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Diss Factsheets

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2016-06-14 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

Constituent 1
Chemical structure
Reference substance name:
Gadolinium trinitrate
EC Number:
233-437-2
EC Name:
Gadolinium trinitrate
Cas Number:
10168-81-7
Molecular formula:
Gd(NO3)3
IUPAC Name:
gadolinium trinitrate
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in the report): gadolinium trinitrate
- CAS = 19598-90-4 (i.e. the hexahydrate form Gd(NO3)3.6H2O). According to Annex V, point 6 of the REACH regulation (CE) No. 1907/2006, this form will be covered by the registration of the anhydrous form (CAS number: 10168-81-7).
- Physical state: crystalline solid
- Further information on test item is 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 1.31.

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Albino 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): 5 weeks
- Weight at randomisation (day -1): 265-316 g
- Housing: Animals were housed in Macrolon cages size IV, with up to 5 animals/cage to allow socialisation. Lignocel® 3/4-S Hygienic Animal Bedding, J. Rettenmaier & Söhne GmbH+CO.KG (D-73494 Rosenberg, 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: 6 days before start of treatment under laboratory conditions
- Indication of any skin lesions: Not specified, only healthy animals used in study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 - 23.7°C
- Humidity (%): 27 - 87%
- 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)

Inductionopen allclose all
Route:
other: intradermal (main study part I)
Vehicle:
physiological saline
Concentration / amount:
0.1 mL of 2.5% 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
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 the test item at 25% concentration in vehicle; saline in the right side
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: 8 male animals
main test: 10 animals in the test group, 5 animals in the control group
Details on study design:
RANGE FINDING TESTS:
- The dose levels for the main study are 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 in saline were used for intradermal injection and 25, 50, 75% (w/v) in saline and 100% (as supplied, dampened with saline) for dermal application. The test item was powdered for an accurate measurement of concentrations.
- 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 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.
- For the dermal application, the volume of the concentrations was 0.5 mL. For the 100% treatment, 0.6550 g test item was dampened with saline, and then applied to the skin. 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 the animals. Two animals per concentration were used.
- 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.

MAIN STUDY
A. INDUCTION EXPOSURE
A1 INTRADERMAL INDUCTION EXPOSURE (day 1)
- No. of exposures: 3 pairs of intradermal injections per animal (0.1 mL),
- Exposure period: 24 h for dermal
- Test groups: 2 injections of Freund's Complete Adjuvant and physiological saline solution (1:1), 2 injections of 2.5% test item in saline, 2 injections of 2.5% test item in a 1:1 mixture of Freund's Complete Adjuvant and physiological saline solution
- Control group: 2 injections of Freund's Complete Adjuvant and physiological saline (1:1), 2 injections of saline, 2 injections of vehicle (saline), formulated in a 1:1 mixture (v/v) of Freund's Complete Adjuvant and physiological saline solution
- Site: Scapular region. On the day of treatment, an area 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 the patch removal.

A2 DERMAL INDUCTION EXPOSURE (day 8)
- Since the 75% concentration of the test item was skin irritant in the dermal dose range finding study, the test area was not painted with 10% 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 sterile gauze patch (4 layers of porous gauze pads) was saturated with approximately 0.5 mL of the test item at a 75% concentration and placed over the injection sites (scapular region). 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). 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 0.5 mL vehicle (saline) with the same method.
- Frequency of applications: one application
- Skin reactions were observed and recorded as follows: 24 hours after treatment

B. CHALLENGE EXPOSURE (day 22)
- 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 and control group: A 2.5x2.5 cm patch of sterile gauze was saturated with approximately 0.5 mL of the test item at a 25% 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).
- 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. As part of this observation, detailed clinical observation was made weekly 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 cases of dermal induction exposure) were evalauted according to the scoring system of 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 25% 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:
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)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none observed
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none observed
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none observed
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none observed
Remarks on result:
no indication of skin sensitisation
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
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 test

- The 100% treatment caused moderate to severe erythema at 1 hour and no more than mild-to-moderate at 24, 48 and 72 hour observations. Moreover, in one animal a yellowish-brown lesion could be observed which did not clear during the preliminary study. Concentrations from 50% to 75% caused no more than mild-to-moderate erythema, whereas the 25% concentration was free of symptoms. No lesion was observed.

- On the basis of the results of the preliminary dose range finding study, the following treatments were used in the main study:

*intradermal induction: 2.5% 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 the 75% test item caused generally very slight erythema 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 25% test item as a challenge exposure on the left flank of the animals (as this concentration caused no irritation) and with physiological saline on the right flank.

Main study

- Test group: after the challenge with the test item at a concentration of 25% 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 25% 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 and 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 is classified as a non-sensitiser, according to current EU-regulations.