Tris[oxalato(2-)]dilutetium

Endpoint summary

Description of key information

Two in vitro studies were performed with the substance Lutetium oxalate. First a DPRA study was conducted according to the OECD test Guideline 442C and in compliance with GLP requirements. However, under the experimental conditions of this study, the test item Lutetium oxalate peptide reactivity analysis was inconclusive due to its insolubility, and therefore the test item was considered incompatible with DPRA test. Then, the in vitro potential for skin sensitisation of Lutetium oxalate was tested using the KeratinoSens test following the OECD Guideline 442D and in agreement with GLP requirements. Three runs were done. However, since contradictory results were observed in the 3 runs (positive, negative and equivocal in the 1st, 2d and 3rd run, respectively), the test item was considered as not applicable to the KeratinoSens assay and the results were thus considered inconclusives.

Thus, to cover this endpoint a read-across approach was done using lutetium oxide silicate and gadolinium oxalate, 2 rare earth substances as source substances with similar physico-chemical properties to cover both moieties of the target substance Lutetium oxalate.

In an in vivo skin sensitisation study in guinea pigs (Tarcai, 2017; GPMT, study performed according to OECD guideline 406, Klimisch 1), the related substance Lutetium oxide silicate demonstrated to not induce a skin sensitisation response in the guinea pig after challenge exposures in animals previously exposed to the test item during the experiment. In the control and treated animals, the mean of the scores was 0.00 according to the 24- and 48-hour results. Therefore, lutetium oxide silicate does not need classification for skin sensitisation according to the current EU-regulations. The same is assumed for Lutetium oxalate.

In an in vivo skin sentisation study in guinea pigs performed on the related substance gadolinium oxalate (Tarcai, 2016, GPMT study done according to OECD guideline 406, scored Klimisch 1), no sign of contact sensitisation were detected after the challenge exposure in guinea pigs previously exposed to the test item during the experiment. In the control and treated animals, the mean of the scores was 0.00 according to the 24- and 48-hour results. Under the conditions of the present assay, gadolinium oxalate was shown to have no skin sensitisation potential and is classified as a non-sensitiser, according to current EU-regulations. The same is assumed for Lutetium oxalate.

The read across justification is attached to IUCLID Section 13.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 23 February 2017 to 19 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliance

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

yes

Remarks:

The test item was found not soluble in the recommended vehicles. Thus, it was suspended at 100 mM in acetonitrile.

GLP compliance:

yes (incl. QA statement)

Remarks:

2 November 2016

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Correction factor: 1.16

Details on the study design:

TEST ITEM FORMULATION PREPARATION
The test item was pre-weighed and stored under appropriate conditions until ready to perform testing.
Based on its molecular weight, it was suspended at 100 mM (expressed as active matter) in the selected vehicle (acetonitrile) without sonication, then maintained under magnetic stirring (for 3 minutes) until the test item samples preparation.
This formulation was a fine suspension and was used just after its preparation.

DESIGN OF THE DIRECT PEPTIDE REACTIVITY ASSAY
The test item was tested in one run. The run was processed as described below.
- Preparation of the samples:
The following samples were prepared in triplicate except for the co-elution control samples for which only one sample was prepared per peptide buffer.

- Co-elution control samples preparation:
For the co-elution control with cysteine peptide: 50 μL of test item formulation was incubated with 750 μL of cysteine peptide dilution buffer (without cysteine peptide) and 200 μL of acetonitrile.
For the co-elution control with lysine peptide: In parallel, 250 μL of test item formulation was incubated with 750 μL of lysine peptide dilution buffer (without lysine peptide).

- Reference control samples preparation:
* Reference control A and B samples: In a vial, acetonitrile was added to a volume of peptide solution (cysteine or lysine) to achieve a nominal concentration of 0.500 mM.
* Reference control C samples: Reference control C samples were prepared for each solvent used to dissolve the test and positive control items.
For the reference control C prepared with cysteine peptide: 50 μL of vehicle (acetonitrile) was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
For the reference control C prepared with lysine peptide: In parallel, 250 μL of vehicle (acetonitrile) was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate buffer at pH 10.2).
* Cinnamaldehyde (positive control) depletion control samples preparation:
For the reactivity of cinnamaldehyde with cysteine peptide: 50 μL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
For the reactivity of cinnamaldehyde with lysine peptide: In parallel, 250 μL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).
* Test item samples preparation:
For the reactivity of test item with cysteine peptide: 50 μL of test item formulation was incubated with 750 μL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 μL of acetonitrile.
For the reactivity of test item with lysine peptide: In parallel, 250 μL of test item formulation was incubated with 750 μL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).
Note: the formulation at 100 mM was maintained under magnetic stirring during the whole test item samples preparation.

- Incubation of the samples:
All samples (co-elution controls, reference controls, test item and positive control samples) were then incubated during 24 (± 2) hours at 25°C and protected from light before injection into the HPLC/UV system.
The test item samples were incubated under magnetic stirring.
At the end of the incubation period, a visual inspection of the samples was performed prior to HPLC analysis to detect precipitate or phase separation (see § Results).
Samples presenting precipitate were centrifuged at 400g for a period of 5 minutes at room temperature and only supernatants were then injected onto the HPLC/UV system. Otherwise, the vials were directly transferred onto the HPLC/UV system.

- Preparation of the calibration curve samples
One set of calibration standards was prepared with each analytical sequence by spiking each peptide (lysine and cysteine) in separate solutions of 20% acetonitrile peptide dilution buffer to obtain at least six different concentration levels ranging from 0.0167 to 0.534 mM. A dilution buffer blank was also included in the standard calibration curve.
The calibration curves were defined by the relationships between the peak area signal of the peptide versus the nominal concentration. These curves were obtained by using the appropriate mathematical model.

