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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
13 Jun - 02 Jul 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP- Guideline study. The ear thickness was not measured in the range-finding or main study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted Jul 2010
Deviations:
yes
Remarks:
ear thickness was not measured in the range-finding or main study
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): PM-5927
- Physical state: off-white powder
- Lot/batch No.: 07-02
- Expiration date of the lot/batch: 05 Feb 2008
- Storage condition of test material: at room temperature protected from light
- Other: pH 5.2 - 6.0 (1% in water, indicative range)

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L'Arbresle Cedex, France
- Age at study initiation: approximately 11 weeks
- Weight at study initiation: 20 - 25 g (range)
- Housing: animals were housed individually in labelled Macrolon cages (MI type, height 12.5 cm) containing sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argeteuil, France)
- Diet: pelleted rodent diet, SM R/M-Z (SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.6 - 23.3 (actual range)
- Humidity (%): 41 - 78 (actual range)
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5, 25 and 50%
No. of animals per dose:
5 females
Details on study design:
RANGE FINDING TESTS: 2 animals were tested with a 25 and 50% solution of the test substance in order to select the highest concentration to be used in the main study. The study protocol was the same as for the main study for Day 1 - 3, with induction treatment daily. Approximately 3 - 4 hours after the last exposure, the ear was cleaned of residual test substance with tap water and the irritation was assessed.
- Compound solubility: the highest concentration of 50% was the maximum concentration that could technically be applied
- Irritation: no skin irritation reactions were observed on the left or right ear

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: ³H-methyl thymidine incorporation determined by ß-scintillation
- Criteria used to consider a positive response: if the results indicate a SI ≥ 3, the test substance is considered to be a skin sensitiser

TREATMENT PREPARATION AND ADMINISTRATION:
A formulation of the test substance in acetone/olive oil (4:1 v/v) was prepared within 4 h before application, homogeneity was obtained to visually acceptable levels. The formulation was mixed thouroughly with a vortex mixer prior to application. 25 µl was applied to the dorsal surface of both ears for 3 consecutive days. On Day 6, each animal was injected via the tail vein with 0.25 mL sterile phosphate buffered saline containing 20 µCi of ³H-methyl thymidine (GE Health care, Buckinghamshire, UK). After approximately 5 h, the mice were sacrificed and the draining lymph nodes of the ears were excised. The relative size was estimated by visual examination and any abnormalities of the nodes and surrounding areas were recorded. The nodes were pooled for each animal in PBS. A single cell suspension of lymph node cells was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 µm). Lymph node cells were washed twice with an excess of PBS and the DNA precipitated with 5% trichloroacetic acid (TCA) at 4 ºC overnight. The precipitate was recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL Ultima Gold cocktail as the scintillation fluid. The counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes, whichever came first. The scintillation counter automatically subtracted the background and converted Count Per Minute (CPM) to Disintegrations Per Minute (DPM).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
A reliability check was performed in March 2007 in order to check the sensitivity of the test system and the reliability of the experimental techniques of the testing laboratory (NOTOX project 484706). Groups of 5 female CBA mice were exposed to 5, 10 and 25% alpha-hexylcinnamicaldehyde in acetone:olive oil (4:1), or the vehicle only. The SI values calculated for the substance concentrations 5, 10 and 25% were 1.3, 1.5 and 5.5, respectively. The negative control had an SI value of 1.0. An EC3 value of 15.6% was calculated using linear interpolation. The calculated EC3 value was in the acceptable range of 2-20%. The results of the previous 6-monthly reliability checks were EC3 values of 7.3, 10.3, 9.5 and 13.1%.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The SI values calculated for the 5, 25 and 50% groups were 1.0, 1.1 and 1.3, respectively (see Table 2). It was not possible to extrapolate an EC3 value as the SI values were consistantly below 3.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The mean DPM/animal values for the 5, 25 and 50% groups were 350, 389 and 471, respectively. The mean DPM/animal value for the control group was 352 (see Table 1 for individual values and Table 2 for mean values with standard deviation).

Any other information on results incl. tables

 All the nodes of the animals in the control and treatment groups were normal in size. No macroscopic abnormalities were noted in the surrounding area. No erythema and no edema was observed at the application sites. The body weights and body weight gains were comparable between the control and the treatment groups and no clinical signs were observed.

Table 1: Radioactivity measurements, individual results

Dose group (%)

Animal

DPM/animal

Control (vehicle)

1

292

Control (vehicle)

2

610

Control (vehicle)

3

84

Control (vehicle)

4

494

Control (vehicle)

5

280

5

1

334

5

2

317

5

3

680

5

4

148

5

5

270

25

1

573

25

2

555

25

3

256

25

4

152

25

5

407

50

1

455

50

2

329

50

3

351

50

4

897

50

5

323

 

Table 2: Disintegrations per minute and stimulation index

Dose group (%)

DPM (mean ± SD)

SI ( mean ± SD)

5

350 ± 89

1.0 ± 0.4

25

389 ± 82

1.1 ± 0.4

50

471 ± 109

1.3 ± 0.5

Control

352 ± 91

1.0

 

Applicant's summary and conclusion

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