Silsesquioxanes, 3-phosphonopropyl, Et Me esters, sodium salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-04-04 to 2008-05-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

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:

225375-65-7

IUPAC Name:

225375-65-7

Details on test material:

Name of test substance: Silicophosphonat
Test-substance No.: 08/0014-1
Batch identification: Partie 21/05
CAS No.: 225375-65-7
Purity: Aqueous preparation (for details see analytical report No.:
08L00034)
Homogeneity: The test substance was homogeneous by visual inspection.
Storage stability: The stability under storage conditions over the study period
was guaranteed by the manufacturer.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
Test species / strain / quality: Mouse / CBA/J
Age on day 0: 6 – 12 weeks
Sex: Female
Supplier: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld
Arrival in the testing facility: Acclimatization period (12 days before the first test-substance application)
Identification: The single housed animals were identified by cage cards
Body weight on day 0: 19.1 g – 22.6 g

HOUSING CONDITIONS
Room temperature / relative humidity:
The animals were housed in fully air-conditioned rooms. Central air-conditioning guaranteed a range of 20 – 24°C for temperature and of 30 – 70% for relative humidity. There were no deviations from these ranges, which influenced the results of the study.
Day / night rhythm: 12 h / 12 h (6.00 a.m. – 6.00 p.m. / 6.00 p.m. – 6.00 a.m.)
Type of cage: Makrolon cage, type II
Enrichment: Nest-building material: wood wool (Typ NBF E-011); Abedd ® Lab. and Vet. Service GmbH Vienna, Austria. PLEXX mouse tunnel (red, transparent) EMSICON Jung GmbH, Forstinning, Germany.
No. of animals per cage: 1
Feeding: Kliba-Labordiät (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
Drinking water: Tap water ad libitum
Bedding: Lignocel FS14; SSNIFF

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

10%, 30% and 50%, vehicle control

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: good solubility in water and polypropylene glycol
- Irritation: separate study
- Lymph node proliferation response: no


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Murine Local Lymph Node Assay
Criteria used to consider a positive response:
In order to reveal a possible induction of sensitization, the response in the draining lymph node after epicutaneous application of several concentrations of the test substance to the skin of the ear backs is determined. The parameters used to characterize the response are lymph node cell count, 3H-thymidine incorporation into the lymph node cells and to a certain extent lymph node weight. Because not only sensitization induction but also irritation of the ear skin by the test substance may induce lymph node responses, the weight of ear punches taken from the area of test-substance application is determined as a parameter for inflammatory ear swelling serving as an indicator for the irritant action of the test substance. The increase SI of cell count by a factor of ≥ 1.5 and/or of 3H-thymidine incorporation by a factor of ≥ 3 as compared to the concurrent vehicle control group is generally considered as
indicating a sensitizing potential of a test substance.

TREATMENT PREPARATION AND ADMINISTRATION:
The study comprised three treatment groups and a vehicle control group. Each group consisted of 5 mice.
Randomization: Prior to first application, the animals were distributed to the individual groups, received their animal numbers and were allocated to the respective cages according to the randomization instructions of „Nijenhuis, A. and Wilf, H.S.: Combinatorial Algorithms, Academic Press, New York, San Francisco, London, 1978, pp. 62 – 64“.
Body weight determination: Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
Signs and symptoms: No detailed clinical examination of the individual animals was performed but any obvious signs of systemic toxicity and/or local inflammation at the application sites were noted in the raw data.
Form of application: Epicutaneous application is simulating dermal contact with the compound which is possible to occur under practical use conditions.
Application volume: 25 μL per ear
Site of application: Dorsal part of both ears
Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
Mortality: Twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.

³H-thymidine injection
On study day five (about 66 to 72 hours after the last application of test substance to the ears) the mice were injected intravenously with 20 μCi of 3H-t ymidine in 250 μl of sterile saline into a tail vein.

