(3R,3aR,6S,6aR)-Hexahydrofuro[3,2-b]furan-3,6-diyl bis(4-hydroxybenzoate)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Jun 2004 to 15 Jul 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

April 24, 2002

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March, 2003

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

(3R,3aR,6S,6aR)-Hexahydrofuro[3,2-b]furan-3,6-diyl bis(4-hydroxybenzoate)

EC Number:

606-059-4

Cas Number:

185756-31-4

Molecular formula:

C20H18O8

IUPAC Name:

(3R,3aR,6S,6aR)-Hexahydrofuro[3,2-b]furan-3,6-diyl bis(4-hydroxybenzoate)

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Solid, powder / white
- Homogeneity: The test substance was homogeneous by visual inspection.

Specific details on test material used for the study:

TEST MATERIAL
- Name of the test substance: DKDS - Reinkristallisat
- Batch No.: CP 203-362-04-02
- Appearance: Solid, powder / white
- Purity: 96.8 w/w%
- Homogeneity: The test substance was homogeneous by visual inspection.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature. The stability under storage conditions was confirmed by reanalysis.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Remarks:

CaOIaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, FRG
- Age at study initiation: About 6 - 8 weeks
- Weight at study initiation: 16.8 - 20.5 g
- Housing: One animal per cage (type: Makrolon 1)
- Bedding: Granulat Typ ¾ (staubfrei); SSNIFF
- Diet: Kliba-Labordiät (Maus / Ratte Haltung "GLP"), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 15 days before the first test substance application.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Humidity: 30 - 70 %
- Photoperiod: 12/12 h

Study design: in vivo (LLNA)

Vehicle:

other: Acetone

Concentration:

- Control group: vehicle alone
- Test group 2: 25 µL of 3% in acetone
-Test group 3: 25 µL of 10% in acetone
- Test group 4: 25 µL of 30% in acetone

The selection of concentrations took into account available information on the chemical/physical properties and the composition of the test substance, the acute toxicity and on primary irritation/corrosion potential.

No. of animals per dose:

6

Details on study design:

TEST SUBSTANCE PREPARATION
The test substance preparations were produced on a weight by weight (w/w) basis shortly before the application by stirring with a magnetic stirrer. The test substance was applied as a solution for all applications. the highest test substance concentration (30%) was found to be the maximum soluble concentration. The 30% preparation was soluble after stirring tor a prolonged period, only. Suspensions would have posed difficulties concerning homogeneity and application.

GENERAL
The study comprised three treatment groups and a vehicle control group. Each group consisted of 6 mice. A check for dead or moribund animals was made twice each workday and once on Saturdays, Sundays and on public holidays. No detailed clinical examination of the individual animais was performed but any obvious signs of systemic toxicity and/or local inflammation at the application sites were noted in the raw data.

BODY WEIGHT
Body weights of the individual animals were determined on study day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.

TEST SUBSTANCE APPLICATION
Epicutaneous application is simulating dermal contact with the compound which is possible to occur under practical use conditions.
- Application volume: 25 µL per ear
- Site of application: Dorsal part of both ears
- Frequency of application: 3 consecutive applications (day 0 - day 2) to the same application site
The animals of groups 1-4 were treated with vehicle or test substance preparation.

TERMINAL PROCEDURES
The animals were sacrificed on study day 5 by cervical dislocation

EAR WEIGHT
Immediately after the death of each animal a circular piece of tissue (diameter of 0.8 cm) was punched out of the apical part of each ear. The weight of the pooled punches was determined for each animal.

LYMPH NODE WEIGHT
Immediately after removal of the ear punches the left and right auricular lymph nodes were dissected. The weight of the pooled Iymph nodes from both sides was determined for each animal.

PREPARATION OF CELL SUSPENSIO AND DETERMINATION OF CELL COUNT
After weight determination, the pooled lymph nodes of each animal were stored in phosphate buffered saline in an ice-water bath until further preparation. A single ceIl suspension was prepared as soon as possible after dissection by carefully passing the lymph nodes through an iron mesh (mesh size 200 µm) into 6 mL of phosphate buffered saline. For determination of cell count the standard suspension was further diluted with Casy®ton in a ratio 1:500. The cell count was determined using a Casy®-Counter. Each suspension was measured in triplicate (3 dilutions of the standard suspension). The mean of the triplicate measurements was used for further evaluation.

Positive control substance(s):

other: Alpha-Hexylcinnamaldehyde, techn. 85%

Statistics:

The statistical evaluations were performed using the WILCOXON test.

Results and discussion

Positive control results:

A concurrent positive control (reliability check) with a known sensitizer was not included into this study.
No signs of systemic toxicity were noticed. The positive control substance induced a statistically significant and biologically relevant response in the auricular lymph nodes, when applied as 3% or 10% preparations in acetone. The Iimited but statistically significant increases in ear weights indicate some irritation of the ear skin at all concentrations.
The increase in cell count index induced by the 3% test substance preparation was at the border of biological significance. The marked increase at 10% cannot be explained by skin irritation alone.
Based on the results discussed above it is concluded that Alpha-Hexylcinnamaldehyde, techn. 85% has a sensitizing effeot in the Murine Local Lymph Node Assay under the test conditions chosen.
The threshold of skinsensitization induction of the compound is >1% <3% under the test conditions used.

In vivo (LLNA)

Results

Results 1

Parameter:

other: Lymph node weight index

Value:

0.93

Test group / Remarks:

3% in acetone

Results 2

Parameter:

other: Lymph node weight index

Value:

1.06

Test group / Remarks:

10% in acetone

Results 3

Parameter:

other: Lymph node weight index

Value:

1.15

Test group / Remarks:

30% in acetone

Results 4

Parameter:

other: Cell Count Index

Value:

0.99

Test group / Remarks:

3% in acetone

Results 5

Parameter:

other: Cell Count Index

Value:

1.13

Test group / Remarks:

10% in acetone

Results 6

Parameter:

other: Cell Count Index

Value:

1.24

Test group / Remarks:

30% in acetone

Results 7

Parameter:

other: Ear Weight Index

Value:

1

Test group / Remarks:

3% in acetone

Results 8

Parameter:

other: Ear Weight Index

Value:

1.02

Test group / Remarks:

10% in acetone

Results 9

Parameter:

other: Ear Weight Index

Value:

0.99

Test group / Remarks:

30% in acetone

Cellular proliferation data / Observations:

LYMPH NODE WEIGHTS AND CELL COUNTS
Treatment of the mice with a 30% test substance preparation induced a slight statistically significant increase in Iymph node ceII counts but not in Iymph node weights as compared to the vehicle control group. Neither the 3% nor the 10% test substance solution caused any statistically significant Iymph node response.

EAR WEIGHTS
Treatment of the mice with 3% - 30% test substance preparations induced no statistically significant increase in ear weights as compared to the vehicle control group.

BODY WEIGHTS
The expected body weight gain was generally observed in the course of the study

OTHER FINDINGS
Rests of test substance were noted in the animals treated with the 30% test substance concentration on the day of the 2nd and 3rd application.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met