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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:
2018-08-23 to 2018-10-xx
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
17 July 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
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 LLNA (OECD 429) since previous experience with water soluble rare earth compounds learned that there is an increased risk for false positive results when performing the LLNA.

Test material

Constituent 1
Chemical structure
Reference substance name:
Yttrium chloride hexahydrate
Cas Number:
10025-94-2
Molecular formula:
YCl3.6H2O
IUPAC Name:
Yttrium chloride hexahydrate
Test material form:
solid: particulate/powder
Details on test material:
- Name of the test material (as cited in the report): yttrium trichloride hexahydrate
- Physical state: solid
- Appearance: white to yellowish crystalline powder
- Further information on test material confidential.
Specific details on test material used for the study:
- Correction for purity of the test item was performed applying a correction factor of 1.59 considering the composition. Concentration was calculated to anhydrous form.
- The test item was formulated using 1% methylcellulose as vehicle.
- The formulations were used approximately within 4 hours after adding the vehicle to the test item and the test item preparation was performed following approved procedures and documented in detail. The formulation was a suspension.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Remarks:
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, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: young adult, ~ 6 weeks old (main test)
- Weight at study initiation: 327-372 g
- Housing: Animals were housed in Macrolon cages size IV, with up to 3 animals/cage to allow socialisation; “Lignocel® 3/4-S Hygienic Animal Bedding” produced by J. Rettenmaier & Söhne GmbH+CO.KG (D-73494 Rosenberg, Holzmühle 1, Germany) was available to animals during the study.
- Diet (e.g. ad libitum): Animals received Cunigra Diet for Rabbits, ad libitum, in the preliminary study (produced by Bonafarm-Bábolna Takarmány Ltd., Hungary). The animals received ssniff® "Complete feed for Guinea pigs – maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany, ad libitum, in the main study (batch number 240 31022, expiry date: 30 November 2018).
- 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 where Cunigra Diet for Rabbits was used. Animals received tap water from municipal supply without addition of ascorbic acid when ssniff® "Complete feed for Guinea pigs – maintenance" was used for feeding, as the food contained the necessary level of ascorbic acid for guinea pigs. 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: 15 days before start of treatment under laboratory conditions (main test)
- Indication of any skin lesions: None

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.5 - 25.8 °C
- Humidity (%): 22 - 78%
- 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:
intradermal
Vehicle:
other: 1% methylcellulose
Concentration / amount:
concentration: 0.5% (w/v)
volume: 0.1 mL
Day(s)/duration:
day 1 of treatment
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
other: 1% methylcellulose
Concentration / amount:
concentration: 75% (w/v)
amount: 0.5 mL
Day(s)/duration:
day 8 of treatment; 48 hours of exposure
Adequacy of induction:
highest technically applicable concentration used
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: 1% methylcellulose
Concentration / amount:
concentration: 75% (w/v)
amount: 0.5 mL
Day(s)/duration:
day 22 of treatment; 24 hours of exposure
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Preliminary test: 12 animals
Confirmatory test: 2 animals
Main test: 10 in the test group, 5 in the cotrol
Details on study design:
RANGE FINDING 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 (all using 1% methyl cellulose as vehicle) was tested to identify the primary irritation following intradermal injection and topical application: 0.01, 0.05, 0.1, 0.5, 1, 2.5 and 5% (w/v) concentrations were used for intradermal injection and 10, 25, 50 and 75% (w/v) concentrations were used for dermal application. Local effects were examined and scored approximately at 1, 24, 48 and 72 hours after the treatment or after patch removal. Skin effects were scored for erythema and oedema, and any other observations of changes to the skin were recorded as well (if present).
- For the intradermal application, 0.1 mL per concentration was injected intradermally into the hair free skin of the animals. Two concentrations were injected per animal, on the right and 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 saline. Each concentration was injected in duplicate. Two animals were used per concentration.
- In the intradermal application, the concentrations between 0.5% and 5% (w/v) caused very slight to well defined erythema and 5% with FCA caused well defined to moderate to severe erythema. In addition, yellowish, whitish-grey or brownish alteration of the skin surface was observed during the preliminary test. The concentrations between 0.01% and 0.1% (w/v) caused only very slight erythema without additional alteration. The size/severity of the alteration between 1-5% (w/v) was too high to be acceptable for a main study. Erythema scores at concentrations between 0.01-0.1% (w/v) were too low for intradermal induction. Based on the condition on the last observation day of the treated areas, the 0.5% (w/v) concentration was considered to be acceptable for a main test.
- 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. One concentration was used on the right side and another concentration on the left side of animals. Two animals per concentration were used.
- In the dermal application, well defined erythema and dry skin were observed at concentration levels of 50 and 75% (w/v) during the 72-hour observation period. Only the 75% (w/v) treatment after 24 hours in one animal showed moderate to severe erythema, however this improved shortly to well defined erythema at the 48-hour observation. No symptoms were noted at concentration levels of 10% and 25% (w/v) during the preliminary test.

