Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04.08.2005 to 25.08.2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, D-33l78 Borchen
- Age at study initiation: 7-12 weeks
- Weight at study initiation: 18-22 g
- Housing: In groups in Makrolon cages
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: described as 'adequate'

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

IN-LIFE DATES: From: 10.08.2005 To: 25.08.2005

Study design: in vivo (LLNA)

Vehicle:
other: acetone/olive oil 3+1 v/v (AOO)
Concentration:
10, 50 and 100% in vehicle
No. of animals per dose:
Five (two in preliminary test)
Details on study design:
RANGE FINDING TESTS:
A preliminary test was performed using 2 animals. 100 % (in AOO (v/v) of the test item was applied to one animal daily, the other animal with
50% (in AOO (v/v) of the test item over 3 consecutive days. Neither signs of systemic toxicity nor signs of irritation at the application site could be
detected in any of the animals. Due to results of the preliminary test the test item was assayed at three concentrations: 10%,50%, 100%. The preparations were made immediately prior to each dosing.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymph node assay
- Criteria used to consider a positive response: A substance is regarded as a 'sensitizer' 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 lymph nodes 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).
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPMlNODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPMlNODE values were determined, background values were subtracted.
EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation {EC3 = c + [(3-d) / (b-d)] x (a-c)}, between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three If all measured points are above or below the stimulation index of three, no EC3 value can be stated.
Results of the ear thickness measurement could be used to differentiate between irritating and sensitating properties of the test item.

TREATMENT PREPARATION AND ADMINISTRATION: Topical Application: Directly prior to the first application the thickness of the ears of all animals
was measured. Each mouse was tested by topical application of 25 µl of the selected solution to the entire dorsal surface of each ear. Topical applications were performed once daily over three consecutive days.
Administration of 3H-methyl thymidine: Five days after the first topical application treatment, all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250 µl of 3H-methyl thymidine, diluted to a working concentration of 80 µCi/ml
Preparation of cell suspension: Before sacrificing the animals the thickness of the ears of all animals was determined again. Approximately 5 hours after 3H-methyl thymidine injection all mice were sacrificed. The draining "auricular Iymph nodes" were excised, pooled for each animal (2 Iymph nodes per animal, if technically possible) and collected in PBS. A single cell suspension of pooled Iymph node cells was prepared by gentle mechanical disaggregation through polyamide ganze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated. After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4 °C overnight for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 10 mL scintillation fluid and transferred into scintillation vials.
Determination of incorporated 3H-methyl thymidine: The 3H-methyl thymidine - incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.
Positive control substance(s):
other: none
Statistics:
No data

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
0.7
Test group / Remarks:
10% concentration
Key result
Parameter:
SI
Value:
0.8
Test group / Remarks:
50% concentration
Key result
Parameter:
SI
Value:
0.5
Test group / Remarks:
100% concentration
Parameter:
EC3
Remarks on result:
other: An EC3 value (derived by linear interpolation) could not be calculated.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Group mean DPMs: vehicle: 454.8 (±197.5) 10%: 314.6 (±108.0) 50%: 378.5 (±98.5) 100%: 257.7 (±57.4)

Any other information on results incl. tables

Table 1 Ear thickness results

Mean ear thickness 

Day 1 

Day 6 

10% groups 

0.21 mm 

0.21 mm 

50% group 

0.21 mm 

0.22 mm 

100% group 

0.22 mm 

0.23 mm 

negative control 

0.22 mm 

0.22 mm 

 

Table 2 Stimulation index results

Stimulation index at 10% 

0.7 

Stimulation index at 50%  

0.8 

Stimulation index at 100%  

0.5 

 

The proliferative response of lymph node cells was calculated as the ratio of 3H-methylthymidine- incorporation into lymph node cells of test group animals relative to that recorded for control group animals. A stimulation index, ratio of test item / negative control, was calculated for each concentration.

All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study.

At the daily clinical observation the animals did not show any visible clinical symptoms.

Table 3: Results of LLNA: Disintegrations per minute per animal (group means)

Sample Description

Test or Control Group

 Animal #

Individual Animal
DPMa

Group Mean
DPM (+/- SE)

Stimulation Index (SI)*

Vehicle

1

381.2

454.8 (±197.5)

1.0

2

308.7

3

437.6

4

837.6

5

308.7

Low (10%)

1

212.1

314.6 (±108.0)

0.7

2

314.1

3

177.2

4

434.9

5

434.9

Medium (50%)

1

381.2

378.5 (±98.5)

0.8

2

475.2

3

445.6

4

193.3

5

397.3

High (100%)

1

179.9

257.7 (±57.4)

0.5

2

357.0

3

247.0

4

238.9

5

265.8

* SI = Group meanDPM)Vehiclecontrol mean DPM

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In a Local Lymph Node Assay conducted using a protocol similar to OECD 429 and to GLP (reliability score 1) D5 gave a negative result and is therefore not expected to be a skin sensitiser.