Tris[oxalato(2-)]dilutetium

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 14 June 2016 to 16 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliance

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

22 September 2015

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 various rare earth compounds learned that there is an increased risk for false positive results when performing the LLNA. Additionally, insoluble inorganic substances, such as gadolinium oxalate, are often not able to penetrate the skin.

Test material

Test material information

Constituent 1

Reference substance name:

Tris[oxalato(2-)]digadolinium

EC Number:

212-766-5

EC Name:

Tris[oxalato(2-)]digadolinium

Cas Number:

867-64-1

Molecular formula:

C6Gd2O12

IUPAC Name:

gadolinium(3+);oxalate

Test material form:

other: wet powder

Details on test material:

- Name of test material (as cited in study report): digadolinium oxalate
- CAS nr: 867-64-1
- Physical state: solid
- Appearance: wet white powder

Specific details on test material used for the study:

- Correction for purity of the test item was applied using a correction factor of 2.05. Concentration was calculated to the anhydrous form.

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: male albino guinea pigs, LAL/HA/ BR; LAB-ÁLL Bt., Budapest, 1174 Hunyadi u. 7., Hungary
- Age at first dosing (Day 1): young adult, ~ 7 weeks old
- Weight at randomisation (Day-1): 376-404 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” produced by J. Rettenmaier & Söhne GmbH+CO.KG (D-73494 Rosenberg, Germany) was available to the 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: 13 days before start of treatment under laboratory conditions. Animals were examined on arrival and only healthy animals were used for the test. The health status was certified by the veterinarian.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4-24.5°C
- Humidity (%): 27-80%
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12/12, light from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

other: intradermal (main study part I)

Vehicle:

other: 1% methyl cellulose

Concentration / amount:

1%

Day(s)/duration:

day 1 of treatment

Induction 2

Route:

other: dermal (main study part II)

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100% (as supplied)

Day(s)/duration:

day 8 of treatment; 48 hours of exposure

Challenge

No.:

#1

Route:

epicutaneous, semiocclusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100% (as supplied); saline in right side

Day(s)/duration:

day 22 of treatment, 24 hours of exposure

No. of animals per dose:

Preliminary test: 8 male animals
Main test: 10 in the test group, 5 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 were 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 1% methyl cellulose were used for intradermal injection and 25, 50, 75% (w/v) and 100% (as supplied, dampened with saline) for dermal application.
- 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 used in a 1:1 mixture (v/v) of Freund's Complete Adjuvant and physiological saline solution. Each concentration was injected in duplicate where applicable. Two animals were used per concentration.
- For the topical application, the volume of the concentrations was 0.5 mL. For the 100% (undiluted) treatment, 0.5 g test item applies as described in OECD 404 for solids, so in this study with purity correction, 1.025 g test item was used, 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 animals. Two animals per concentration were used.
- Skin reactions were observed and recorded as follows: 1, 24, 48 and 72 hours after the patch removal and/or treatment
- Skin effects were scored for erythema and oedema, any other observation of changes to the skin was recorded.

MAIN STUDY
A. INDUCTION EXPOSURE
A.1 INTRADERMAL INDUCTION EXPOSURE (day 1)
- No. of exposures: three pairs of intradermal injections (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 1% test item in 1% methyl cellulose; 2 injections of 1% 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 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

A.2 DERMAL INDUCTION EXPOSURE (day 8)
- Since the undiluted test item was not a skin irritant in the dermal dose range-finding study, the test area was painted with 0.5 mL of 10% sodium dodecyl sulphate in Vaseline 24 hours prior to the topical induction application, in order to create a local irritation.
- Site: The same scapular region that received the intradermal injections, was used for dermal induction exposure.
- Test groups: Seven days after the intradermal injections, the same hair-free scapular area was treated. 100% (1.025 g) of the test item was placed on a 2.5x2.5 cm sterile gauze patch (4 layers of porous gauze pads) and dampened with saline, then applied 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). 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 22)
- Day(s) of challenge: 22
- Exposure period: 24 hours
- 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 groups: 100% (1.025 g) of the test item was dampened with saline on a 5x5 cm sterile gauze patch, then applied to the left side of all animals (both the test and the control). 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 24 hours with a fully occlusive foil (Closed Patch Test). After patch removal, any remaining test item was removed with a wet gauze swab. The right shaved side of all animals was treated with 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 exposures) were evaluated according to the scoring system by Draize (1959).

