Tetraammonium decachloro-μ-oxodiruthenate(4-)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 June - 28 July 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted according to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Tetraammonium-decachloro-mu-oxodiruthenate

IUPAC Name:

Tetraammonium-decachloro-mu-oxodiruthenate

Constituent 2

Reference substance name:

Tetraammonium decachloro-μ-oxodiruthenate(4-)

EC Number:

286-924-7

EC Name:

Tetraammonium decachloro-μ-oxodiruthenate(4-)

Cas Number:

85392-65-0

Molecular formula:

Cl10ORu2.4H4N

IUPAC Name:

tetraammonium decachloro-μ-oxodiruthenate(4-)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): tetraammonium-decachloro-mu-oxodiruthenate
- Substance type: brown crystalline solid, powder
- Physical state: solid
- Analytical purity: 98.0%
- Impurities (identity and concentrations): metallic impurities 0.3%
- Composition of test material, percentage of components: (NH4)4Ru2Cl10O x H2O 98.0%, NH4Cl 0.7%, H2O 1.0%
- Isomers composition: no data
- Purity test date: 15-Jul-2015
- Lot/batch No.: 71495159
- Expiration date of the lot/batch: 14-Apr-2017
- Stability under test conditions: no data
- Storage condition of test material: store in a cool, dry place in tightly closed contatiners at 5-25 deg C; avoid exposure to sunlight and high humidity

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories S.r.l., San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy
- Age at study initiation: 10 weeks
- Weight at study initiation: 19.2-20.8 g (range)
- Housing: type II. polypropylene/polycarbonate cages; individual caging, mice were provided with glass tunnel-tubes; bedding was available to animals during the study
- Diet (e.g. ad libitum): ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice”, ad libitum
- Water (e.g. ad libitum): tap water (considered fit for human consumption) from the municipal supply, ad libitum
- Acclimation period: 21 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.0-27.5
- Humidity (%): 24-86
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 22-Jul-2015 To: 28-Jul-2015 (main experiment)

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

10, 25 and 50% (w/v)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Due to the physical characteristics of the test item, 100 % (w/v) concentration was not achievable in any of the vehicles tested. At 50 % (w/v), the formulations were still not suitable for treatment except Propylene glycol, where a suspension was formed with slow sedimentation. Therefore, Propylene glycol was considered most suitable. The highest achievable concentration based on the physical characteristics of the test item, was 50 % (w/v).
- Irritation: no irritation at the site of application.
- Lymph node proliferation response: draining auricular lymph nodes were larger than normal in the 50% (w/v) group, by visual inspection, and normal in the 25% (w/v) group.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: local lymph node assay
- Criteria used to consider a positive response: the test item is regarded as a sensitizer if both of the following criteria are fulfilled:
- that exposure to at least one concentration of the test item resulted in an incorporation of 3H-methyl thymidine at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION: formulated in propylene glycol; topical application of 25 µL of the appropriate formulation, using a pipette, on the dorsal surface of each ear. Animals were dosed once daily for 3 consecutive days (days 1,2,3); no treatment on days 4,5,6.
On Day 6, animals were intravenously injected via the tail vein with 250 μL of sterile PBS (phosphate buffered saline) containing approximately 20 μCi of 3H-methyl thymidine and left for 5 hours (± 30 minutes).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

None.

Results and discussion

Positive control results:

No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study.
A significant lymphoproliferative response (stimulation index value of 9.5) was noted for 25% hexyl cinnamic aldehyde (HCA) in propylene glycol in the main experiment.
This value was considered to confirm the appropriate performance of the assay.
Furthermore, the DPN values observed for the positive control substance in this experiment were within the historical control range.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Negative control (propylene glycol): 1.0 Tetraammonium-decachloro-mu-oxodiruthenate 10%: 1.2 Tetraammonium-decachloro-mu-oxodiruthenate 25%: 1.5 Tetraammonium-decachloro-mu-oxodiruthenate 50%: 7.9 Positive control (HCA 25% in propylene glycol): 9.5

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Negative control (propylene glycol): mean 116.6 dpn (disintegrations per node, i.e. disintegrations per minute (dpm) divided by 2 (2 lymph nodes per animal)) (range 63.3-161.3) Tetraammonium-decachloro-mu-oxodiruthenate 10%: mean 141.4 dpn (range 85.3-241.3) Tetraammonium-decachloro-mu-oxodiruthenate 25%: mean 175.1 dpn (range 57.3-322.3) Tetraammonium-decachloro-mu-oxodiruthenate 50%: mean 925.9 dpn (range 72.3-2255.8) Positive control (HCA 25% in propylene glycol): mean 1102.7 dpn (range 817.3-1675.8)

Any other information on results incl. tables

EC3 (the estimated concentration needed to produce a stimulation index of 3) is 30.9% (w/v).

GHS criteria for classification are EC3 ≤2% 1A, EC3 >2% 1B.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

In a guideline LLNA, to GLP, tetraammonium-decachloro-mu-oxodiruthenate induced a stimulation index of above 3 (calculated EC3 value of 30.9%) indicating a skin sensitising potential.

Executive summary:

The skin sensitisation potential of tetraammonium-decachloro-mu-oxodiruthenate was evaluated in a mouse LLNA, conducted in accordance with OECD Test Guideline 429 and to GLP.

 

Following a preliminary irritation and toxicity test, in which a maximum concentration of 50% (w/v) was established for the main test, female mice (5/group) were topically treated on three consecutive days with 25 µl/ear of vehicle (propylene glycol), test material at 10, 25 or 50%, or positive control (hexyl cinnamaldehyde (HCA)). After three days without treatment, cell proliferation was measured by incorporation of 3H-methyl thymidine and the values obtained were used to calculate stimulation indices.

 

There was no mortality, no overt toxicity and no sign of irritation at the application sites. Stimulation indices were 1.2, 1.5 and 7.9 for 10, 25 and 50% (w/v) tetraammonium-decachloro-mu-oxodiruthenate, respectively. In the absence of confounding effects (irritation or systemic toxicity), the proliferation values are considered to reflect sensitising potential. The EC3 value for tetraammonium-decachloro-mu-oxodiruthenate was 30.9% (w/v). The stimulation index for the positive control was 9.5. Hence, under the conditions of this assay, tetraammonium-decachloro-mu-oxodiruthenate was considered to have skin sensitising potential.

 

Based on the results of this study, the test material should be classified as a skin sensitiser, category 1B under EU CLP criteria (EC 1272/2008).