- HPLC/UV analysis of the samples:
The study samples were assayed in batches using HPLC/UV analysis.
For each peptide, the analytical sequence included at least:
. one blank sample (peptide dilution buffer),
. one calibration curve injected at the beginning of the analytical batch,
. three reference control A samples,
. the co-elution control sample,
. three reference control B samples,
. reference control C samples (replicate 1),
. positive control sample (replicate 1),
. test item study sample (replicate 1).
The injection order of the reference control C, positive control and test item study samples were reproduced identically for replicate 2 and then replicate 3:
. three reference control B samples.

Key result

Run / experiment:

other: cysteine peptide

Parameter:

other: mean depletion

Value:

0.07

Key result

Run / experiment:

other: lysine peptide

Parameter:

other: mean depletion

Value:

0

Other effects / acceptance of results:

EVALUATION OF THE RESULTS
The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.
Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide using the formula described in § Data analysis and calculation (Any other information on materials and methods incl. tables).
. for the cysteine peptide, the mean depletion value was 0.07%,
. for the lysine peptide, the mean depletion value was 0.00%.
The mean of the percent cysteine and percent lysine depletions was equal to 0.03%. However, since precipitate/suspension was observed at the end of the incubation with the peptides, the peptides depletion may be underestimated.

Since the mean of the percent cysteine and percent lysine depletions was < 6.38%, the test item could have been considered to have no or minimal reactivity. However, since the test item was not completely dissolved in the vehicle prior the test item samples preparation (deviation to the OECD guideline No. 442C) and since precipitate/suspension was observed at the end of the incubation with both peptides, no firm conclusion on the lack of reactivity can be drawn from these results.

SOLUBILITY RESULTS

Several vehicles were tested during the solubility assay. The test item was found not soluble at 100 mM in acetonitrile, milli-Q water, 1:1 mixture of acetonitrile:milli-Q water, isopropanol, acetone, 1:1 mixture of acetonitrile:acetone, 1:9 mixture of DMSO:acetonitrile and 1:1 mixture of DMSO:acetonitrile, even after a sonication step of 1 minute.

Based on complementary solubility results, a fine suspension was obtained at 100 mM in acetonitrile (without sonication) but forming a deposit when stopping agitation. In order to avoid sedimentation of the suspension of test item, the formulation was maintained under magnetic stirring from its preparation to its incubation with the peptides. The test item samples were maintained under magnetic stirring until the end of the incubation with the peptides.

EVALUATION OF THE PRESENCE OF PRECIPITATE AT THE END OF THE INCUBATION WITH PEPTIDES

At the end of the incubation period, a visual inspection of all samples (co-elution controls, reference controls, test item and positive control samples) was performed prior to HPLC analysis.

As precipitate was observed in the test item, positive control and reference control samples incubated with the cysteine and lysine peptides, and in co-elution samples prepared with the lysine and cysteine dilution buffer, these vials were centrifuged at 400g for 5 minutes at room temperature to force precipitate to the bottom of the vial. Thus, only supernatants were injected into the HPLC/UV system.

TABLES OF RESUTLS: Percent peptide depletion for the test item samples

Table 1: Determination of cysteine peptide and lysine peptide depletion in samples spiked with a solution at 100 mM of lutetium oxalate

Sample number	Cysteine peptide		Lysine peptide		Mean depletion rate (%) of Lutetium oxalate	Depletion classification
	Peak area (µV/sec)	% depletion	Peak area (µV/sec)	% depletion
1 2 3	2545628 2507549 2496400	0.00* 0.00* 0.21	2349757 2350224 2365017	0.00* 0.00* 0.00*
Mean SD % CV	- - -	0.07 0.12 173.2	- - -	0.00* nc nc	0.03	Inconclusive
Precipitate:	Yes		Yes
Micelle	No		No

*: Value set to 0 due to negative depletion

-: not applicable

nc: not calculated

Table 2: Determination of cysteine peptide and lysine peptide concentration in reference control C samples prepared in degassed acetronitrile

Sample number	Cysteine peptide			Lysine peptide
	Peak area (µV/sec)	Concentration (mM)	% Dev	Peak area (µV/sec)	Concentration (mM)	% Dev
1 2 3	2532711 2470468 2501550	0.474 0.462 0.468	(-5.2) (-7.5) (-6.4)	2348303 2313793 2357013	0.497 0.490 0.499	(-0.5) (-2.0) (-0.2)
Mean SD % CV	2501576 - -	0.468 0.006 1.2	(-6.4) - -	2339703 - -	0.495 0.005 1.0	(-0.9) - -

Table 3: Determination of % interference due to co-elution of luetium oxalate with cysteine or lysine peptides

	Peak detected at the cysteine retention time		Peak detected at the lysine retention time
Sample number	Peak area (µV/sec)	% Interference	Peak area (µV/sec)	% Interference
1	0	(0.0)	0	(0.0)
Precipitate:	Yes		Yes
Micelle	No		No

Interpretation of results:

other: Inconclusive

Conclusions:

Under the experimental conditions of this study, the test item Tris[oxalato(2-)]dilutetium peptide reactivity analysis was inconclusive due to its insolubility, and therefore the test item was considered incompatible with the DPRA test.

Executive summary:

The objective of this study was to evaluate the reactivity of the test item to synthetic cysteine and lysine peptides. This test is part of a tiered strategy for skin sensitization assessment.

Methods

The reactivity of the test item was evaluated in chemico by monitoring peptide depletion following a 24-hour contact between the test item and synthetic cysteine and lysine peptides. The method consisted of the incubation of a diluted solution of cysteine or lysine with the test item for 24 hours. At the end of the incubation, the concentrations of residual peptides were evaluated by HPLC with Ultra-Violet detection at 220 nm.

Peptide reactivity was reported as percent depletion based on the peptide peak area of the replicate injection and the mean peptide peak area in the three relevant reference control C samples (in the appropriate solvent).

Results

Based on solubility trials, the test item was found not soluble in the recommended vehicles. Thus, it was suspended at 100 mM in acetonitrile, leading to a deviation to the OECD guideline No. 442C.

