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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
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
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)
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
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).
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.

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

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
CLP: not classified
DSD: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:
Migrated from Short description of key information:
Skin sensitisation (OECD 429, LLNA): not sensitising

Justification for selection of skin sensitisation endpoint:
There is only one study available.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Skin

A GLP-compliant skin sensitisation study (LLNA) was performed with silicon orthophosphate according to OECD 429 (Lütkenhaus, 2012).

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.

Therefore, 3.125, 6.25 and 12.5% of the test substance (in acetone/olive oil (4:1, v/v) were applied in the main study in a total dose of 25 µl to each ear of 5 female CBA/CaOlaHsD mice each on three consecutive days. On day 6 of the experiment, 3H-methyl thymidine (3H-TdR) was injected into the tail vein of each experimental mouse. Approximately five hours later all animals were killed and the draining auricular lymph node of each ear was excised into PBS and a single cell suspension was prepared which was precipitated with trichloroacetic acid in preparation for scintillation. The mean DPM/animal value determined for the vehicle control group was 962 DPM. For the experimental groups treated with test substance concentrations of 3.125, 6.25 and 12.5%, values of 1463, 2081 and 2431 DPM, respectively, were found revealing SI values of 1.5, 2.2 and 2.6, respectively. An EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3. The means of the ear thickness per group showed no significant difference compared to the negative control. Under the conditions of this test, the test substance was considered to be not skin sensitising.

Justification for classification or non-classification

The available data on skin sensitisation of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.