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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 August 19th, 2016 to November 28th, 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
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)
Remarks:
September 22th, 2015
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The skin sensitisation potential test method (OECD Guideline 406) was preferred above LLNA (OECD Guideline 429) since previous experience with several water soluble Rare Earth compounds learned that their irritating potential may confound the results of LLNA tests.

Test material

Constituent 1
Chemical structure
Reference substance name:
Ytterbium trinitrate
EC Number:
237-384-6
EC Name:
Ytterbium trinitrate
Cas Number:
13768-67-7
Molecular formula:
HNO3.1/3Yb
IUPAC Name:
ytterbium trinitrate
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Ytterbium trinitrate

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: CRL:HA
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
- Justification of species: The guinea pig is the standard species used for skin sensitisation studies.
- Femalesnulliparous and non-pregnant: yes
- Age at study initiation: Young adult
- Weight at study initiation: 349 - 416 g
- Housing: Animals were housed in macrolon cages size IV, with 5 animals/cage to allow socialization
- Diet: Animals received Cunigra Diet for Rabbits (produced by Bonafarm-Bábolna Ta karmány Ltd., Hung ary), 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: 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: 27 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 - 21.9 °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: August 30th To: October 15th, 2016

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
- Control group: vehicle only
- Test group: 0.1% w/v test item formulated in saline
Day(s)/duration:
Day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
- Control group: vehicle only
- Test group: 100 % w/v test item formulated in saline
Day(s)/duration:
Started on D8 with a 48h application
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:
Challenge exposure:
- control group: 10% w/v test item formulated in saline
- test group: 10% w/v test item formulated in saline
Day(s)/duration:
Started on D22 with a 24h application
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Preliminary test: 10 female animals
Main test: 10 in the test group and 5 in the control group
Details on study design:
RANGE FINDING TESTS:
Test item concentrations:
- intradermal injection: 0.1, 0.5, 1, 2.5 and 5% (w/v)
- dermal application: 5, 10, 25, 50, 75 and 100% (w/v)

Time of observations for local effects:
- 1, 24, 48 and 72 hours after the treatment or after patch removal

Test item application and observations:
- For the intradermal application, 0.1 mL per concentration was injected intra-dermally 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.
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.
- For the dermal application, 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.
Slight or well defined erythema (scores 1 and 2) was observed at concentrations of 25, 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 5% and 10% (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 10% (w/v) test item formulated in saline was decided to be used for the challenge treatment.

MAIN STUDY
A. INTRADERMAL INDUCTION EXPOSURE
- Day of treatment: 1
- No. of exposures: 1
- Test group:
• 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
- Control group:
• 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.
- Site: scapular region
- Time of observations: 24 hours after the treatment

B. DERMAL INDUCTION EXPOSURE
- Day of treatment: 8
- No. of exposures: 1
- Exposure period: 48 hours
- Test group : 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 control group was treated with saline only.
The gauze patches were kept in contact with the skin by a patch with a surrounding adhesive hypoallergenic plaster. The treated areas were covered with a fully occlusive foil (Closed Patch Test). After the patch removal any remaining test item was removed with a wet gauze swab.
- Site: scapular region
- Time of observations: 1, 24, 48 and 72 hours after the patch removal

B. CHALLENGE EXPOSURE
- Day of challenge: 22
- No. of exposures: 1
- Exposure period: 24 hours
- Test group and control group: A 2.5x2.5 cm patch of sterile gauze patch was saturated with 10% (w/v) test item in saline and applied to the left side of all animals (both the test and the control). 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 in the previous chapter (Closed Patch Test). After the patch removal any remaining test item was removed with a wet gauze swab.
- Site: left and right sides
- Evaluation (hr after challenge): 24 and 48 hours after the patch removal
Challenge controls:
As described in details on study design
Positive control substance(s):
yes
Remarks:
2-Mercaptobenzothiazole

Results and discussion

Positive control 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.
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 chemical
Dose level:
left side: 10% (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 chemical
Dose level:
left side: 10% (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
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Left side: 10% (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.
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Left side: 10% (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.
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

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 433.2 g and 475.4 g, respectively.

Clinical observations and mortality:No signs of systemic or local toxicity were observed in any animals.

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.

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 five 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 two animals; giving a mean of the scores of 0.2. 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
patch removal

48 hours after
patch 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 10% (w/v) test item formulated in saline, **: Right side was treated with saline only.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Challenge with test item (Ytterbium trinitrate) evoked no positive responses in the test animals 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 the test item Ytterbium trinitrate was shown to have no sensitisation potential and classified as a non-sensitizer.
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. The study was performed according to OECD Guideline No. 406 (adopted in 1992) 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 10% (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.

 

Results

No signs of systemic toxicity were observed in any animal.

 

Incidence rate:

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.

 

Intensity of sensitisation response:

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 Ytterbium trinitrate was shown to have no sensitisation potential and classified as a non-sensitizer.