The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. The study was therefore considered to be valid.

Analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with either the lysine or the cysteine peptides. As a result, the mean percent depletion values were calculated for each peptide using the formula described in § Data analysis and calculation:

* for the cysteine peptide, the mean depletion value was 0.07%,

* for the lysine peptide, the mean depletion value was 0.00%.

The mean of the percent cysteine and percent lysine depletions was equal to 0.03%. However, since precipitate/suspension was observed at the end of the incubation with the peptides, the peptides depletion may be underestimated.

Since the mean of the percent cysteine and percent lysine depletions was < 6.38%, the test item could have been considered to have no or minimal reactivity. However, since the test item was not completely dissolved in the vehicle prior the test item samples preparation (deviation to the OECD guideline No. 442C) and since precipitate/suspension was observed at the end of the incubation with both peptides, no firm conclusion on the lack of reactivity can be drawn from these results.

Conclusion

Under the experimental conditions of this study, the test item Tris[oxalato(2-)]dilutetium peptide reactivity analysis was inconclusive due to its insolubility, and therefore the test item was considered incompatible with the DPRA test.

Reference 2

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

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.

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.

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.

Route:

other: intradermal (main study part I)

Vehicle:

other: 1% methyl cellulose

Concentration / amount:

1%

Day(s)/duration:

day 1 of treatment

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

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

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.

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

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

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.

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.

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

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.

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.

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Report date: 2 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliance

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.

Specific details on test material used for the study:

Correction for purity of the test item was applied with a correction factor 1.05 considering the composition. Concentrations were calculated to anhydrous form as requested by the Sponsor.

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, recognised by the international guidelines as recommended test system..
- Femalesnulliparous and non-pregnant: yes
- Age at study initiation: Young adult, 9 weeks old
- Weight at study initiation: 363 - 435 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., Hungary), 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: 19 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): 16.5 - 21.6 °C
- Humidity (%): < 20 - 60%
- 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: 11 October 2016 To: 13 January 2017

Route:

intradermal

Vehicle:

other: 1% methylcellulose

Concentration / amount:

- control group: 1% methylcellulose
- test control: 1% w/v test item formulated in 1% methylcellulose

Day(s)/duration:

Not applicable

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

Route:

epicutaneous, occlusive

Vehicle:

other: 1% methylcellulose

Concentration / amount:

- control group: 1% methylcellulose
- test group: 100% w/v test item formulated in 1% methylcellulose

Day(s)/duration:

48 h

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

No.:

#1

Route:

epicutaneous, semiocclusive

Vehicle:

other: 1% methylcellulose

Concentration / amount:

Control group and test group: 100% w/v test item formulated in 1% methylcellulose

Day(s)/duration:

24 h

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Preliminary test: 6 female animals
Main test: 10 in the test group, 5 in the control group

Details on study design:

RANGE FINDING TESTS:
Test item concentrations:
- intradermal injection: 1, 2.5 and 5% (w/v)
- dermal application: 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 intradermally into the hair free skin of the animals where the format properties made it possible. Two concentrations were injected on the right side and another two concentrations on the left side of the animals. The concentrations of 5% for intradermal treatment were also tested in a 1:1 mixture (v/v) of Freund's Complete Adjuvant and 1% methyl cellulose (1% MC) solution. Each concentration was injected in duplicate. Two animals were used per concentration.
The concentration of 2.5%, 5% and 5% with FCA caused maximum of well-defined erythema (score 2); 1% caused very slight erythema (score 1). The format of 5% with FCA could not be administered as a whole (the skin resisted to the injection), thus two additional animals were used after the end of the first preliminary study to check the maximum concentration (with saline or 1% methyl cellulose) that is administrable as a whole injection (0.1 mL). Concentrations of 5%, 2.5% and 1% with FCA:saline; and 5% and 2.5% FCA:1% methyl cellulose were not appropriate for intradermal treatment (not possible to inject or not possible to inject as a whole). The highest applicable concentration was 1% in FCA:1% methyl cellulose, therefore this concentration and vehicle was considered to be appropriate for the Main Study intradermal treatment.

- 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. In the preliminary study one concentration was used on the right side and another concentration on the left side of animals. Two animals per concentrations were used in the preliminary study.
Adverse effects were not observed during the dermal treatment of the preliminary study in each concentration (25%, 50%, 75% and 100%).

MAIN STUDY
A. INTRADERMAL INDUCTION EXPOSURE
- Day of treatment: 1
- No. of exposures: 1
- Test group:
• 2 injections of Freund's Complete Adjuvant and 1% MC in a 1:1 (v/v) mixture,
• 2 injections of 1% MC,
• 2 injections of 1% test item formulated in a 1:1 mixture (v/v) of Freund's Complete Adjuvant and 1% MC
- Control group:
• 2 injections of Freund's Complete Adjuvant and 1% MC in a 1:1 (v/v) mixture,
• 2 injections of 1% MC,
• 2 injections of vehicle (1% MC), formulated in a 1:1 mixture (v/v) of Freund's Complete Adjuvant and 1% MC.
- Site: scapular region
- Time of observations: 24 hours after the treatment

B. DERMAL INDUCTION EXPOSURE
- Day 7: treatment with 10% Sodium Dodecyl Sulphate in vaseline
[Since the 100% test item was not skin irritant in the dermal dose range-finding study, the induction 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.]
- Day of treatment: 8
- No. of exposures: 1
- Exposure period: 48 hours
- Test group and contro group: A 2.5 x 2.5 cm sterile gauze patch (approximately 4 layers of porous gauze pads) was saturated with approximately 0.5 mL of the test item at 100% concentration and placed over the injection sites (scapular region). The control group was treated with 0.5 mL vehicle (1% MC) with 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 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

