Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Jul - 11 Aug 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2010

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:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, 65189 Wiesbaden, Germany
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Lanthanum chloride hexahydrate
IUPAC Name:
Lanthanum chloride hexahydrate
Constituent 2
Reference substance name:
17272-45-6
Cas Number:
17272-45-6
IUPAC Name:
17272-45-6
Details on test material:
- Name of test material (as cited in study report): Lanthanum Trichloride
- CAS No. 17272-45-6
- Molecular formula: LaCl3*6H2O
- Molecular weight: 353.3578 g/mol
- Physical state: white powder
- Analytical purity: 99.99% (content of Lanthanum Oxide in Total Rare Earth Oxides)
- Lot/batch No.: 290 000 0990
- Date of production: 30 Mar 2009
- Expiration date of the lot/batch: 29 Sep 2010
- Storage condition of test material: Room temperature (20 ± 5 °C), dry
- other: water content: 30.5%

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., 5960 AD Horst, The Netherlands
- Strain: CBA/CaOlaHsd
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 21.0 ± 1.1 g
- Housing: single
- Diet (ad libitum): pelleted standard diet
- Water (ad libitum): tap water
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Relative humidity (%): 45 - 70
- Photoperiod (hrs dark / hrs light): artificial light, 12 h/12 h

Study design: in vivo (LLNA)

Vehicle:
other: ethanol:deionised water (30:70)
Concentration:
Main study: 0 (vehicle), 5, 10 and 25% (w/v)
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration, which could be technically used, was 25% test item solution (w/v%) in ethanol:deionised water (30:70)
- Two mice were treated with concentrations of 10 and 25% each on three consecutive days.
- Irritation: clinical signs were recorded within 1 hour and 24 ± 4 hours after each application as well as on day 7. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: local lymph node assay (LLNA)
- Criteria used to consider a positive response: First, the exposure to at least one concentration of the test item resulted in an incorporation of [³H]TdR (³H-methyl thymidine) at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index. Second, the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 5, 10, and 25% (w/v) in ethanol:deionised water (30:70). The application volume (25 µL) was spread over the entire dorsal surface (diameter: approx. 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
Five days after the first topical application, all mice were administered with 250 µL of 80.0 µCi/mL [³H]TdR (corresponding to 20.0 µCi 3HTdR per mouse) by intravenous injection via a tail vein. Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium.
The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared, washed gently and incubated at approximately +4 °C for at least 18 hours.
The proliferative capacity of pooled lymph node cells was determined by the incorporation of [³H]TdR measured in a beta-scintillation counter.

OBSERVATIONS:
- Mortality / Viability: once daily (week day) from experimental start to necropsy.
- Body weights: prior to the first application and prior to treatment with [³H]TdR.
- Clinical signs (local / systemic): within 1 hour after each application, and 24 ± 4 hours after the first and second application as well as on the day of preparation. Especially the treatment sites were observed carefully.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Body weight: mean values and standard deviations were calculated.
[³H]TdR incorporation was calculated as mean cpm ± SD, and stimulation indices of lymph node cell proliferation between substance-treated and vehicle-treated groups were calculated.

Results and discussion

Positive control results:
DPM per lymph node:
0 (vehicle group, acetone:olive oil (4:1)): 727.6
5 % hexyl cinnamic aledhyde: 1303.6
10 % hexyl cinnamic aledhyde: 1518.4
25 % hexyl cinnamic aledhyde: 4976.6

Stimulation index:
5 % hexyl cinnamic aledhyde: S.I. = 1.79
10 % hexyl cinnamic aledhyde: S.I. = 2.09
25 % hexyl cinnamic aledhyde: S.I. = 6.84

The positive control substance showed the expected positive result (S.I. > 3).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
5 % test item: S.I.= 0.82 10 % test item: S.I.= 1.25 25 % test item: S.I. = 3.38 As the exposure to the highest test concentration resulted in a greater than 3-fold increase in incorporation of [³H]TdR compared to the concurrent control (indicated by the Stimulation index), the test item was found to be a skin sensitiser.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM per lymph node: 0 % (vehicle control, ethanol:deionised water (30:70)): 311.3 5 % test item: 253.9 10 % test item: 388.1 25 % test item: 1052.3

Any other information on results incl. tables

Mortality, clinical signs and body weights:

No death occurred during the study period. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

Body weights of the animals were within the range commonly recorded for animals of this strain and age (see Table 3).

Table 1: Calculation and Results of the individual test item data:

Test item concentration % (w/v)

Group

Measurement DPM

Calculation: DPM per node

S.I.

-

BG 1

406

-

-

-

BG 2

14

-

-

0

1

2700

311.3

 

5

2

2241

253.9

0.82

10

3

3315

388.1

1.25

25

4

8628

1052.3

3.38

BG = background (1 mL trichloroacetic acid) in duplicate

1 = control group

2 -4 = test groups

S.I. Stimulation Index

Calculation: DPM - BG (mean value) divided by number of lymph nodes (8)

EC3 (estimated concentration for S.I. of 3) = 22.3 % (w/v)

Table 2: Calculation and results of the individual positive control data:

Test item concentration % (w/v)

Group

Measurement DPM

Calculation: DPM per node

S.I.

-

BG 1

23

-

-

-

BG 2

19

-

-

0

1

5842

727.6

 

5

2

10450

1303.6

1.79

10

3

12168

1518.4

2.09

25

4

39834

4976.6

6.84

BG = background (1 mL trichloroacetic acid) in duplicate

1 = control group

2 -4 = test groups

S.I. Stimulation Index

Calculation: DPM - BG (mean value) divided by number of lymph nodes (8)

EC3 (estimated concentration for S.I. of 3) = 12.9 % (w/v)

Table 3: Body weights:

Dose group

Mean±SD (g)

animal weights prior to first application

Vehicle control

20.6± 1.0

5% test item

21.6± 0.4

10% test item

20.7± 1.7

25% test item

21.5± 1.4

summary

21.0± 1.1

animal weights prior to first application

Vehicle control

22.3± 1.1

5% test item

22.1± 0.8

10% test item

21.8± 1.3

25% test item

22.4± 0.3

summary

22.1± 0.9

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information at the highest concentration tested with a stimulation index just higher than 3 fold the control, indicating a weak sensitizing potential