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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:
July 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Additionally the study was reviewed by an external expert (R.J. Dearman, University of Manchester, Division of Infection, Immunity & Respiratory Medicine)

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(2002)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
no further information
Specific details on test material used for the study:
Purity: 99.8%, a certificate of analysis was provided to the test institute by the sponsor
- Stability under test conditions: No information on testing of stability of the test substance in the vehicle is provided in the report, however, all dose preparations were prepared freshly before dosing (all dose preparations were used within 24 hours of preparation).

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: CBA/Ca/Ola/Hsd
- Source:Harlan Interfauna UK Limited, Blackthorne, Bicester, Oxon, UK
- Age at study initiation: 8–12 weeks
- Weight at study initiation: 17.3-22.8 g
- Housing: a maximum of 4 mice was housed per cage
- Diet and water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): a minimum of 15 changes/hour
- Photoperiod (hrs dark / hrs light):12/12

Study design: in vivo (LLNA)

Vehicle:
other: 1:3 Ethanol : Diethyl phthalate
Remarks:
Rational for the vehicle chosen: Betts CJ, Contact Dermatitis 56, 70-75, 2007
Concentration:
2.5, 5, 10, 25, and 50 % w/v
No. of animals per dose:
4
Details on study design:
Approximately 25µL of a 2.5, 5, 10, 25 or 50% w/v preparation of the test substance in 1:3 EtOH:DEP was applied, using a variable volume micro-pipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using 1 : 3 EtOH:DEP alone. The procedure was repeated daily for 3 consecutive days.
Three days after the third application, all the animals were injected, via the tail vein, with approximately 250µL of phosphate buffered saline (PBS) containing 20 µCi of a 2.0 Ci/mmol
specific activity 3H-methyl thymidine. Approximately 5 hours later, the animals were humanely killed by inhalation of 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 lymph nodes through a 200-mesh stainless steel gauze. The cell suspensions were then washed three times by centrifugation with approximately 10 ml of PBS. Approximately 3 ml of 5 % w/v trichloroacetic acid (TCA) was added and, after overnight precipitation at 4 °C, the samples were pelleted by centrifugation and the supematant was discarded. The cells were then resuspended in approximately 1ml of TCA.
The lymph node suspensions were transferred to scintillation vials and 10 ml of scintillant (Optiphase) was added prior toP-scintillation counting using a Packard Tri-Carb Liquid Scintillation Counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

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

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1
Test group / Remarks:
2.5 % (w/v)
Parameter:
SI
Value:
0.9
Test group / Remarks:
5 % (w/v)
Parameter:
SI
Value:
0.5
Test group / Remarks:
10 % (w/v)
Parameter:
SI
Value:
0.6
Test group / Remarks:
25 % (w/v)
Parameter:
SI
Value:
1.2
Test group / Remarks:
50 % (w/v)
Parameter:
EC3
Remarks:
(%)
Value:
> 50
Test group / Remarks:
Estimated EC3, since test substance concentration up to 50 % caused no SI value increase greater than 3-fold.
Cellular proliferation data / Observations:
Benzyl alcohol in 1:3 EtOH:DEP did not have the capacity to cause skin sensitization at any of the doses applied. The EC3 value giving rise to a 3-fold increase in lymphocyte proliferation was estimated to be greater than 50 % w/v (greater than 12500 µg/cm²).

Effects on body weight/body weight gain or on general condition were not reported.

Any other information on results incl. tables

 Conc. of test substance (% w/v)  Number of lymph nodes assayed  dpm  dpm per lymph node  Test:control ratio
 0 (vehicle only)  8  2248  281  N/A
 2.5  8  2289  286  1.0
 5  8  1975  247  0.9
 10  8  1191  149  0.5
 25  8  1420  178  0.6
 50  8  2635  329  1.2
 EC3 Estimated to be greater than 50 % w/v (> 12500 µg/cm²)

Applicant's summary and conclusion

Executive summary:

A LLNA on female CBA/Ca/Ola/Hsd-mice revealed no 3 -fold increase in lymphocytes after substance application at concentrations of 2.5, 5, 10, 25, and 50 % and thus no indication for a sensitising potential of the substance.

Hexyl cinnamaldehyde as positive control showed the expected positive result after application of a 10 or 25 % formulation in acetone:olive oil 4:1, confirming thus the validity of the protocol used for this study.