C. CHALLENGE EXPOSURE
- Day of challenge: 22
- No. of exposures: 1
- Exposure period: 24 hours
- Test group and control group: A 2.5 x 2.5 cm patch of sterile gauze patch was saturated with approximately 0.5 mL of the test item at 100% concentration (highest achievable and 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 vehicle (1% MC).
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

Positive control results:

Challenge with the test item 2-Mercaptobenzothiazole elicited discrete erythema (score 1 ) on the skin surface of previously sensitised guinea pigs. The mean of the scores were 0.80 (80% of animals) at the 24 hours observation and 0.70 (70% of animals) at the 48 hours observation. In the control group the mean of the scores was 0.00.
On the basis of the results of the present study, the test item 2-Mercaptobenzothiazole was classified as a skin sensitiser. This demonstrates that the Magnusson and Kligman method (OECD 406) in this laboratory is considered to be reliable.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

left side: 100% (w/v) test item formulated in 1% MC; right side: 1% MC 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: 100% (w/v) test item formulated in 1% MC; right side: 1% MC 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:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

left side: 100% (w/v) test item formulated in 1% MC; right side: 1% MC 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

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

left side: 100% (w/v) test item formulated in 1% MC; right side: 1% MC 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

Key result

Reading:

1st reading

Hours after challenge:

24

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

Key result

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

Remarks on result:

positive indication of skin sensitisation

Body weight

There were no notable differences between the test animal group and the control group.

Clinical observations and mortality

No signs of systemic or local toxicity were observed.

No mortality was observed during the study.

Skin effects after the induction exposure

* Test group: After the challenge with the test item at a concentration of 100% formulated in 1% MC, 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-hour results. The right shaved side of test animals was treated with 1% MC and no reaction was noted.

* Control group: After the challenge with the test item at a concentration of 100% formulated in 1% MC, no visible changes were found at the 24 and 48 hours examinations on the left flank. The right shaved side of control animals were treated with 1% MC and no reaction was noted.

Table 1: Scores of erythema after treatment of challenge exposure

Groups	Animal number	Day 24		Day 25
		Left side	Right side	Left side	Right side
1. Control group	8	0	0	0	0
	5	0	0	0	0
	7	0	0	0	0
	20	0	0	0	0
	17	0	0	0	0
2. Dosed group	14	0	0	0	0
	13	0	0	0	0
	12	0	0	0	0
	10	0	0	0	0
	21	0	0	0	0
	9	0	0	0	0
	19	0	0	0	0
	11	0	0	0	0
	6	0	0	0	0
	16	0	0	0	0

Interpretation of results:

GHS criteria not met

Conclusions:

Challenge with test item (Dilutetium oxide silicate) 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 Dilutetium oxide silicate was shown to have no sensitisation potential and need not be classified as a skin sensitiser, according to current GHS criteria and EUregulations.

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, 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 1% (w/v) for intradermal injections and at a concentration of 100% (w/v) for topical sensitisation treatment formulated in 1% methylcellulose (1% MC). Five control guinea pigs were simultaneously exposed to 1% MC only during the sensitisation phase. Since the 100% test item was not skin irritant in the dermal dose range-finding study, the induction 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.

Two weeks after the last induction exposure, a challenge dose at a concentration of 100% in 1% MC was administered on the left side of all animals. The right side of the animals were treated with 1% MC. Challenge was performed by dermal application of the test item for 24 hours with a fully occlusive foil (Closed Patch Test). 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 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 Dilutetium oxide silicate was shown to have no sensitisation potential and need not be classified as a skin sensitiser, according to current GHS criteria and EUregulations.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From to 26 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliance

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

yes

Remarks:

The cytotoxicity, measured by a MTT reduction test, was not taken into consideration in the interpretation of the sensitisation results.

GLP compliance:

yes (incl. QA statement)

Remarks:

2 November 2016

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

Correction factor: 1.16

Details on the study design:

The test item was tested in three independent runs using cells from a different passage number. The third run was performed since non concordant results were obtained between the first two runs. The plates were processed as described below in the § Method.

- Solubility assay
A solubility assay was performed prior the first treatment in order to select the vehicle (among DMSO, water for injections or treatment culture medium). Sonication for 10 minutes and heating at 60°C for 10 minutes were used in order to improve the solubility of the test item.
No visual inspection was performed to evaluate the presence or absence of precipitate/emulsion of the test item formulation diluted in culture medium at the final concentration of 1000 μM.

- Method for a run of KeratinoSens assay:
* Cell seeding for testing:
. Cells were grown using general culture procedures up to 80-90% confluence,
. the day prior to treatment, cells were washed twice with D-PBS containing 0.05% EDTA, harvested, re-suspended in Maintenance medium No. 2 and counted using Trypan Blue dye. Cell concentration was adjusted to a density of 8 x 104 cells/mL,
. cells were then distributed into four 96-well plates (three white plates and one transparent plate), by adding 125 μL (representing 1 x 104 cells) per well taking care to avoid sedimentation of the cells during seeding,
. after seeding, the cells were grown for 24 (± 1) hours in the 96-well microtiter plates prior to test item addition.

* Treatment:
. After the 24-hour growing period, the medium was removed by aspiration and replaced by 150 μL of treatment medium,
. from the Master tubes or flasks 4x maintained under magnetic stirring, a volume of 50 μL was added to each well of the three white assay plates and 50 μL to the transparent plate for the cytotoxicity evaluation,
. all plates were covered by a sealing membrane to avoid evaporation of volatile test items and to avoid cross-contamination between wells,
. the plates were then incubated for 48 (± 2) hours at 37°C, 5% CO2, 90% humidity.

- Endpoint measurements:
* Microscopic observation to evaluate the presence or absence of precipitate - transparent plate:
. After the 48 (± 2) hours incubation period, the presence or absence of precipitate/emulsion was determined in each well by microscopic inspection.

