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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Aug - 23 Sep 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
no ear thickness measurement in the pre-screen test

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
23 Jul 2010
Deviations:
yes
Remarks:
No ear thickness measurement in the pre-screen test
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Silicic acid (H4SiO4), tetraethyl ester, reaction products with bis(acetyloxy)dioctylstannane
EC Number:
300-346-5
EC Name:
Silicic acid (H4SiO4), tetraethyl ester, reaction products with bis(acetyloxy)dioctylstannane
Cas Number:
93925-43-0
Molecular formula:
C4H8O2, C20H44O4SiSn, C24H52O6SiSn, C40H84O8SiSn2, C60H128O12Si2Sn3, C80H172O16Si3Sn4, C100H216O20Si4Sn5
IUPAC Name:
Silicic acid (H4SiO4), tetraethyl ester, reaction products with bis(acetyloxy)dioctylstannane

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Bicester, UK
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: Individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet: 2014 Teklad Global Rodent diet (Harlan Teklad, Bicester, UK), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): Approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
other: Butanone
Concentration:
25, 50 and 100% (v/v)
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
1 female mouse was treated by daily application of 25 μl of the undiluted test substance to the dorsal surface of the ear, for 3 consecutive days. The body weight was recorded on Day 1 prior to dosing and on Day 6.
- Systemic toxicity: The animal was observed for signs of toxicity twice on Day 1 and 2, and once on Day 4, 5 and 6. The body weight was recorded on Day 1 prior to dosing and on Day 6.
Irritation: The animal was observed for local skin irritation to the application site twice on Day 1 and 2, and once on Day 4, 5 and 6.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by β-scintillation and γ-counting
- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a “non-sensitiser”.
- Other: The animals were observed for signs of toxicity twice on Day 1 and 2, and once on Day 4, 5 and 6. The body weight was recorded on Day 1 prior to dosing and on Day 6 prior to termination.

TREATMENT PREPARATION AND ADMINISTRATION: 25 µl of the test material was applied to the entire dorsal surface of each ear of each mouse on Day 1, 2 and 3 in concentrations 25% and 50% in butanone, and undiluted. The irritation effects on the treatment site were assessed daily. On Day 6 an injection of 250 µl phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine (3H-TdR) was made into the tail vein of each experimental mouse. Five hours later, the draining auricular lymph node of each ear was excised into PBS and pooled per experimental group. A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze and rinsed with PBS. The precipitates were incubated for approximately 18 h at approximately 4 °C, centrifuged, resuspended in 1 mL TCA and transferred to 10 mL scintillation fluid before β-scintillation counting.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
A study (study dates: 16 - 22 Apr 2009; project number: 0039/1083) was performed to assess the sensitivity of the strain of mouse used at the testing facility to a known sensitizer and show the sensitivity and reproducibility of the test. The positive control substance hexyl cinnamic aldehyde (15% (v/v) in butanone) was considered to be a sensitizer under the conditions of the test (SI 4.08).

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.16
Test group / Remarks:
25% (v/v)
Key result
Parameter:
SI
Value:
1.56
Test group / Remarks:
50% (v/v)
Key result
Parameter:
SI
Value:
1.47
Test group / Remarks:
100% (v/v)
Cellular proliferation data / Observations:
CLINICAL OBSERVATIONS: No mortality and no signs of systemic toxicity were noted in the test or control animals during the test. Fur loss was noted in 4/4 animals treated with undiluted test material or the test material at a concentration of 50% (v/v). The area of the fur loss was not reported.

DETAILS ON STIMULATION INDEX CALCULATION
The SI of the 25, 50 and 100% treatment group was 1.16, 1.56 and 1.47, respectively. None of the test substance concentrations produced a 3-fold increase in 3HTdR incorporation.

EC3 CALCULATION
None of the SI values were above 3 and it is therefore not possible to determine a EC3 concentration.

BODY WEIGHTS: Body weight changes were comparable to those observed in the corresponding control group animals over the same period.

Any other information on results incl. tables

Table 1: Individual body weights and the results of the radioactive disintegrations

Concentration in butanone (% (v/v))

Body weight (g)

Radioactivity incorporated

Day 1

Day 6

Gains

DPM

dpm/node

SI

Control

18

18

0

9834.09

1229.26

-

19

19

0

21

20

-1

21

22

1

25

18

18

0

11428.97

1428.62

1.16

20

21

1

17

16

-1

20

20

0

50

20

20

0

15303.39

1912.92

1.56

19

19

0

22

22

0

19

19

0

100

19

19

0

14486.57

1810.82

1.47

21

20

-1

21

20

-1

20

21

1

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008.