w/v) test item concentrations caused extensive skin lesions at the treatment site, with significant redness (erythema) around the edges, but no erythema in the coloured area (apparently with no blood flow). At these concentrations it was considered that the degree of local effect was more than “mild-to-moderate erythema”. However, 0.1% (w/v) test item in the vehicle caused only no more than mild-to-moderate erythema (score 0 or 1), without any observable skin lesions during the observation period, therefore this concentration could be used in the main study.

- Dermal application:
* Tested concentrations: A series of test item concentrations formulated in saline was tested to identify the primary irritation following dermal application: 25, 50, 75% and 100% (w/v). The volume of the formulations was 0.5 mL. A closed patch exposure was performed by means of an occlusive bandage using similar treatment procedures as for the main study. The time of exposure for the dermal application was 48 hours. One concentration was used on the right side and another concentration on the left side of animals. Two animals per concentrations were used.
* Obtained results: Local effects were examined and scored 1, 24, 48 and 72 hours after patch removal. Skin effects were scored for erythema and oedema, any other observations of changes to the skin was recorded. Slight or well defined erythema (scores 1 and 2) was observed at concentrations of 50, 75 and 100% (w/v). As the highest tested concentration (100%) caused no more than mild-to moderate skin irritation, this was used for the dermal induction. However, at a concentration of 25% (w/v), no reaction on the skin of guinea pigs was seen. The concentration used for the challenge exposure should be the highest non-irritant dose; therefore 25% (w/v) test item formulated in saline was decided to be used for the challenge treatment.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: Two exposures (one intradermal induction exposure and one dermal (epicutaneous, occlusive) induction exposure).
- Exposure period:
* Intradermal induction exposure: Ponctual injection.
* Dermal (epicutaneous, occlusive) induction exposure: 48-hour exposure.
- Test group:
* Intradermal induction exposure: Three pairs of intradermal injections (0.1 ml/site) were made in as follows (the first listed nearest the head):
- 2 injections of Freund's Complete Adjuvant and physiological saline solution in a 1:1 (v/v) mixture.
- 2 injections of 0.1% test item in saline.
- 2 injections of 0.1% test item formulated in a 1:1 mixture (v/v) of Freund's Complete Adjuvant and physiological saline solution.
* Dermal (epicutaneous, occlusive) induction exposure: Seven days after the intradermal injections, the same hair-free scapular area used for dermal induction exposure was treated. A 2.5x2.5 cm sterile gauze patch (4 layers of porous gauze pads) was saturated with approximately 0.5 mL of 100% (w/v) test item in saline and placed over the injection sites.The gauze patches were kept in contact with the skin by a patch with a surrounding adhesive hypoallergenic plaster. The treated areas were covered for 48 hours with a fully occlusive foil (Closed Patch Test).
- Control group:
* Intradermal induction exposure: Three pairs of intradermal injections (0.1 ml/site) were made in as follows (the first listed nearest the head):
- 2 injections of Freund's Complete Adjuvant and physiological saline solution in a 1:1 (v/v) mixture.
- 2 injections of saline.
- 2 injections of saline formulated in a 1:1 mixture (v/v) of Freund's Complete Adjuvant and physiological saline solution.
* Dermal (epicutaneous, occlusive) induction exposure: Seven days after the intradermal injections, the same hair-free scapular area used for dermal induction exposure was treated. A 2.5x2.5 cm sterile gauze patch (4 layers of porous gauze pads) was saturated with approximately 0.5 mL of saline only and placed over the injection sites. The gauze patches were kept in contact with the skin by a patch with a surrounding adhesive hypoallergenic plaster. The treated areas were covered for 48 hours with a fully occlusive foil (Closed Patch Test)..
- Site:
* Intradermal induction exposure: Scapular region (on the day before treatment, an area approximately 5x5 cm on the scapular region of the animals was clipped free of hair and was carefully shaved).
* Dermal (epicutaneous, occlusive) induction exposure: Scapular region (the same scapular region which received the intradermal injections, were used for dermal induction exposure).
- Time of observation:
* Intradermal induction exposure: 24 hours after injection.
* Dermal (epicutaneous, occlusive) induction exposure: 1, 24, 48 and 72 hours after the patch removal.

B. CHALLENGE EXPOSURE
- No. of exposures: Only one challenge exposure.
- Day(s) of challenge: Two weeks after the dermal induction application, the animals were exposed to a dermal challenge dose (on Day 22).
- Exposure period: 24-hour exposure.
- Test groups and control groups: A 2.5x2.5 cm patch of sterile gauze patch was saturated with 25% (w/v) test item in saline and applied to the left side of all animals. The right shaved side of all animals was treated with vehicle only. The volume of formulated test item was 0.5 mL. Treatment was performed as described for dermal induction exposure (Closed Patch Test).
- Site: Sides (approximately 24 hours before the treatment, the hair was removed from an area of approximately 5x5 cm on the left and right sides of each animal).
- Concentrations: 25% (w/v) test item formulated in saline.
- Evaluation (hr after challenge): 24 and 48 hours after the patch removal.
Challenge controls:
Challenge controls were treated exactly as test group (see the above field: "Details on study design").
Positive control substance(s):
yes
Remarks:
2-Mercaptobenzothiazole