* Luminescence flash signal to evaluate induction signal - white plates:
. After incubation, the supernatants from the white assay plates were discarded,
. the cells were washed once with D-PBS,
. a volume of 20 μL of passive lysis buffer was added to each well and the cells were incubated for 20 (± 2) minutes at room temperature and under orbital shaking,
. the plates containing the passive lysis buffer were then placed in the luminometer for reading using the following program:
- 50 μL of the luciferase substrate was added to each well,
- 1 second after this addition, the luciferase signal was integrated for 2 seconds.

* Absorbance signal to evaluate the cytotoxicity - transparent plate:
. For the cell viability assay plate, the medium was replaced by 200 μL of treatment medium,
. a volume of 27 μL of a MTT solution at 5 mg/mL in D-PBS was then added to each well of the transparent 96-well plate,
. the plates were covered with a sealing membrane and returned at 37°C in the incubator in humidified atmosphere for 4 hours (± 10 minutes),
. at the end of the incubation period, the medium was removed and a volume of 200 μL of a 10% SDS solution was added to each well,
. the plates were covered with a sealing membrane and placed at 37°C in the incubator in humidified atmosphere for an overnight period to extract the formazan from cells,
. after the overnight incubation, the absorption of each well was determined at 600 nm using the plate reader.

- Acceptance criteria:
Each run was considered valid if the following criteria were met:
. the positive control results should be positive, thus the gene induction should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations,
. the average EC1.5 value for the positive control should be within two standard deviations of the historical mean. In addition, the average induction (Imax) in the three replicate plates for the positive control at 64 μM should be between 2 and 8. If the latter criterion was not fulfilled, the dose-response of cinnamic aldehyde was carefully checked, and the run was accepted if there was a clear dose-response with increasing luciferase activity at increasing concentrations for the positive control,
. the average coefficient of variation of the luminescence reading in the negative control wells of the triplicate plates should be < 20%.

- Evaluation criteria of the test item:
The results of each run are analyzed individually and if the test item is classified as positive in two runs, the final outcome is considered positive. If the test item is classified as negative in two runs, the final outcome is negative. In case, the first two runs were not concordant, a third run was performed and the final outcome was that of the two concordant runs.
The test item is considered as positive if the following four conditions are all met in two of two or in two of three runs, otherwise the KeratinoSens prediction is considered as negative:
. the Imax is > 1.5-fold and statistically significantly different as compared to the negative control (as determined by a two-tailed, unpaired Student’s T-test),
. at the lowest concentration with a gene induction > 1.5-fold (i.e. at the EC1.5 determining value), the cell viability is > 70%,
. the EC1.5 value is < 1000 μM (or < 200 μg/mL for test item without MW),
. there is an apparent overall dose-response for luciferase induction (or a reproducible biphasic response).
In cases of precipitate/emulsion in wells after the 48 (± 2) hours incubation period, luciferase activity may be underestimated and a negative conclusion cannot be drawn with sufficient confidence.

Key result

Run / experiment:

other: First run

Parameter:

other: Imax

Value:

21.64

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: First run

Parameter:

other: EC1.5

Value:

14.32

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Second run

Parameter:

other: Imax

Value:

1.45

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Since precipitate was observed in the test item-treated wells at concentrations ≥ 7.81 μM at the end of the 48-hour treatment period, the luciferase activity may be underestimated. Therefore, the lack of activity cannot be drawn with sufficient confidence.

Key result

Run / experiment:

other: Third run

Parameter:

other: Imax

Value:

2.69

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No EC1.5 was calculated in the absence of statistical significance.

Remarks:

Besides the absence of reliable cell viability values, the criteria for a positive response were only partially met in this third run. Since there were gene inductions > 1.5 with an apparent dose-response relationship, these results cannot be considered as clearly negative. Thus, these results were considered as equivocal.

Other effects / acceptance of results:

KERATINOSENS RUN
- First run
All acceptance criteria were met for the positive and negative controls, this run was therefore considered to be valid.
The criterion “the average induction (Imax) in the three replicate plates for the positive control at 64 μM should be between 2 and 8” was not fulfilled (i.e. Imax of 8.48). However, since a clear dose-response with increasing luciferase activity at increasing concentrations was obtained for the positive control, this was considered not to have any impact on the validity of the results of this run.
This run was performed using the following concentrations: 0.49, 0.98, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 μM in culture medium containing 1% DMSO.
At these tested concentrations:
* the cytotoxicity, measured by a MTT reduction test, was not taken into consideration in the interpretation of the sensitisation results (see § Evaluation of the MTT results in Any other information on results incl. tables),
* precipitate was observed in test item-treated wells at concentrations ≥ 3.91 μM at the end of the 48-hour treatment period leading to a potential underestimation of the luciferase activity,
* statistically gene-fold inductions above the threshold of 1.5 in comparison to the negative control was noted at concentrations ≥ 15.6 μM with an apparent dose-response relationship up to 250 μM, followed by a decrease of these gene-fold inductions, probably due to the high test item precipitation observed at 500 and 1000 μM and/or to a cytotoxicity that might be hidden by the potential enzymatic interaction (see § Evaluation of the MTT results in Any other information on results incl. tables),
* the Imax was 21.64 and the calculated EC1.5 was 14.32 μM.
Despite the absence of reliable cell viability values, the criteria for a positive response were considered to be met in this first run.

- Second run
All acceptance criteria were met for the positive and negative controls, this run was therefore considered to be valid.
The same concentrations as those tested in the first run were used and the following results were obtained:
* the cytotoxicity, measured by a MTT reduction test, was not taken into consideration in the interpretation of the sensitisation results (see § Evaluation of the MTT results in Any other information on results incl. tables),
* precipitate was observed in test item-treated wells at concentrations ≥ 7.81 μM at the end of the 48-hour treatment period leading to a potential underestimation of the luciferase activity,
* no statistically significant gene-fold induction above the threshold of 1.5 in comparison to the negative control was noted at any tested concentrations. Moreover, the Imax value was < 1.5 (i.e. 1.45).
Despite the absence of reliable cell viability values, the criteria for a negative response were considered to be met in this second run.
However, since precipitate was observed in the test item-treated wells at concentrations ≥ 7.81 μM at the end of the 48-hour treatment period, the luciferase activity may be underestimated. Therefore, the lack of activity cannot be drawn with sufficient confidence.

