Dysprosium trinitrate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2016-09-06 to 2017-04-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The GPMT method (OECD 406) was preferred above the LLNA (OECD 429) since previous experience with several water soluble rare earth compounds containing an acid residue learned that their irritating potential may confound the results of LLNA tests.

Test material

Test material information

Constituent 1

Reference substance name:

Dysprosium trinitrate

EC Number:

233-410-5

EC Name:

Dysprosium trinitrate

Cas Number:

10143-38-1

Molecular formula:

Dy.3HNO3

IUPAC Name:

dysprosium trinitrate

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): dysprosium trinitrate (the hydrated form was used as test item)
- Physical state: crystalline solid
- Appearance: white or pale/slightly yellow crystal deliquescent
- Further details on test material confidential.

Specific details on test material used for the study:

Correction factor: Concentration was calculated to anhydrous form. The correction factor for this study was 1.35.

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Albino guinea pigs, LAL/HA/BR

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: LAB-ÁLL Bt., Budapest, 1174 Hunyadi u. 7., Hungary
- Females (if applicable) nulliparous and non-pregnant: not applicable
- Microbiological status of animals, when known: not specified
- Age at first dosing (day 1): young adult, approx. 12-13 weeks
- Weight at randomisation (day -1): 568-715 g
- Housing: Animals were housed in Macrolon cages size IV, with up to 5 animals/cage to allow socialization. Lignocel® 3/4-S Hygienic Animal Bedding, from J. Rettenmaier & Söhne GmbH+CO.KG (D-73494 Rosenberg, Holzmühle 1, Germany) was available to animals during the study.
- Diet (e.g. ad libitum): ad libitum, Cunigra Diet for Rabbits (produced by Bonafarm-Bábolna Takarmány Ltd., Hungary)
- Water (e.g. ad libitum): Ad libitum, tap water from municipal supply as for human consumption, containing at least 50 mg/100 mL ascorbic acid. 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: 56 days before start of treatment under laboratory conditions
- Indication of any skin lesions: Not specified. Only healthy animals used for testing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.2–22.5°C
- Humidity (%): 30–80%
- Air changes (per hr): 15-20 air exchange/hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

other: intradermal (main study part I)

Vehicle:

physiological saline

Concentration / amount:

0.1 mL of 1% test item in vehicle

Day(s)/duration:

Day 1 of treatment

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Induction 2

Route:

other: dermal (main study part II)

Vehicle:

physiological saline

Concentration / amount:

0.5 mL of 75% test item in vehicle

Day(s)/duration:

Day 8 of treatment/48 h of 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:

0.5 mL of 10% (w/v) test item in saline

Day(s)/duration:

Day 22 of treatment/24 h of exposure

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

preliminary test: 10 male animals
main test: 10 aminals in the test group, 5 animals in the control group

Details on study design:

RANGE FINDING TESTS:
- The dose levels for the main study were selected based on the results of the Preliminary Test.
- A day prior to the test, the hair was removed from the right and left sides of the animals (approximately 5x5 cm). The hair removal was performed carefully to ensure animals are closely shaven.
- A series of test item concentrations was tested to identify the primary irritation following intradermal injection and dermal application: 0.01, 0.05, 0.1, 0.5, 1, 2.5, and 5% (w/v) concentrations were used for intradermal injection and 5, 10, 25, 50, 75 and 100% (w/v) for dermal application formulated in saline.
- For the intradermal application, 0.1 mL per concentration was injected intradermally into the hair free skin of the animals. Two concentrations were injected on the right side an 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.
- Local effects were examined and scored 1, 24, 48 and 72 hours after the treatment or after patch removal.
- Skin effects were scored for erythema and oedema; any other observations of changes to the skin were recorded.
- For the dermal application, the volume of the concentrations 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. In the first preliminary study, one concentration was used on the right side and another concentration on the left side of the animals. In the second preliminary study, three concentrations were tested on the right side, back and left side of the animals. Two animals per concentration were used in each preliminary study.
- Skin reactions were observed and recorded as follows: 1, 24, 48 and 72 hours after patch removal and/or treatment.
- Skin effects were scored for erythema and oedema, any other observations of changes to the skin were recorded.

MAIN STUDY
A. INDUCTION EXPOSURE
A1 INTRADERMAL INDUCTION EXPOSURE
- No. of exposures: 3 pairs of intradermal injections per animal (0.1 mL)
- Test groups (the first listed nearest to the head): 2 injections of Freund's Complete Adjuvant and physiological saline solution in a 1:1 (v/v) mixture, 2 injections of 1% test item in saline, 2 injections of 1% test item in a 1:1 (v/v) mixture 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 vehicle (saline) formulated in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline solution
- Site: Scapular region. On the day before treatment, an area of approximately 5x5 cm on the scapular region of the animals was clipped free of hair and was carefully shaved.
- Frequency of applications: one application
- Skin reactions were observed and recorded as follows: 1, 24, 48 and 72 hours after patch removal.