Results and discussion

Positive control results:
- Name of the positive control substance: 2-Mercaptobenzothiazole.
- Tested concentrations:
* Intradermal induction exposure: 1% (w/v).
* Dermal induction exposure: 75% (w/v).
* Challenge treatment: 50% (w/v).
- Obtained results: Challenge with reference item 2-Mercaptobenzothiazole resulted in a positive response in test animals previously sensitised. The net response values at the 24 and 48 hours observations represented an incidence rate of 80% and 70% and net score values of 0.80 and 0.70 respectively. In the control animals no visible changes were found either at the 24 or 48 hours examinations following challenge with the reference item. The dermal scores represented discrete erythema (score 1) developed on the skin of sensitised guinea pigs.
- Conclusion: On the basis of the results of the reliability check study, the reference item 2-Mercaptobenzothiazole was classified as a skin sensitizer. This demonstrated that the experimental procedure and the test system were appropriate.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Left side: 25% (w/v) test item formulated in saline, right side: saline only.
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Dermal response scores (for erythema) equal to 0 for all animals and for both sides.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Left side: 25% (w/v) test item formulated in saline, right side: saline only.
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Dermal response scores (for erythema) equal to 0 for all animals and for both sides. No notable body weight difference between test / control group.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Left side: 25% (w/v) test item formulated in saline, right side: saline only.
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
Dermal response scores (for erythema) equal to 0 for all animals and for both sides.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Left side:25% (w/v) test item formulated in saline, right side: saline only.
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
Dermal response scores (for erythema) equal to 0 for all animals and for both sides.

Any other information on results incl. tables

SKIN EFFECTS AFTER THE INDUCTION EXPOSURE

Test group

At the 24-hour examination after the intradermal induction exposure to 0.1% (w/v) test item formulated in saline, very slight (barely perceptible) erythema (Draize score: 1) were observed in two animals. No oedema was observed in any animals.

At the 1-hour examination after the dermal induction exposure to 100% (w/v) test item, very slight (barely perceptible) erythema (Draize score: 1) were observed in five animals; giving a mean of the scores of 0.5. No oedema was observed in any animals examined at this time. At the 24-, 48- and 72-hour examinations after the dermal induction exposure to 100% (w/v) test item, no erythema was observed anymore in test animals and no oedema was observed.

Control group

At the 24-hour examination after the intradermal induction exposure to saline, neither erythema nor oedema was observed in control animals.

At all examinations (1, 24, 48 and 72 hours) after the dermal induction exposure to saline, neither erythema nor oedema was observed in control animals.

Table 1: Summary of the result after the challenge exposure

Groups and animals

24 hours after removal

48 hours after removal

Left side*

Right side**

Left side*

Right side**

Control – Animal 1

0

0

0

0

Control – Animal 2

0

0

0

0

Control – Animal 3

0

0

0

0

Control – Animal 4

0

0

0

0

Control – Animal 5

0

0

0

0

Mean scores

0

0

0

0

Dosed – Animal 6

0

0

0

0

Dosed – Animal 7

0

0

0

0

Dosed – Animal 8

0

0

0

0

Dosed – Animal 9

0

0

0

0

Dosed – Animal 10

0

0

0

0

Dosed – Animal 11

0

0

0

0

Dosed – Animal 12

0

0

0

0

Dosed – Animal 13

0

0

0

0

Dosed – Animal 14

0

0

0

0

Dosed – Animal 15

0

0

0

0

Mean scores

0

0

0

0

*: Left side was treated with 25% (w/v) test item formulated in saline, **: Right side was treated with saline only.

BODY WEIGHT

There were no notable differences between the test animal group and the control group. On Day 25 prior to euthanasia, control and test animals presented a mean body weight of 463.2 g and 471.5 g, respectively.

CLINICAL OBSERVATIONS AND MORTALITY

No signs of systemic or local toxicity were observed in any animal. No mortality was observed during the study. There were no overt signs of an adverse clinical response to treatment with the test item during the course of the study.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
No indication of skin sensitisation.
Conclusions:
Challenge with the test item terbium trinitrate evoked no positive responses in Guinea pigs previously sensitised with the test item or in the control group. The net response value represented an incidence rate of 0% and the net score value of 0.00. In conclusion, under the conditions of the present assay, terbium trinitrate was shown to have no sensitisation potential.
Executive summary:

A skin sensitisation study was performed in the guinea pig according to the Magnusson and Kligman method, using a maximisation method with Freund's Complete Adjuvant to evaluate the sensitisation potential of test item (OECD Guideline No. 406) and in compliance with GLP guidelines.

Based on the results of a preliminary test, ten test animals were subjected to sensitisation procedures in a two-stage process, named induction phase: i.e. an intradermal treatment and a 48-hour topical application (dermal treatment under an occlusive dressing). The test item was used at a concentration of 0.1% (w/v) in saline for intradermal injections and at a concentration of 100% (w/v) in saline for topical sensitisation treatment. Five control guinea pigs were simultaneously exposed to vehicle only during the sensitisation phase; saline being used for both intradermal and dermal treatments.

Two weeks after the last induction exposure, a challenge dose at a concentration of 25% (w/v) test item formulated in saline was administered on the left side of all animals for 24 hours. The right side of the animals was treated with vehicle only (saline). Challenge was performed by dermal application of the test item. Test and control animals were treated in the same way. Skin reactions were measured 24 and 48 hours after patch removal.

No signs of systemic toxicity were observed in any animal. After the challenge exposure, no signs of contact sensitisation were detected in guinea pigs previously exposed to the test item during the induction phase of the experiment. In the control and treated animals the mean of the scores was 0.00 according to the 24 and 48-hour results.

In conclusion, under the conditions of the present assay the test item Terbium trinitrate was shown to have no sensitisation potential.