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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

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)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Silicon orthophosphate
EC Number:
234-858-4
EC Name:
Silicon orthophosphate
Cas Number:
12037-47-7
Molecular formula:
O16P4Si3
IUPAC Name:
silicon(4+) tetraphosphate
Details on test material:
- Name of test material (as cited in study report): silicon orthophosphate
- Physical state: solid, light grey
- Analytical purity: >80% (w/w)
- Batch No.: 5
- Storage condition of test material: at room temperature, dry atmosphere, protected from light

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 Winkelmann, 33178 Borchen, Germany
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 19-24 g
- Housing: groups of five in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding
- Diet (ad libitum): Altromin 1324 maintenance diet for rats and mice
- Water (ad libitum): tap water, sulphur acidified to a pH value of approx. 2.8
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
3.125, 6.25, 12.5%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
Preliminary to the main test, a solubility test was performed to define the maximum concentration which was technically applicable to the animals. The maximum technically applicable concentration of the test item was found to be 12.5% in acetone/olive oil.
In order to determine the highest tolerated and not excessively irritant test concentration a prescreen test was performed in 6 animals which was conducted under the same conditions as the main study, except there was no assessment of lymph node proliferation.Two animals each were treated with 12.5, 25 and 50% test substance concentration, respectively. Three further animals were treated with 100% vehicle alone (acetone/olive oil). No signs of systemic toxicity, but signs of excessive local irritation at the application site were observed in animals treated with 25 and 50% test substance concentration. Neither signs of systemic toxicity nor signs of excessive irritation at the application site could be detected in any other animal. No changes in ear thickness measurements were observed over the six day treatment. All animals showed the expected weight development.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by beta-scintillation
- Criteria used to consider a positive response: criteria given in Commission Regulation (EU) No 286/2011 (A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).

TREATMENT PREPARATION AND ADMINISTRATION:
Immediately before the first application the thickness of both ears of all animals was measured. 25 μl of the test compound was applied to the entire dorsal surface of each ear of each mouse. A second measurement of the ear thickness of all animals was carried out approximately 48 hours after the first application. The application was repeated on days 2 and 3; local irritation reactions were assessed. 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. A single cell suspension of lymph node cells was prepared from pooled lymph node cells by gentle mechanical disaggregation through polyamide gauze (200 mesh size). Cells were precipitated with 5% trichloroacetic acid at 4 °C for 18 hours.
Positive control substance(s):
other: P-Phenylenediamine (CAS 106-50-3, Sigma, purity > 98%; Lot 060M0186V6), 1% solution. The recent reliability check was performed in May 2012. The raw data of this study are kept in the BSL archives (BSL Project ID 114612 I).

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
None of the three tested concentrations of the test item reached the stimulation index of 3: The stimulation index at a concentration of 3.125, 6.25 or 12.5% was 1.5, 2.2 and 2.6, respectively. The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Significant lymphoproliferatic responses were not noted for the test substance. Mean DPM/animal values were 961.8, 1462.8, 2080.5 and 2431.3 for the control, 3.125, 6.25 and 12.5% groups, respectively.

Any other information on results incl. tables

All animals survived throughout the test period without showing any clinical sings. Body weights developed as expected, which includes a weight loss of up to 2 g throughout the study. The means of the ear thickness per group showed no significant difference compared to the negative control (see Table 1).

Table 1: Ear thickness measurements (mean values out of 5 animals/group in mm)

Group

Day 1

Day 3

Day 6

Negative Control

0.19

0.19

0.19

3.125% TS

0.20

0.19

0.20

6.25% TS

0.20

0.20

0.21

12.5% TS

0.20

0.20

0.20

Table 2: Radioactive determination of the test substance groups and control group

Test Item

Animal number

DPM

DPM – mean background

DPM/node

Stimulation Index

Negative control

16

790.0

772.0

386.0

 

 

17

903.0

885.0

442.5

 

 

18

1789.0*

n.d.

n.d.

 

 

19

890.0

872.0

436.0

 

 

20

1264.0

1246.0

623.0

 

 

MV

961.8

943.8

471.9

1.0

 

SD

179.9

179.9

89.9

 

3.125% TS

1

1386.0

1368.0

684.0

1.4

 

2

2361.0*

n.d

n.d.

n.d.

 

3

1499.0

1481.0

740.5

1.6

 

4

1237.0

1219.0

609.5

1.3

 

5

1729.0

1711.0

855.5

1.8

 

MV

1462.8

1444.8

722.4

1.5

 

SD

179.6

179.6

89.8

0.2

6.25% TS

6

2074.0

2056.0

1028.0

2.2

 

7

2072.0

2054.0

1027.0

2.2

 

8

3054.0*

n.d.

n.d.

n.d.

 

9

2378.0

2360.0

1180.0

2.5

 

10

1798.0

1780.0

890.0

1.9

 

MV

2080.5

2062.5

1031.3

2.2

 

SD

205.2

205.2

102.6

0.2

12.5% TS

11

1253.0*

n.d.

n.d.

n.d.

 

12

2682.0

2664.0

1332.0

2.8

 

13

2089.0

2071.0

1035.5

2.2

 

14

2679.0

2661.0

1330.5

2.8

 

15

2275.0

2257.0

1128.5

2.4

 

MV

2431.3

2413.3

1206.6

2.6

 

SD

257.8

257.8

128.9

0.3

DPM: disintegration per minute, SD: standard deviation, MV: mean value, *: outlier, failed Nalimov; TS: test substance

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
CLP: not classified
DSD: not classified