A2 DERMAL INDUCTION EXPOSURE
- Since 75% concentration of the test item was skin irritant in the dermal dose range finding study, the test area was not painted with 10% of sodium dodecyl sulphate.
- Site: The same scapular region which received the intradermal injections was used for dermal induction exposure.
- Test groups: A 2.5x2.5 cm gauze patch (4 layers of porous gauze pads) was saturated with approximately 0.5 mL of the test item at 75% concentration and placed over the injection sites (scapular region). The control group was treated with 0.5 mL vehicle (saline) with the same method. The gauze patches were kept in contact with the skin by a patch with surrounding adhesive hypoallergenic plaster. The treated areas were covered for 48 hours with a fully occlusive foil (Closed Patch Test). After the patch removal, any remaining test item was removed with a wet gauze swab. Following the dermal induction treatment, the animals were left untreated for 14 days prior to challenge applications.
- Control group: The control group was treated with saline only.
- Frequency of applications: one application
- Skin reactions were observed and recorded as follows: 24 hours after treatment

B. CHALLENGE EXPOSURE
- Day(s) of challenge: day 22
- Exposure period: 24 h
- Site: Left and right 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.
- Test groups: A 2.5x2.5 cm patch of sterile gauze was saturated with approximately 0.5 mL of the test item at a 10% concentration in saline (highest non-irritant dose) and applied to the left side of all animals (both the test and the control). The right shaved side area of all animals was treated with the vehicle (saline). After the patch removal, any remaining test item was removed with a wet gauze swab.
- Control group: The right shaved side area of all animals (treated and controls) was treated with the vehicle (saline).
- Skin reactions were observed and recorded as follows: 24 and 48 hours after the patch removal.

OTHER
BODY WEIGHT:
Body weight was recorded with a precision of 1 g at randomisation (day -1), then at least weekly, including day 25 prior to euthanasia. The mean values and the standard deviations were calculated and reported.

OBSERVATIONS
- Mortality/clinical signs: daily during the test
- Detailed clinical observations were made on all animals outside the home cage in a standard arena before the first treatment (on the day of randomisation) and at least weekly thereafter. The dermal irritation scores (in case of dermal induction exposure) were evaluated according to the scoring system by Draize (1959).

TERMINAL PROCEDURE
Terminally animals were sacrificed under pentobarbital anaesthesia.

Challenge controls:

A 2.5x2.5 cm patch of sterile gauze was saturated with approximately 0.5 mL of the test item at a 10% concentration in saline (highest non-irritant dose) and applied to the left side of all animals (both the test and the control). The right shaved side area of all animals was treated with the vehicle (saline) .

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 90% and 80% and net score values of 0.90 and 0.80 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 sensitiser. This demonstrated that the experimental procedure and the test system were appropriate.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No signs of systemic or local toxicity were observed. No mortality was observed.

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No signs of systemic or local toxicity were observed. No mortality was observed.

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

10%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No signs of systemic or local toxicity were observed. No mortality was observed.

Remarks on result:

no indication of skin sensitisation

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

10%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No signs of systemic or local toxicity were observed. No mortality was observed.

Remarks on result:

no indication of skin sensitisation

Results 5

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

50% w/v (2-mercaptobenzothiazole)

No. with + reactions:

9

Total no. in group:

10

Clinical observations:

Discrete erythema (score 1) on the skin of the animals.

Remarks on result:

positive indication of skin sensitisation

Results 6

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

50% w/v (2-mercaptobenzothiazole)

No. with + reactions:

8

Total no. in group:

10

Clinical observations:

Discrete erythema (score 1) on the skin of the animals.

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

Preliminary study

- The treatments between 0.01% and 5%, and 5% with FCA caused no more than mild-to-moderate erythema. However, yellowish, whitish-grey or brownish alteration of the skin surface was observed (except at the 0.05% treatment) during the preliminary study. The size/severity of the alteration at a concentration of 5% was too high to accept for a main study. Slight recovery was observed at the concentrations of 2.5%, 1% and 0.01%. Despite of the slight recovery at a concentration of 2.5% it was considered inappropriate for treatment because of the area of brownish alteration. Based on the condition on the last observation day of the treated areas, a 1% concentration was considered to be acceptable for the main study.

- Very slight/well defined erythema and very slight oedema were observed at all concentrations at 1 or 24 hours which were reversible within 48 hours. During the observation period erosion (only at the 100% treatment and at 1 hour in one animal), fissured skin (50-75%) and peeling (25-100%) were noted. Erosion is considered to be excessive, so the 100% treatment is excluded from the main study.

- On the basis of the results of the Preliminary Dose Range Finding Study, the following treatments were used in the main study:

*intradermal induction: 1% test item formulated in physiological saline was used in the test group for intradermal injections. The control group was treated with injections of physiological saline only.

*dermal induction: 75% test item formulated in physiological saline was used for dermal treatments. Control animals were treated with physiological saline only. Since 75% test item caused erythema, oedema, fissured skin and peeling in the dermal dose range finding study, the test area was not painted with 10% sodium dodecyl sulphate.

*challenge phase: all animals of the treatment and control group were treated with 10% test item formulated in saline as a challenge exposure on the left flank (as this concentration was the highest which caused no irritation) and with physiological saline on the right flank, respectively.

Main study

- Test group: after the challenge with the test item at a concentration of 10% formulated in saline, no positive response was observed in the treated animals on the left flank. The mean of the scores was 0.00 according to the 24 and 48-h results. The right shaved side of all animals was treated with saline and no reaction was noted.

- Control group: after the challenge with the test item at a concentration of 10% formulated in saline, no visible changes were found at the 24 and 48-h examinations on the left flank. The right shaved side of control animals was treated with saline and no reaction was noted.

- Clinical observations/mortality: no signs of systemic or local toxicity were observed. No mortality was observed during the study.

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Challenge with the test item 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 was shown to have no sensitisation potential and classified as a non-sensitiser, according to current EU-regulations.