CONFIRMATORY TEST
- A main test was carried out after the preliminary test. During review of raw data of the main test, an inconsistency was revealed in the test item formulation data for the topical induction phase. After the review of the results of the main test it was recognised that although the irritating effect of the test item after the intradermal exposure was similar to that observed in the preliminary test, the level of irritation after topical (dermal) induction was significantly different compared to that observed in the preliminary test (all scores were 0 in the main test, while scores of 2 or 3 were recorded in the preliminary experiment). Based on the information and raw data inconsistency from the main test, the validity of the results could not be guaranteed. Therefore, a confirmatory test to confirm previous results and the rerun of the main test was considered necessary. The results of the first main test are not included in the study report, but archived with the raw data.
- In order to guarantee the optimal concentrations for each phase of the main study, the chosen concentrations (based on the results of the preliminary study) were tested using 2 animals. One animal was used for topical (dermal) treatment and another one for intradermal treatment. Each concentration was tested on the right and left side of the animals. The tested concentrations were 0.5% (w/v) in 1% methyl cellulose for intradermal treatment, and 75% (w/v) in 1% methyl cellulose for topical treatment.
- The concentration of 0.5% (w/v) caused very slight to well defined erythema after intradermal treatment. In addition, white and brown alteration of the skin surface were observed during the confirmatory test. Based on earlier experiences this still fell into an acceptable level of alteration for a main test. Based on the condition on the last observation day of the treated areas, the 0.5% (w/v) concentration was considered to be acceptable for a main study.
- The concentration of 75% (w/v) caused erythema only at 1 hour after patch removal and only on one side of the animal. No erythema, or any other symptoms were observed at 24, 48 and 72 hours. Although the results were not identical to the earlier preliminary test, since this was the highest feasible concentration, it was considered to be applicable for topical induction and challenge treatment.
- In conclusion, based on the results of the Preliminary Dose Range Finding Study and the confirmatory test, the following treatments were chosen for the main study:
- Intradermal induction: 0.5% (w/v) test item formulated in 1% methyl cellulose in the test group for intradermal injections. Injection of 1% methyl cellulose only in the control group.
- Topical induction: 75% (w/v) test item formulated in 1% methyl cellulose for dermal treatment, being the highest achievable concentration suitable for application to the animals. Treatment of 1% methyl cellulose only in the control group. No sodium dodecyl sulphate pre-treatment was considered to be required before topical induction as the chosen concentration for the intradermal induction phase caused erythema and skin alteration.
- Challenge phase: All animals of the treatment and control group: 75% (w/v) test item (in 1% methyl cellulose) as a challenge exposure on the left side (as this concentration was the highest which caused no irritation in the confirmatory test between 24-72 hours observation) and with 1% methyl cellulose on the right side.

MAIN STUDY
The dose levels for the main study were selected based on the results of the preliminary and confirmatory tests.
Control animals were treated similarly to test animals, except that during the induction phase, the test item was omitted.
Induction involved two main procedures: intradermal treatment (Main Test I) and dermal exposure (Main Test II) with closed patch technique.

A1. INTRADERMAL INDUCTION EXPOSURE (day 1)
- No. of exposures: Three pairs of injections per animal (0.1 mL/site).
- Test groups (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 0.5% (w/v) test item in 1% methyl cellulose; 2 injections of 0.5% (w/v) test item, formulated in a 1:1 mixture (v/v) of Freund's Complete Adjuvant and physiological saline solution.
- Control group (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 1% methyl cellulose; 2 injections of 1% methyl cellulose, formulated in a 1:1 mixture (v/v) of Freund's Complete Adjuvant and physiological saline solution.
- Site: Scapular region. On the day before treatment, an area of approx 5x5 cm was clipped free of hair and was carefully shaved
- Frequency of applications: 1 application
- Skin reactions were observed and recorded as follows: 24 hours after treatment

A2. DERMAL INDUCTION EXPOSURE (day 8)
- Site: The same scapular region that received the intradermal injections was used for dermal induction exposure.
- Test groups: A 2.5x2.5 cm sterile gauze patch (approximately 4 layers of porous gauze pads) was saturated with approximately 0.5 mL of the test item at 75% (w/v) concentration (the highest dose found to cause no more than mild-to-moderate skin irritation in the preliminary dose range finding study) and placed over the injection sites (scapular region). The control group was treated with 0.5 mL vehicle (1% methyl cellulose) according to the same method. 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 1% methylcellulose only.
- Frequency of applications: 1 application
- Skin reactions were observed and recorded as follows: 1, 24, 48 and 72 hours after patch removal

