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:
June 16, 2006 to June 27, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Bourgeonal
- Physical state: liquid
- Analytical purity: Not specified
- Impurities (identity and concentrations):
- Lot/batch No.: 02759TT
- Expiration date of the lot/batch: Not specified

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK Limited, Shaw's Farm, Blackthorne, Bicester, Oxon, UK
- Age at study initiation: Young adults(8-12 weeks old)
- Weight at study initiation: Ranged from 16.6 to 20.6 g
- Housing: maximum of 4 mice housed per cage, in cages suitable for animal sof thi sstrain and weight range. Environemntal enrichment provided included tents, bases and nestlets.
- Diet (e.g. ad libitum): RM1, supplied by Special Diet Services Limited, Within, Essex, UK.
- Water (e.g. ad libitum): From mains supply
- Acclimation period: At least 5 day sprior to start of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30-70%
- Air changes (per hr): Minimum of 15 changes per hour
- Photoperiod (hrs dark / hrs light): Artificial light, giving 12 hours light, 12 hours dark.

IN-LIFE DATES: From: June 21, 2006 To: June 27, 2006

Study design: in vivo (LLNA)

Vehicle:
other: 1:3 Ethanol/ Diethylphthalate
Concentration:
1, 2.5, 5, 10 and 25 % w/v
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Not reported
- Irritation: Not reported
- Lymph node proliferation response:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
- Criteria used to consider a positive response:

TREATMENT PREPARATION AND ADMINISTRATION:
RANGE FINDING TESTS:
- Compound solubility:
- Irritation:
- Lymph node proliferation response:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
- Criteria used to consider a positive response: The results are expressed as a disintegration per minute (dpm) value per each group. The activity of each test group is then divided by the activity of the vehicle control group to give a test: control ratio known as the stimulation index (SI) for each concentration. The criteria for a positive response is that one or more concentrations of the test substance should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group. The assay is able to identify those materials that elicit responses in a standard Magnusson and Kligman maximisation guinea pig test for sensitisation. Consequently, a test substance which does not fulfi the crierion is designated as unlikely to be a skin sensitiser.

TREATMENT PREPARATION AND ADMINISTRATION: Groups of four female mice were used for this study. Approximately 25µL of a 1,2.5, 5, 10 or 25% 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 final application, all the animals were injected, via the tail vein, with approximately 250µL of phosphate buffered saline (PBS) containing 20µCi of a 2.0Ci/mmol Specific activity 3H-methyl thymidine. Approximatley 5 hours later, the animals were humanely killed by inhalation of halothane 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 with PBS.
A single cell suspension was prepared by mechanical disaggregation of lymph nodes through 200-mesh stainless steel gauze. The cell suspensions 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 4°C, the samples were pelleted by centrifugation and the supernatant was discarded. The cells 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 ß-scintillation counting using a Packard Tri-Carb 3100TR Liquid Scintillation Counter
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
In a positive control study, hexylcinnamaldehyde was shown to have the capacity to cause skin sensitisation when applied as a 25% (w/v) preparation in acetone: olive oil (4:1), confirming the validity of the protocol used for this study.
The reliability of the protocol using a positive control was conducted between January 25, 2006 and January 31, 2006.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Concentration of test substance (% w/v) Stimulation index (SI) 0 (vehicle only) N/A 1 0.9 2.5 1.4 5 3.6 10 4.2 25 9.8
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Concentration of test substance (% w/v) Disintegrations per minute (dpm) Dpm per lymph node 0 (vehicle only) 4538 567 1 3930 491 2.5 6293 787 5 16372 2047 10 18948 2369 25 44444 5556

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Remarks:
Migrated information
Conclusions:
The test item has the capacity to cause skin sensitisation when applied as dose levels of 5, 10 and 25% w/v preparations in 1:3 EtOH:DEP. The EC3 value giving rise to a three-fold increase in lymphocyte proliferation was calculated to be 4.3% w/v (1075µg/cm2).
Executive summary:

The test item, Bourgeonal, was tested for potential skin sensitisation according to OECD guideline 429.

The application of the test substance at concentrations of 1, 2.5, 5, 10 and 25 % w/v in 1:3 EtOH:DEP resulted in an isotope incorporation, which was greater than 3 -fold at the 5, 10 and 25 % w/v concentrations. Consequently, the test substance is likely to be a skin sensitiser under the conditionsof the test. The concentration giving rise to a 3 -fold increase in lymphocyte proliferation (EC3) was calculated to be 4.3% w.v (1075 µg/cm2).

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

In conclusion, Bourgeonal in 1:3 EtOH:DEP vehicle is a skin sensitiser under the conditions of the test with an EC3 value of 4.3% (1075 µg/m2).