Since the results of the first two runs were non concordant (first run positive and second run negative though with limitations due to test item precipitation), a third run was performed in order to draw a solid final outcome.

- Third run
All acceptance criteria were met for the positive and negative controls, this run was therefore considered to be valid.
The same concentrations as those tested in the first two runs were used and the following results were obtained:
* the cytotoxicity, measured by a MTT reduction test, was not taken into consideration in the interpretation of the sensitisation results (see § Evaluation of the MTT results in Any other information on results incl. tables),
* precipitate was observed in test item-treated wells at concentrations ≥ 7.81 μM at the end of the 48-hour treatment period leading to a potential underestimation of the luciferase activity,
* gene-fold inductions above the threshold of 1.5 were noted at concentrations ≥ 250 μM but without any statistically significance in comparison to the negative control. The Imax was 2.69 but no EC1.5 was calculated in the absence of statistical significance.
Besides the absence of reliable cell viability values, the criteria for a positive response were only partially met in this third run. Since there were gene inductions > 1.5 with an apparent dose-response relationship, these results cannot be considered as clearly negative. Thus, these results were considered as equivocal.

DISCUSSION
Since the first run was considered as positive (based on the high statistically significant inductions observed), the second run as negative (though with limitations due to test item precipitation) and the third run as equivocal (non statistically significant but dose-related inductions > 1.5), the test item was considered as not applicable to the KeratinoSens assay and the results are thus inconclusive. Moreover, the luciferase activity may be underestimated due to the presence of precipitate from lower dose-levels, the results were found heterogeneous between runs and the cytotoxicity was not taken into consideration in the interpretation of the results due to the potential impact of the test item being an oxalate on the MTT reduction.

SOLUBILITY TEST

In the solubility test, the test item did not form a stable dispersion at 200 mM in any recommended vehicles (DMSO, water for injections or treatment culture medium) even after 10 minutes of sonication and 10 minutes of heating at 60°C. Then, a fine homogeneous suspension was found at 100 mM in DMSO after 10 minutes of sonication but forming a deposit when stopping agitation (solubility not improved by 10 minutes of heating at 60°C). In order to avoid sedimentation of the suspension of test item, the stock formulation (at 100 mM in DMSO) and further dilutions were maintained under magnetic stirring from their preparation and until treatment. Since the master plates technically cannot be agitated during sampling, they were replaced by master tubes or flasks.

No visual inspection was performed to evaluate the presence or absence of precipitate/emulsion of the test item formulation diluted in culture medium at the final concentration of 1000 μM.

EVALUATION OF THE MTT RESULTS

Cell viability determination is based on cellular mitochondrial succinate dehydrogenase activity measured (within the mitochondria of viable cells) by the reduction and conversion of a yellow dye, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], into a blue formazan salt.

Competitive inhibitor, which shares common characteristics with the substrate, thus allowing it to compete with that substrate for available enzymatic active sites, may interfere with cell viability measurements.

In the present study, the cell viability values were < 70% at all tested concentrations reproductively in the three runs performed. Such values were found at all tested concentrations, without any dose-response relationship. However, the test item being an oxalate, it may have been a competitive inhibitor to the succinate deshydrogenase and thus may have an impact on the MTT reduction (see publications of Hernandez and Stephanopoulos). Therefore, the calculated cell viability values were considered as no reliable and thus were not taken into account in the interpretation of the results, leading to a deviation to the OECD Guideline No. 442D.

VIABILITY VALUES, INDUCTION VALUES, Imax, IC30, IC50 AND EC1.5 VALUES OBTAINED AFTER TREATMENT WITH THE TEST ITEM IN EACH RUN AS WELL AS THE MEAN AND SD VALUES

Table 1: Evaluation of the viability (%) of cultures treated with the test item for each run

Viability results	Concentration (µM)
Tris[oxalate(2-)]dilutetium	0.49	0.98	1.95	3.91	7.81	15.6	31.3	62.5	125	250	500	1000
Viability (%) in Run 1	28	29	24	28	23	23	22	21	19	19	30	39
Viability (%) in Run 2	20	19	20	19	19	20	19	21	19	24	24	24
Viability (%) in Run 3	23	23	24	23	25	24	26	21	22	30	30	31
Mean viability (%)	24	24	23	23	22	22	22	21	20	24	28	31
Geometric mean (%)	24	23	23	23	22	22	22	21	20	24	28	31
SD	4	5	2	4	3	2	4	0	2	5	4	7

Table 2: Gene induction values, I_max, IC₃₀, IC₅₀and EC_1.5 values, mean and SD values obtained after treatment with the test item in each run

Induction values individual replicated	Concentration (µM)
Tris[oxalate(2-)]dilutetium	0.49	0.98	1.95	3.91	7.81	15.6	31.3	62.5	125	250	500	1000
Induction values in Run 1	1.0	0.9	1.0	0.9	1.1	1.6	3.0	8.0	21.6	19.8	5.8	2.6
Induction values in Run 2	0.8	0.9	1.1	0.8	1.1	0.9	0.9	0.9	1.1	1.0	1.1	1.4
Induction values in Run 3	1.10	1.07	1.05	1.00	1.13	1.17	1.03	1.47	1.20	1.52	2.04	2.69
Mean induction	1.0	1.0	1.0	0.9	1.1	1.2	1.6	3.5	8.0	7.4	3.0	2.2
SD	0.1	0.1	0.0	0.1	0.0	0.3	1.2	4.0	11.8	10.7	2.5	0.7

Table 3: I_maxand EC_1.5results

Tris[oxalate(2-)]dilutetium	Imax	EC1.5 (µM)	IC50 (µM)	IC30 (µM)
Run 1	21.64	14.32	-	-
Run 2	1.45	-	-	-
Run 3	2.69	-	-	-
Mean	8.59	n.r.	n.r.	n.r.
Geometric mean	n.r.	n.c.	-	-
SD	11.32	n.c.	-	-

-: no data available

n.c.: not calculated

n.r.: not requested by the OECD Guideline

Interpretation of results:

other: Inconclusive

Conclusions:

As a consequence, and under the experimental conditions of this study, the test item Tris[oxalato(2-)]dilutetium was considered as not applicable to the KeratinoSens assay and the results are thus inconclusive.

