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Toxicological information

Skin sensitisation

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Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 July 2015 - 05 August 2015
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
according to guideline
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
dicalcium bis(oxosilanebis(olate)) silanedione
EC Number:
Molecular formula:
Not applicable for inorganic UVCB
dicalcium bis(oxosilanebis(olate)) silanedione
Test material form:

In vivo test system

Test animals

other: CBA/Ca (CBA/CaOlaHsd)
Details on test animals and environmental conditions:
- Source: Envigo RMS B.V., Inc Horst, Netherlands.
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: Animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: Food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Ltd., Oxon, UK), ad libitum
- Water: Mains tap water, ad libitum
- Acclimation period: at least 5 days

- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: 15 changes per hour
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: 2015-07-16 to 2015-08-05

Study design: in vivo (LLNA)

propylene glycol
Preliminary Test - 10% w/w in proplylene glycol
Main Test - 10%, 5% or 2.5% w/w in proplylene glycol
No. of animals per dose:
Preliminary Study : One animal
Main Study : 4 females/dose
Details on study design:
Preliminary Screening Test
- Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse at the maximum concentration that was suitable for dosing.
- The mouse was treated by daily application of 25 µl of the test item at concentrations of 10% w/w in propylene glycol , to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3).
- The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6.
- Local skin irritation was scored daily according to a scale of 0 to 4, (See Appendix 4).
- Any clinical signs of toxicity, if present, were also recorded.
- The bodyweight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.

- The ear thickness was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation), pre dose on Day 1, post dose on Day 3 and on Day 6.
- Any changes in the ear thickness were noted.
- Mean ear thickness changes were calculated between Days 1 and 3 and Days 1 and 6.
- A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

Main Test
Test Item Administration
- Groups of four mice were treated with the test item at concentrations of 10%, 5% or 2.5% w/w in proplylene glycol.
- The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration.
- The mice were treated by daily application of 25 µl of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3).
- The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
- A further group of four mice received the vehicle alone in the same manner.
- A routine positive control study using 5 animals treated with 50 µL of Phenylacetaldehyde (>90%) as a solution in proplyene glycon at a concentration of 5%v/vwas used (See Appendix 1 & 2)

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item, vehicle control (Day 6) all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR:80µCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 µCi to each mouse.

Clinical Observations:
- All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures

- Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation.
- The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group.
- For each group 1 mL of PBS was added to the pooled lymph nodes.

Preparation of Single Cell Suspension:
- A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200 mesh stainless steel gauze.
- The lymph node cells were rinsed through the gauze with 4 ml of PBS into a petri dish labelled with the study number and dose concentration.
- The lymph node cells suspension was transferred to a centrifuge tube.
- The petri dish was washed with an additional 5 ml of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube.
- The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes.
- The pellet was resuspended in 10 mL of PBS and re-pelleted.
- To precipitate out the radioactive material, the pellet was resuspended in 3 ml of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation:
- After approximately eighteen hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid
- 3HTdR incorporation was measured by ß-scintillation counting.
- The "Poly QTM" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes in darkness
- The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. - After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).
Positive control substance(s):
other: Phenylacetaldhyde (>90%) 5%v/v in propylene glycol
No data

Results and discussion

Positive control results:
Stimulation Index for Phenylacetaldehyde (>90%) at 5% iv/v in propylene glycol was 14.82 and classified as a sensitiser.

In vivo (LLNA)

Resultsopen allclose all
Key result
Key result
Key result
Key result
other: disintegrations per minute (DPM)
Remarks on result:
other: The radioactive disintegrations per minute per group are given in Table 4

Any other information on results incl. tables


- Clinical observations, body weight and mortality data are given in Table 1

- Local skin irritation is given in Table 2

- Ear thickness measurements and mean ear thickness changes are given in Table 3

- There were no signs of systemic toxicity



- Clincial observations and mortality data are given in Table 5 (attached)

- Individual body weights and body weight change are given in Table 6 (attached)

- There were no deaths

- There were no signs of systemic toxicity in the test or control animals

- Body weight change of the test animals between Day 1 and Day 6 was comparible to that observed in the corresponding control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Migrated information Criteria used for interpretation of results: EU
The skin sensitisation potential of the test material was assessed in accordance with OECD Guideline 429. The test item was considered to be a non-sensitiser under the conditions of the test.
Executive summary:


A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear, according to the OECD Guideline 429 and EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay).


Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 10% w/w, this concentration was selected as the highest dose level to be investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 uL (25 uL per ear)) of the test item as a suspension in propylene glycol at concentrations of 2.5, 5.0 and 10% w/w. A further group of four animals was treated with propylene glycol .


No mortality and no clinical signs were observed during the observation period. Body weight of the animals was unaffected by the test item treatment. The stimulation index for concentrations 2.5, 5.0 and 10% were 1.16, 1.30 and 0.94, respectively. The positive control (5%v/v phenylacetaldehyde) exhibited evidence of sensitisation (SI = 14.82) indicating the validity of the study. In this study. The substance is not a skin sensitizer in mice.


The test item was considered to be a non-sensitizer under the conditions of the test.