B. CHALLENGE EXPOSURE (day 22)
- Site: 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 and control groups: A 2.5x2.5 cm patch of sterile gauze was saturated with approximately 0.5 mL of the test item at 75% (w/v) concentration (the highest dose found to cause no skin irritation in the confirmatory test) and applied to the left side of all animals (both the test and the control animals). The right shaved side area of all animals was treated with the vehicle (1% methyl cellulose). Treatment was performed as described for the Closed Patch Test (see dermal induction exposure). The time of exposure was 24 hours. After the patch removal, any remaining test item was removed with a wet gauze swab.
- Frequency of applications: 1 application
- Skin reactions were observed and recorded as follows: 24 and 48 hours after patch removal

OBSERVATIONS AND SCORING
- 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.
- Mortality/clinical signs: Daily recorded 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 intradermal and dermal irritation scores (in case of induction exposure) were evaluated according to the scoring system by Draize (1959).

SCORING
- Erythema and eschar formation:
0 = No erythema
1 = Very slight erythema (barely perceptible)
2 = Well defined erythema
3 = Moderate to severe erythema
4 = Severe erythema (beef redness) to slight eschar formation (injuries in depth)

- Oedema formation:
0 = No oedema
1 = Very slight oedema (barely perceptible)
2 = Slight oedema (edges of area well defined by definite raising)
3 = Moderate oedema (raised approx. 1 mm)
4 = Severe oedema (raised more than 1 mm and extending beyond area of exposure)

After the challenge exposure, each animal was examined and scored 24 and 48 hours after the end of the exposure period.

Grading was performed according to the following system (classification of skin sensitisation):
0 = no visible change
1 = discrete or patchy erythema
2 = moderate and confluent erythema
3 = intense erythema and swelling

TERMINAL PROCEDURE
Terminal animals were sacrificed under pentobarbital anaesthesia.
Challenge controls:
The right shaved side area of all animals was treated with the vehicle (1% methyl cellulose).
Positive control substance(s):
yes
Remarks:
2-mercaptobenzothiazole

Results and discussion

Positive control results:
Challenge with the test item 2-mercaptobenzothiazole elicited discrete and moderate erythema (score 1 or 2) on the skin surface of previously sensitised guinea pigs. The mean of the scores was 0.90 (80% of animals) at the 24-hour observation and 0.70 (70% of animals) at the 48-hour observation. In the control group the mean of the scores was 0.00.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
75% (w/v) formulated in 1% methylcellulose
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
4 animals out of 10 had erythema score 1 or 2 (mean score 0.50)
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
75% (w/v) formulated in 1% methylcellulose
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
5 animals out of 10 had erythema score 1 or 2 (mean score 0.60)
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
1% methylcellulose
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no visible changes
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
1% methylcellulose
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no visible changes
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 in 1% methylcellulose
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
Discrete and moderate erythema (score 1 or 2) on the skin surface of previously sensitised guinea pigs (mean score 0.90).
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
50% w/v (2-mercaptobenzothiazole)
No. with + reactions:
7
Total no. in group:
10
Clinical observations:
Discrete and moderate erythema (score 1 or 2) on the skin surface of previously sensitised guinea pigs (mean score 0.70).
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

SKIN EFFECTS AFTER THE CHALLENGE EXPOSURE

- Test group: After challenge with the test item at a concentration of 75% (w/v) formulated in 1% methyl cellulose, a positive response was observed in the treated animals on the left flank as follows:

*At the 24-hour observation, sensitisation was observed in 40% of the test group animals (4 animals out of 10 had erythema score 1 or 2).

*At the 48-hour observation, sensitisation was observed in 50% of the test group animals (5 animals out of 10 had erythema score 1 or 2).

- Test group: At 24- and 48-hour observations, the right side of all animals treated with the vehicle was symptom-free.

- Control group: After the challenge with the test item at a concentration of 75% (w/v) formulated in 1% methyl cellulose (the same mixture formulated for the Test group above, at the same time), no visible changes were found at the 24- and 48-hour examinations on the left flank. The right shaved sides of control animals were treated with 1% methyl cellulose and no reaction was noted. In the animals of the control group, the mean of the scores was 0.00 according to the 24- and 48-hour observations.

CLINICAL OBSERVATIONS/MORTALITY

- No signs of systemic or local toxicity were observed in any animal.

- 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:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Challenge with the test item (yttrium trichloride hexahydrate) evoked positive responses in the test animals previously sensitised with the test item. The net response value represented an incidence rate of 40% and 50% and net score values of 0.50 and 0.60 at 24- and 48-hour observations, respectively. In conclusion, under the conditions of the present assay the test item yttrium trichloride hexahydrate was shown to have a skin sensitisation potential and needs to be classified as a skin sensitiser, according to current EU-regulations.