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:
24th May 2006 to 6th June 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Test material form:
solid: particulate/powder
Details on test material:
Light pink tinted powder
Specific details on test material used for the study:
Light pink powder

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca/Ola/Hsd
Sex:
female
Details on test animals and environmental conditions:
A maximum of 4 mice were housed by cage, in cages suitable for animals of this strain and weight range. Environmental enrichment was provided including tents, bases and nestlets.

The animal room was designed to give the following environmental conditions:
Temperature - 22 +/- 3 degrees centigrade
Relative humidity - 30 - 70%
Air - Minimum of 15 changes per house
Light cycle - artifical, giving 12 hours light and 12 hours dark

Both temp and relative humidity were recorded daily. The recorded values were within the specified ranged.

Diet (RM1), supplied by Special Diet Services Limited, Witham, Essex, UK and mains water, supplied by an automatic system, were available ad libitum.

Each batch of diet is routinely analyses for composition and contaminants. Water is also periodically analysed for contaminants. Not contaminants were found in the diet or water at levels considered likely to interfere with the pirpose or outcome of the study. Certificates of analyses are retained in the CTL archives.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 50 and 60% w/v along with 0% controls
No. of animals per dose:
4
Details on study design:
Hexylcinnamaldehyde was selected as the positive control as it has known sentising properties.

Groups of four female mice were used for this study. Approximately 25µl or a 25, 50 or 60% w/v preparation of the test substance in acetone in olive oil (4:1) was applied, using a variable volume micro-pipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using acetone in olive oil (4:1) alone. the procedure was repeated daily for 3 consecutive days.

Three days after the third application, all the animals were injectied, via the tail vein, with approximately 250µl of phosphate buffered sale (PBS) containing 20µCi of a 2.0Ci/mmol specific activity 3H-methyl theymidine. Approximately 5 hours later, the animals were humanely killed by inhalation of the halothane vapour followed by cervical dislocation. the draining auricular lymph nodes were removed from each animal and, together with the nodes from the other animals in the group, were placed in a container of PBS.

A single cell suspension was prepared by mechanical disaggregation of lumph nodes through a 200-mesh stainless steel gauze. the cell suspesntions were then washed three times by centrifugation with approximately 10ml of PBS. Approximately 3ml of 5% w/v trichloroacetic acid (TCA) was added and, after overnight precipitation at 4oC, the samples were pelleted by centrifugations and the supernatant was discarded. The celles were then resuspended in approximately 1ml of TCA.

the lymph node suspensions were transferred to scintillation vials and 10ml of scintillant (Optiphase) was added prior to β-scintillation counting using a Packard Tri-Card 3100TR Liquid Scintillation Counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The application of hexycinnamaldehyde at concentrations of 5%, 10% and 25% w/v in acetone in olive oil (4:1) resulted in a greater than 3-fold increase in isotope incorporation at the 25% w/v concentration. There, hexylcinnamaldehyde was shown to be a skin senstiser, confirming the validity of the protocol used for the study.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
Conc of test substance 0% (w/v) - Vehicle only
Key result
Parameter:
SI
Value:
2.2
Test group / Remarks:
Conc of test substance 25% (w/v)
Key result
Parameter:
SI
Value:
3.2
Test group / Remarks:
Conc of test substance 50% (w/v)
Key result
Parameter:
SI
Value:
3.5
Test group / Remarks:
Conc of test substance 60% (w/v)
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
Conc of hexylcinnamaldehyde 0% (w/v) - vehicle only
Key result
Parameter:
SI
Value:
2.1
Test group / Remarks:
Conc of hexylcinnamaldehyde 5% (w/v)
Key result
Parameter:
SI
Value:
2.4
Test group / Remarks:
Conc of hexylconnamaldehyde 10% (w/v)
Key result
Parameter:
SI
Value:
7.2
Test group / Remarks:
Conc of hexylcinnamaldehyde 25% (w/v)
Cellular proliferation data / Observations:
The application of the test substance at concentrations of 25, 50 and 60% w/v in acetone and olive oil (4:1) resulted in an increase in isotope incorporation which was greater than 3 -fold at the 50 and 60% w/v concentrations.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In conclusion, UL125 is a skin sensitiser under the conditions of the test.