TERMINAL PROCEDURE:
Animals were euthanised with an intraperitoneal injection of Euthanimal 40% (sodium pentobarbital) followed by cervical dislocation to confirm death. No examination was performed and the carcass discarded.

Challenge controls:

100% (1.025 g as anhydrous Gd oxalate) of test item was dampened with saline on a 5x5 cm sterile gauze patch, then applied to the left side of all animals. The right shaved side of all animals was treated with saline.

Positive control substance(s):

yes

Remarks:

2-mercaptobenzothiazole

Results and discussion

Positive control results:

The dermal scores represented discrete erythema (score 1) that 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:

100% (1.025 g as anhydrous Gd oxalate)

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:

100% (1.025 g as anhydrous Gd oxalate)

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:

100% (1.025 g as anhydrous Gd oxalate)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

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

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

100% (1.025 g as anhydrous Gd oxalate)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

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

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 test

- The two highest concentrations (5% and 2.5%) were not injectable intradermally due to the physical properties of the liquid formulation and the very fine injection needle required. A concentration of 1% caused only very slight erythema (score 1) at the 24-hour observation point. As the two highest concentrations were not applicable in this test, 1% was used for intradermal application in the main study, as it was the highest achievable concentration.

- The dermal treatments at the tested concentrations produced no reaction on the skin of guinea pigs. 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 1% methyl cellulose was used in the test group for intradermal injections. The control group was treated with injections of 1% methyl cellulose only.

*Dermal induction: 100% (1.025 g as anhydrous Gd oxalate) of test item (as supplied, dampened with saline) was used. Saline only was applied to the control animals. Since the 100% (undiluted) test item was not a skin irritant in the dermal dose range-finding study, the test area was painted with 0.5 mL of 10% sodium dodecyl sulphate in Vaseline 24 hours prior to the topical induction application, in order to create local irritation. Therefore six days after the intradermal injections, the same scapular area (approximately 5x5 cm) was clipped free of hair (if necessary) prior to the application.

*Challenge phase: All animals of the treatment and control group were treated with 100% test item (as supplied, dampened with saline) on the left flank and saline only on the right flank, as a challenge exposure.

Main study

- Test group: After the challenge with the test item at a concentration of 100% (undiluted, dampened with saline), no positive response was observed in the treated animals on the left flank. The mean of the scores for erythema was 0.00 according to the 24- and 48-hour 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 100% (undiluted, dampened with saline), no visible changes were found at the 24- and 48-hour 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: All control and treated animals had lower body weight on Day 21 when compared to Day 14. From Day 21 to Day 25 the body weight increased with the exception of one control and one treated animal. Since control animals also exhibited a body weight decrease, this change was not considered test item-related.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

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

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.

The skin sensitisation potential test method (OECD 406) was preferred above LLNA (OECD 429) since previous experience with several water soluble Rare Earth compounds learned that their irritating potential may confound the results of LLNA tests. Additionally, insoluble inorganic forms are often not able to penetrate the skin.

Based on the results of a preliminary test, ten test animals were subjected to sensitisation procedures in a two-stage process incorporating an induction phase using an intradermal treatment followed by a 48-hour topical application (dermal treatment under an occlusive dressing). The test item was used at a concentration of 1% (w/v) in 1 % methyl cellulose for intradermal injections and at a concentration of 100% (undiluted, dampened with saline) for topical sensitisation treatment. Since the 100% (undiluted) test item was not a skin irritant in the dermal dose range-finding study, the test area was painted with 0.5 ml of 10% sodium dodecyl sulphate in Vaseline 24 hours prior to the topical induction application, in order to create a local irritation. Five control guinea pigs were simultaneously exposed to 1% methyl cellulose only during the sensitisation phase (intradermal treatment) and saline (dermal treatment).

Two weeks after the last induction exposure, a challenge dose at a concentration of 100% (undiluted, dampened with saline) was dermally administered on the left side of all animals for 24 hours. The right side of the animals was treated with saline only. Skin reactions were measured 24 and 48 hours after patch removal.

Results

No test item-related signs of systemic toxicity were observed in any animal.

Incidence rate:

No signs of contact sensitisation were detected after the challenge exposure in guinea pigs previously exposed to the test item during 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.

Conclusion

In conclusion, under the conditions of the present assay the test item Digadolinium oxalate was shown to have no sensitisation potential and is classified as a non-sensitizer, according to current EU-regulations.