Executive summary:

The objective of this study was to evaluate the potential of the test item, Tris[oxalato(2-)]dilutetium, to activate the Nrf2 transcription factor. This test is a part of a tiered strategy for the evaluation of skin sensitisation potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

The design of this study was based on the guideline No. 442D dated February 2015 (except that the cytotoxicity, measured by a MTT reduction test, was not taken into consideration in the interpretation of the sensitisation results) and the study was performed in compliance with CiToxLAB France standard operating procedures and with the OECD Principles of Good Laboratory Practice.

Methods

The KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37°C. Then the medium was removed and the cells were exposed to the vehicle control (DMSO) or to different concentrations of test item (batch 1534498) and of positive control (cinnamic aldehyde). For each run, the test item was suspended in DMSO at 100 mM, sonicated during 10 minutes and then maintained under magnetic stirring until treatment.

The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT (Thiazolyl Blue Tetrazolium bromide) reduction test but was not taken into consideration in the interpretation of the sensitisation results.

Three independent runs were performed.

Results

Evaluation of the MTT results

In the present study, the cell viability values were < 70% at all tested concentrations reproductively in the three runs performed. Such values were found at all tested concentrations, without any dose-response relationship. However, the test item being an oxalate, it may have been a competitive inhibitor to the succinate deshydrogenase and thus may have an impact on the MTT reduction. Therefore, the calculated cell viability values were considered as no reliable and thus were not taken into account in the interpretation of the results, leading to a deviation to the OECD Guideline No. 442D.

First run

All acceptance criteria were met for the positive and negative controls (i.e. the positive control results were positive (Imax = 8.48), the average EC1.5 value for the positive control was within two standard deviations of the historical mean (EC1.5 = 10.81 μM) and the average coefficient of variation of the luminescence reading in the negative control wells of the triplicate plates was < 20% (CV = 12.72%)). This run was therefore considered to be valid.

This run was performed using the following concentrations: 0.49, 0.98, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 μM in culture medium containing 1% DMSO.

At these tested concentrations:

* the cytotoxicity, measured by a MTT reduction test, was not taken into consideration in the interpretation of the sensitisation results,

* precipitate was observed in test item-treated wells at concentrations ≥ 3.91 μM at the end of the 48-hour treatment period leading to a potential underestimation of the luciferase activity,

* statistically gene-fold inductions above the threshold of 1.5 in comparison to the negative control was noted at concentrations ≥ 15.6 μM with an apparent dose-response relationship up to 250 μM, followed by a decrease of these gene-fold inductions, probably due to the high test item precipitation observed at 500 and 1000 μM and/or to a cytotoxicity that might be hidden by the potential enzymatic interaction,

* the Imax was 21.64 and the calculated EC1.5 was 14.32 μM.

Despite the absence of reliable cell viability values, the criteria for a positive response were considered to be met in this first run (i.e. the Imax was > 1.5 fold and statistically significantly different as compared to the negative control, the EC1.5 value was < 1000 μM and there was an apparent overall dose-response for luciferase induction).

Second run

All acceptance criteria were met for the positive and negative controls (i.e. the positive control results were positive (Imax = 5.17), the average EC1.5 value for the positive control was within two standard deviations of the historical mean (EC1.5 = 13.65 μM) and the average coefficient of variation of the luminescence reading in the negative control wells of the triplicate plates was < 20% (CV = 15.20%). This run was therefore considered to be valid.

The same concentrations as those tested in the first run were used and the following results were obtained:

* the cytotoxicity, measured by a MTT reduction test, was not taken into consideration in the interpretation of the sensitisation results,

* precipitate was observed in test item-treated wells at concentrations ≥ 7.81 μM at the end of the 48-hour treatment period leading to a potential underestimation of the luciferase activity,

* no statistically significant gene-fold induction above the threshold of 1.5 in comparison to the negative control was noted at any tested concentrations. Moreover, the Imax value was < 1.5 (i.e. 1.45).

Despite the absence of reliable cell viability values, the criteria for a negative response were considered to be met in this second run (i.e. the Imax was < 1.5).

However, since precipitate was observed in the test item-treated wells at concentrations ≥ 7.81 μM at the end of the 48-hour treatment period, the luciferase activity may be underestimated. Therefore, the lack of activity cannot be drawn with sufficient confidence.

Third run

All acceptance criteria were met for the positive and negative controls (i.e. the positive control results were positive (Imax = 5.26), the average EC1.5 value for the positive control was within two standard deviations of the historical mean (EC1.5 = 7.61 μM) and the average coefficient of variation of the luminescence reading in the negative control wells of the triplicate plates was < 20% (CV = 10.35%)). This run was therefore considered to be valid.

The same concentrations as those tested in the first two runs were used and the following results were obtained:

* the cytotoxicity, measured by a MTT reduction test, was not taken into consideration in the interpretation of the sensitisation results,

* precipitate was observed in test item-treated wells at concentrations ≥ 7.81 μM at the end of the 48-hour treatment period leading to a potential underestimation of the luciferase activity,

* gene-fold inductions above the threshold of 1.5 were noted at concentrations ≥ 250 μM but without any statistically significance in comparison to the negative control. The Imax was 2.69 but no EC1.5 was calculated in the absence of statistical significance.