Terminal procedures
The animals were sacrificed on study day 5 about 5 hours after 3H-thymidine injection by cervical dislocation.
Determination of ear weight: Immediately after the death of each animal a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each test group. These measurements serve
for detecting a potential inflammatory ear swelling.
Removal and weight determination of the lymph nodes: Immediately after removal of the ear punches the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal.
Preparation of cell suspension and determination of cell count: After weight determination, the pooled lymph nodes of each
test group were stored in phosphate buffered saline in an icewater bath until further preparation. A single cell suspension
was prepared as soon as possible after dissection by carefully passing all lymph nodes per test group through an iron mesh
(mesh size 200 μm) into 40 mL of phosphate-buffered physiological saline. For determination of cell counts, an
aliquot of each suspension was further diluted with Casy®ton in a ratio 1:500. The cell count was determined using a Casy®-
Counter.
Measurement of 3Hthymidine incorporation of the lymph node cells: The remaining cell suspensions were washed twice with
phosphate buffered saline (PBS) and precipitated with 5% trichloro-acetic acid (TCA). Each precipitate was transferred to
scintillation fluid and incorporation of 3H-thymidine into the cells was measured in a ß-scintillation counter.

Calculations
The stimulation indices of cell count, 3H-thymidine incorporation, lymph node weight and ear weight were calculated as the ratio of the test group values for these parameters divided by those of the vehicle control group.

Statistics:

If applicable, the EC (estimated concentration) leading to the respective SI values were calculated by linear or semi-logarithmical regression between the data points directly below and above the SI if possible or using the two nearest points below

Results and discussion

Positive control results:

A concurrent positive control (reliability check) with a known sensitizer was not included into this study. Studies using the positive control substance Alpha-Hexylcinnamaldehyde are performed twice a year in the laboratory in order to show that the test system is able to detect sensitizing compounds under the test conditions chosen.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Stimulation Index1 vehicle propylene glycol 1.00 10% in propylene glycol 1.09 30% in propylene glycol 1.55 50% in propylene glycol 1.58

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: [DPM/Lymph Node Pair] vehicle propylene glycol 525.2 10% in propylene glycol 573.2 30% in propylene glycol 815.6 50% in propylene glycol 827.7

Any other information on results incl. tables

Concentration	Cell Counts [Counts/Lymph Node Pair]	Stimulation Index
vehicle propylene glycol	5,568,000	1.00
10% in propylene glycol	5,262,667	0.95
30% in propylene glycol	6,948,000	1.25
50% in propylene glycol	6,608,333	1.19

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

No signs of systemic toxicity were noticed.
When applied as 10% preparation in propylene glycol, the test substance did not induce relevant changes in the auricular lymph node cell counts or ³H-thymidine incorporation. The 30% and 50% test substance preparations caused slight but not concentration related increases in cellularity and 3H-thymidine incorporation into the lymph node cells, which failed to reach the cut off stimulation indices (increase to 1.5 or 3 fold or above of control value =
stimulation index (SI) ≥ 1.5 or 3, respectively) and thus lie below the threshold of immunologic relevance. In addition there was no relevant increase in lymph node weights. The 10%, 30% and 50% test-substance preparations did not cause an increase in ear weights as indication of ear skin irritation whencompared to the vehicle control.
Thus it is concluded that Silicophosphonat does not show a skin sensitizing effect in the
Murine Local Lymph Node Assay under the test conditions chosen.

Executive summary:

The skin sensitizing potential of Silicophosphonat was assessed using the radioactive Murine Local Lymph Node Assay. The assay simulates the induction phase for skin sensitization in mice. It determines the response of the auricular lymph nodes on repeated application of the test substance to the dorsal skin of the ears. Groups of 5 female CBA/J mice each were treated with 10%, 30% and 50% w/w preparations of the test substance in propylene glycol or with the vehicle alone. No signs of systemic toxicity were noticed. When applied as 10% preparation in propylene glycol, the test substance did not induce relevant changes in the auricular lymph node cell counts or ³H-thymidine incorporation. The 30% and 50% test substance preparations caused slight but not concentration related increases in cellularity and 3H-thymidine incorporation into the lymph node cells, which failed to reach the cut off stimulation indices (increase to 1.5 or 3 fold or above of control value = stimulation index (SI) ≥ 1.5 or 3, respectively) and thus lie below the threshold of immunologic relevance. In addition there was no relevant increase in lymph node weights. The 10%, 30% and 50% test-substance preparations did not cause an increase in ear weights as indication of ear skin irritation when compared to the vehicle control. Thus it is concluded that Silicophosphonat does not show a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.