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Description of key information

The skin sensitising potential of Ruthenium(IV) oxide has been assessed in a GLP compliant mouse local lymph node assay (LLNA): BrdU-ELISA according to OECD Test Guideline 442B. The test item ruthenium(IV) oxide did not reveal any skin sensitising properties in the local lymph node assay: BrdU-ELISA.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 May 2018 - 18 December 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Version / remarks:
adopted 25 June 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EU) No. 640/2012 of 6 July 2012 amending Regulation (EC) No. 440/2008; EC method B.51: Skin Sensitisation, Local Lymph Node Assay: BrdU-ELISA.
Version / remarks:
6 July 2012.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA
Specific details on test material used for the study:
Ru content = 75.08% (calculated after loss on reduction = 100% - 24.92%)
Species:
mouse
Strain:
CBA:JN
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier Labs, CS 4105 Le Genest, Saint Isle, Saint Berthevin, Cedex 53941, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at main study initiation: approx 10 weeks
- Weight at study initiation: 19-23 g
- Housing: Before application the animals were housed in groups of 5 animals in MAKROLON cages (type III) with a basal surface of approximately 39 cm x 23 cm and a height of approximately 15 cm. After application the animals were housed singly in order to prevent them licking off the test item from the ears of the other animals. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material. The cages were changed and cleaned once a week.
- Diet: Commercial diet ssniff® R/M-H V1534 (sourced from ssniff Spezialdiäten GmbH, 59494 Soest, Germany) was offered ad libitum. Food residue was removed.
- Water: Tap water was offered ad libitum.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3 (maximum range)
- Humidity (%): 55 ± 15 (maximum range)
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12 (about 150 lux at approx 1.5m room height)
- in-life date: 13-18 December 2018
Vehicle:
propylene glycol
Remarks:
Acetone/olive oil (4:1, v/v) and N,N-dimethylformamide did not provide higher concentrated suitable suspensions. Dimethyl sulfoxide was not considered as vehicle at the request of the Sponsor.
Concentration:
Pre-screening test: 10, 25, 50 and 75% (w/w)
Main study: 0, 2.5, 5 and 10% (w/w)
No. of animals per dose:
5
Details on study design:
PRE-SCREEN (RANGE-FINDING) TESTS:
- Compound solubility: In a preliminary solubility assessment, propylene glycol was selected as it is recommended by the OECD guideline and provided suitable solutions of the test item both for administration and adherence to the mouse ear of such high concentrations. Doses were selected based on the recommendations in the OECD guideline, adjusted to the physical appearance of the test item as solid, with 75, 50, 25 and 10% (w/w). A 10% concentration was the highest concentration of Ruthenium(IV) oxide in propylene glycol to obtain an applicable solution.
- Test item adminstration: The test item formulations were administered to the dorsum of both ears of each animal at an application volume of 25 µL/ear.
- Systemic toxicity and Irritation: All mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to termination (Day 6). Both ears of each mouse were observed for erythema and scored on a 0-4 scale (0: no erythema; 1: very slight erythema (barely perceptible); 2: well-defined erythema; 3: moderate to severe erythema; 4: severe erythema (beet redness) to eschar formation preventing grading of erythema).
The following clinical observation would indicate systemic toxicity: changes in nervous system function (e.g. pilo-erection, ataxia, tremors, and convulsions); changes in behaviour (e.g. aggressiveness, change in grooming activity, marked change in activity level); changes in respiratory patterns (i.e. changes in frequency and intensity of breathing such as dyspnoea, gasping, and rales), and changes in food and water consumption. In addition, signs of lethargy and/or unresponsiveness and any clinical signs of more than slight or momentary pain and distress, or a >5% reduction in body weight from Day 1 to Day 6 and mortality would be considered in the evaluation. Moribund animals or animals showing signs of severe pain and distress would have been humanely killed.
- Ear thickness measurements: Ear thickness measurements were taken using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose), and Day 6. Additionally, on Day 6, ear thickness was determined by ear punch weight determinations, which was performed after the animals were humanely killed. Excessive local irritation is indicated by an erythema score ≥3 and/or ear thickness increase of ≥25% on any day of measurement. The highest dose selected for the main LLNA: BrdU-ELISA study was the next lower dose in the pre-screen concentration series that doses not induce systemic toxicity and/or excessive local skin irritation.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymph node assay BrdU-ELISA
- Criteria used to consider a positive response: Results of each treatment group are expressed as the mean SI. The SI is derived by dividing the mean BrdU luminescence/mouse within each test item group and the positive control group by the mean BrdU luminescence labelling index for the solvent/vehicle control group. The average SI for the vehicle control is then one.
SI = (Mean test item or positive control group)/(Mean negative control group)
The decision process regards a result as positive when SI ≥ 1.6. However, the strength of the dose-response relationship, the statistical significance and the consistency of the vehicle and positive control responses would also be used when determining whether a borderline result (i.e. SI value between 1.6 and 1.9) is declared positive.
For a borderline positive response between an SI of 1.6 and 1.9, it may be necessary to consider additional information such as dose-response relationship, evidence of systemic toxicity or excessive irritation, and where appropriate, statistical significance together with SI values to confirm that such results are positives. Consideration would be also given to various properties of the test item, including whether it has a structural relationship to known skin sensitizers, whether it causes excessive skin irritation in the mouse, and the nature of the dose-response observation.
In addition, the average ear weights per group and the average ear thickness per group were compared to the vehicle control group as an indication for possible irritating properties.