Besides the absence of reliable cell viability values, the criteria for a positive response were only partially met in this third run (i.e. the Imax was > 1.5 fold but not statistically significantly different as compared to the negative control). Since there were gene inductions > 1.5 with an apparent dose-response relationship, these results cannot be considered as clearly negative. Thus, these results were considered as equivocal.

Discussion

Since the first run was considered as positive (based on the high statistically significant inductions observed), the second run as negative (though with limitations due to test item precipitation) and the third run as equivocal (non statistically significant but dose-related inductions > 1.5), the test item was considered as not applicable to the KeratinoSens assay and the results are thus inconclusive. Moreover, the luciferase activity may be underestimated due to the presence of precipitate from lower dose levels, the results were found heterogeneous between runs and the cytotoxicity was not taken into consideration in the interpretation of the results due to the potential impact of the test item being an oxalate on the MTT reduction.

Conclusion

As a consequence, and under the experimental conditions of this study, the test item Tris[oxalato(2-)]dilutetium was considered as not applicable to the KeratinoSens assay and the results are thus inconclusive.

Reference 5

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

Two in vitro studies (DPRA under OECD test Guideline 442C and KeratinoSens under OECD Guideline 442D) were performed with the substance Lutetium oxalate. However, the reuslts of the DPRA study were inconclusives and it was considered that due to its insolubility, the test item was imcompatible with the DPRA study. In the KeratinoSens study, contradictory results were observed in the 3 runs performed. Thus, the test item was considered as not applicable to the KeratinoSens assay and the results were thus considered inconclusives.
Since both in vitro studies are inconclusives, a read-across approach was done using lutetium oxide silicate and gadolinium oxalate, 2 substances of the rare earth family with similar physico-chemical properties to Lutetium oxalate that were used to cover both moieties of the target substance Lutetium oxalate. The justification for read across is attached in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

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

Remarks on result:

no indication of skin sensitisation

Remarks:

This result is considered relevant for Lutetium oxalate too. The justification for read across is attached to IUCLID Section 13.

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

Remarks on result:

no indication of skin sensitisation

Remarks:

This result is considered relevant for Lutetium oxalate too. The justification for read across is attached to IUCLID Section 13.

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

Remarks on result:

no indication of skin sensitisation

Remarks:

The justification for read across is attached to IUCLID Section 13.

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

Remarks on result:

no indication of skin sensitisation

Remarks:

The justification for read across is attached to IUCLID Section 13.

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

Interpretation of results:

GHS criteria not met

Conclusions:

No reliable skin sensitisation study is available for Lutetium oxalate. Two in vitro studies (DPRA and KeratinoSens) were performed on Lutetium oxlate but both were inconclusives. Therefore, reliable data from the related substance Gadolinium oxalate is used to cover this endpoint. With this supporting substance, no signs of contact sensitisation were detected after the challenge exposure in guinea pigs previously exposed to the test item during the experiment. In the control and treated animals, the mean of the scores was 0.00 according to the 24- and 48-hour results. Under the conditions of the present assay, the substance Digadolinium oxalate was shown to have no skin sensitisation potential and is classified as a non-sensitiser, according to current EU-regulations. The same is assumed for Lutetium oxalate. The justification for read across is attached to IUCLID Section 13.

Reference 6

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

Two in vitro studies (DPRA under OECD test Guideline 442C and KeratinoSens under OECD Guideline 442D) were performed with the substance Lutetium oxalate. However, the reuslts of the DPRA study were inconclusives and it was considered that due to its insolubility, the test item was imcompatible with the DPRA study. In the KeratinoSens study, contradictory results were observed in the 3 runs performed. Thus, the test item was considered as not applicable to the KeratinoSens assay and the results were thus considered inconclusives.
Since both in vitro studies are inconclusives, a read-across approach was done using lutetium oxide silicate and gadolinium oxalate, 2 substances of the rare earth family with similar physico-chemical properties to Lutetium oxalate that were used to cover both moieties of the target substance Lutetium oxalate. The justification for read across is attached in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

left side: 100% (w/v) test item formulated in 1% methyl cellulose (1% MC); right side: 1% MC only

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Remarks:

This result is considered relevant for Lutetium oxalate too. The justification for read across is attached to IUCLID Section 13.

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

left side: 100% (w/v) test item formulated in 1% MC; right side: 1% MC

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Remarks:

This result is considered relevant for Lutetium oxalate too. The justification for read across is attached to IUCLID Section 13.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

left side: 100% (w/v) test item formulated in 1% MC; right side: 1% MC

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Remarks:

The justification for read across is attached to IUCLID Section 13.

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

left side: 100% (w/v) test item formulated in 1% MC; right side: 1% MC

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Remarks:

The justification for read across is attached to IUCLID Section 13.

Key result

Reading:

1st reading

Hours after challenge:

24

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

Key result

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

Remarks on result:

positive indication of skin sensitisation

Interpretation of results:

GHS criteria not met

Conclusions:

No reliable skin sensitisation study is available for Lutetium oxalate. Two in vitro studies (DPRA and KeratinoSens) were performed on Lutetium oxalate but both were inconclusives.Therefore, reliable data from the related substance Lutetium oxide silicate is used to cover this endpoint. With this supporting substance, no signs of contact sensitisation were detected after the challenge exposure in guinea pigs previously exposed to the test item during the experiment. In the control and treated animals, the mean of the scores was 0.00 according to the 24- and 48-hour results. Under the conditions of the present assay, the substance Lutetium oxide silicate was shown to have no skin sensitisation potential and is classified as a non-sensitiser, according to current EU-regulations. The same is assumed for Lutetium oxalate. The justification for read across is attached to IUCLID Section 13.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin senstisation:

The test item is considered to be non-sensitising, based on a read across approach using data from the 2 supporting substances lutetium oxide silicate and gadolinium oxalate.

Respiratory sensitisation:

No reliable study is available.