.beta.-D-Ribofuranoside, methyl 2,3-O-(1-methylethylidene)-, 5-(4-methylbenzenesulfonate)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09-02-2005 to 07-04-2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study procedures described in this report meet or exceed the requirements of the following guidelines: OECD Guideline for the Testing of Chemicals, Guideline 429: Skin Sensitization: Local Lymph Node Assay (adopted 24 April 2002). Commision Directive 2004/73/EC, B.42: Skin Sensitization: Local Lymph Node Assay, 29 April 2004.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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:

[(3aS,4S,6S)-6-methoxy-2,2-dimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methyl 4-methylbenzene-1-sulfonate

EC Number:

609-920-2

Cas Number:

4137-56-8

Molecular formula:

C16H22O7S

IUPAC Name:

[(3aS,4S,6S)-6-methoxy-2,2-dimethyl-tetrahydro-2H-furo[3,4-d][1,3]dioxol-4-yl]methyl 4-methylbenzene-1-sulfonate

Details on test material:

Identity Tosylfuranosid
Description White solid
Stability of test item Stable under storage conditions
Expiry date 31-JUL-2005
Storage conditions At room temperature (20 °C ± 5 °C), away from direct
sunlight.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST SYSTEM
Test system Mice, CBA/CaOlaHsd
Rationale Recognized by OECD Guideline 429 as the
recommended test system.
Source Harlan Netherlands
B.V. Postbus 6174
NL - 5960 AD Horst / The Netherlands
Number of animals for 2 females
the pre-test (non-GLP)
Number of animals for
the main study
16 females
Number of animals per group 4 females (nulliparous and non-pregnant)
Number of test groups 3
Number of control (vehicle) group 1
Age 8 - 12 weeks (beginning of acclimatization)
Body weight 16 g - 24 g (ordered)
Identification Each cage by unique cage card.
Randomization Randomly selected by computer algorithm at time of
delivery.
Acclimatization Under test conditions after health examination. Only
animals without any visible signs of illness were used
for the study.

The sensitivity and reliability of the experimental technique employed was assessed by use of
a substance which is known to have skin sensitization properties in CBA/CaOlaHsd mice. The
validation- / positive control study was performed with ALPHA-HEXYLCINNAMALDEHYDE in
acetone/olive oil (4/1, v/v) using CBA/CaOlaHsd mice (RCC Study Number 858384) between
19-JAN-2005 to 02-FEB-2005, results see Appendix D.

HUSBANDRY
Room no. E24 / RCC Itingen
Conditions Standard Laboratory Conditions. Air-conditioned with
ranges for room temperature 22 + 3 °C, relative
humidity 30 - 70 % and 10 - 15 air changes per hour.
Room temperature and humidity were monitored
continuously and values outside of these ranges
occasionally occurred, usually following room cleaning.
These transient variations are considered not to have
any influence on the study and, therefore, these data
are not reported but are retained at RCC. There was a
12 hour fluorescent light / 12 hour dark cycle with at
least 8 hours music during the light period.
Accommodation Individual in Makrolon type-2 cages with standard
softwood bedding ("Lignocel", Schill AG, CH-4132
Muttenz).
Diet Pelleted standard Kliba 3433, batch no. 92/04 mouse
maintenance diet (Provimi Kliba AG,
CH-4303 Kaiseraugst) available ad libitum. Results of
analyses for contaminants are archived at RCC. There
was no contamination of the diet.
Water Community tap water from Itingen, available ad libitum.
Results of representative bacteriological, chemical and
contaminant analyses are archived at RCC. There was
no contamination of water.

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

10 %, 25 % and 50 % (w/v), respectively, in acetone/olive oil (4/1, v/v).

No. of animals per dose:

4

Details on study design:

TOPICAL APPLICATION
Each test group of mice was treated by topical (epidermal) application to the dorsal surface
of each ear lobe (left and right) with different test item concentrations of 10 %, 25 % and
50 % (w/v) in acetone/olive oil (4/1, v/v). The application volume, 25 µl, was spread over the
entire dorsal surface (diameter ~ 8 mm) of each ear lobe once daily for three consecutive days. A
further group of mice was treated with an equivalent volume of the relevant vehicle alone
(control animals). A hair dryer was passed briefly over the ear’s surface to prevent the loss of
any of the test item applied.

ADMINISTRATION OF 3H-METHYL THYMIDINE*
3H-methyl thymidine (3HTdR) was purchased from Amersham International (Amersham
product code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml).
Five days after the first topical application, all mice were administered with 250 µl of
76.13 µCi/ml 3HTdR (equal to 19.0 µCi 3HTdR) by intravenous injection via a tail vein.

DETERMINATION OF INCORPORATED 3HTDR*
Approximately five hours after treatment with 3HTdR all mice were euthanized by inhalation
of CO2 (dry ice).
The draining lymph nodes were rapidly excised and pooled for each experimental group
(8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph
node cells were prepared by gentle mechanical disaggregation through stainless steel gauze
(200 µm mesh size). After washing twice with phosphate buffered saline (approx. 10 ml) the
lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated
at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The
precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass
scintillation vials with 10 ml of ‘Irga-Safe Plus’ scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a ß-scintillation counter. Similarly,
background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid.
* Preparation of 3HTdR solutions and 3HTdR measurements at RCC Ltd, Environmental Chemistry &
Pharmanalytics
No phase report of the results of the 3HTdR level analysis was provided by the Principal Investigator.
The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive
disintegrations per minute (DPM).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:

The sensitivity and reliability of the experimental technique employed was assessed by use of
a substance which is known to have skin sensitization properties in CBA/CaOlaHsd mice. The
validation- / positive control study was performed with ALPHA-HEXYLCINNAMALDEHYDE in
acetone/olive oil (4/1, v/v) using CBA/CaOlaHsd mice (RCC Study Number 858384) between
19-JAN-2005 to 02-FEB-2005

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.7

Test group / Remarks:

Group 2 10% (w/v)

Results 2

Parameter:

SI

Value:

0.5

Test group / Remarks:

Group 3 25% (w/v)

Results 3

Parameter:

SI

Value:

0.4

Test group / Remarks:

Group 4 50% (w/v)

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test
concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared with
concurrent controls, as indicated by the STIMULATION INDEX (S.I.).
In this study STIMULATION INDICES of 0.7, 0.5 and 0.4 were determined with the test item
at concentrations of 10 %, 25 % and 50 % (w/v), respectively, in acetone/olive oil (4/1, v/v).
Tosylfuranosid was therefore found to be a non-sensitizer when tested at up to the highest
applicable concentration of 50 % (w/v) in acetone/olive oil (4/1, v/v).

Executive summary:

In order to study a possible contact allergenic potential of Tosylfuranosid, three groups each

of four female mice were treated daily with the test item at concentrations of 10 %, 25 % and

50 % (w/v) in acetone/olive oil (4/1, v/v) by topical application to the dorsum of each ear lobe

(left and right) for three consecutive days. 50 % was the highest technically applicable

concentration in the vehicle. A control group of four mice was treated with the vehicle

(acetone/olive oil (4/1, v/v)) only. Five days after the first topical application the mice were

injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine).

Approximately five hours after intravenous injection, the mice were sacrificed, the draining

auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node

cells were prepared from pooled lymph nodes which were subsequently washed and

incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node

cells was determined by the incorporation of 3H-methyl thymidine measured in a ß-

scintillation counter.

All treated animals survived the scheduled study period.

No clinical signs were observed in any animals with the exception of animal No.11 (Group 3,

25 %). On the second application day, a slight ear swelling and erythema were observed at

both dosing sites in this animal.