TREATMENT PREPARATION AND ADMINISTRATION: g
•Day 1: The weight of each animal was identified and recorded. Ear thickness of left and right ear of each animal was recorded. Any clinical observation was recorded. 25 µL of the appropriate dilution of the test item, the negative control, the positive control or the vehicle of the positive control were applied to the dorsum of each ear.
•Days 2 and 3: The application procedure carried out on day 1 was repeated.
•Day 4: No treatment.
•Day 5: 0.5 mL (5 mg/mouse) of BrdU (10 mg/mL) solution was injected inter-peritoneally.
•Day 6: The weight of each animal and any clinical observation were recorded. Approximately 24 hours after BrdU injection the animals were humanely killed. The draining auricular lymph nodes from each mouse ear were excised and processed separately in phosphate buffered saline (PBS) for each animal. To further monitor the local skin response in the study, additional parameters such as scoring of ear erythema and ear thickness measurements (obtained by using a thickness gauge and ear punch weight determinations at necropsy) were carried out.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
As no concentration-related increased values were observed, no statistical evaluation was carried out.
Comparison to negative control was carried out using Student's t-test.
Positive control results:
Treatment with the positive control item caused a statistically significant increase in the BrdU labelling index, compared to the negative control (propylene glycol), which reflects a positive sensitisation response. The Stimulation Index of 2.231 exceeded the threshold value of 1.6 over the positive control vehicle group. Therefore, the study can be regarded as valid as the positive control elicited a positive sensitisation response.
Key result
Parameter:
SI
Value:
< 1.6
Test group / Remarks:
2.5, 5 and 10%
Remarks on result:
other: Treatment with the test item at concentrations of 2.5, 5 and 10% (w/w) revealed a dose-related increase in the BrdU labelling index (SI values of 1.316, 1.455 and 1.578, respecitvely), but none of the SI values exceeded the threshold of 1.6.

In a preliminary test employing two animals per dose, concentrations of 2.5%, 5% or 10% (w/w) were used. No signs of irritation (erythema score ≥ 3 and/or an ear thickness increase of more than 25%) were noted.

Parameter

Negative control

2.5% test item

5% test item

10% test tem

Positive control

Vehicle of positive control 

BrdU labelling index

 1.463 1.925   2.129* 2.309*  5.273*  2.363    

Stimulation index (SI

1.000

1.316

1.455*

1.578*

2.231*

 1.000

Ear weight (mg)

14.0

16.7

16.0

16.4*

18.1

 16.7   

Difference of ear thickness (mm, TD6)

226.0

232.0

230.0

234.0

241.0

229.0     

  * statistically significant increase compared to negative control at p ≤ 0.01

Although there was a statistically significant and dose related increase in the BrdU labelling indices of the 5% and 10% (w/w) treated groups, the stimulation indices calculated for the BrdU labelling index, did not exceed the threshold value of 1.6. Hence, the test item is classified as not skin sensitising.

There were no deaths and no systemic clinical signs or effects on body weights were observed during the study.The ear weight (punch biopsies) and the difference of ear thickness on test day 3 and test day 6 compared to the vehicle control were not or only slightly increased, i.e. no skin irritating properties were noted.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the present test conditions, the test item did not reveal any skin sensitising properties in the local lymph node assay: BrdU-ELISA.
Executive summary:

The skin sensitising potential of Ruthenium(IV) oxide has been assessed in a GLP mouse local lymph node assay (LLNA): BrdU-ELISA, conducted according to OECD Test Guideline 442B. Following a preliminary range-finding study to assess irritancy, female mice CBA/JN mice (5/group) were treated topically with 0, 2.5, 5 and 10% test item (in propylene glycol) on three consecutive days. There was no treatment on test days 4, 5 and 6. On day 6, cell proliferation in the local lymph nodes was measured by incorporation of injected 5-bromo-2-deoxyuridine (BrdU) using ELISA. The cell proliferation in the local lymph nodes was determined by measuring the BrdU content with BrdU-ELISA and the values obtained were used to calculate Stimulation Indices (SI).

Although there was a statistically significant and dose-related increase in the BrdU labelling indices of the 5% and 10% (w/w) treated groups, the stimulation indices calculated for the BrdU labelling index did not exceed the threshold value of 1.6. Hence, there was no clear evidence of cell proliferation and the test item is classified as not skin sensitising.

There were no deaths and no systemic clinical signs or effects on body weights were observed during the study.The ear weight (punch biopsies) and the difference of ear thickness on test day 3 and test day 6 compared to the vehicle control were not or only slightly increased, i.e. no skin irritating properties were noted.

Treatment with the positive control item caused a statistically significant increase in the BrdU labelling index, compared to the negative control, and a Stimulation Index in excess of the threshold value of 1.6. Therefore, the study can be regarded as valid.

In conclusion, under the present test conditions, the test item Ruthenium(IV) oxide did not reveal any skin sensitising properties in the local lymph node assay: BrdU-ELISA.

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

Ruthenium (IV) oxide does not meet the criteria for classification as skin sensitizer.

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