D-Glucitol, 1,5-anhydro-1-C-[3-[[5-(4-fluorophenyl)-2-thienyl]methyl]-4-methylphenyl]-, 2,3,4,6-tetraacetate, (1S)-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2008-09-23 to 2008-10-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

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:

[(2R,3R,4R,5S,6S)-3,4,5-tris(acetyloxy)-6-(3-{[5-(4-fluorophenyl)thiophen-2-yl]methyl}-4-methylphenyl)oxan-2-yl]methyl acetate

EC Number:

617-901-5

Cas Number:

866607-35-4

Molecular formula:

C32H33FO9S

IUPAC Name:

[(2R,3R,4R,5S,6S)-3,4,5-tris(acetyloxy)-6-(3-{[5-(4-fluorophenyl)thiophen-2-yl]methyl}-4-methylphenyl)oxan-2-yl]methyl acetate

Test material form:

solid: particulate/powder

Details on test material:

- Physical state: solid
- Appearance: white powder

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: 18 (2 for the pretest and 16 for the main study) female mice (nulliparous and non-pregnant, CBA/CaOlaHsd; from Harlan Laboratories B.V.
- Age at beginning of acclimatisation: 8-12 weeks
- Initial weight: 20.4 - 22.5 g
- Housing: Single housing, in Makrolon Type I cages, with wire mesh top (EHRET GmbH) and granulated soft wood bedding.
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (Harlan Laboratories GmbH)
- Water (e.g. ad libitum): tap water, ad libitum (Gemeindewerke)
- Acclimation period: under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30-70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12 (artificial light from 6.00 a.m. to 6.00 p.m.)

IN-LIFE DATES: From: 2008-09-23 To: 2008-10-08

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

1, 10 and 25 % (v/v) of test article

No. of animals per dose:

4 females per group; 1 control group and 3 test groups

Details on study design:

RANGE FINDING TESTS:
Rationale for dose levels: A solubility experiment was performed. The highest test item concentration, which can be technically used was a 25% suspension in dimethylformamide. To determine the highest non-irritant test concentration or the highest technically applicable concentration, a pretest was performed in two mice. Two mice were treated with concentrations of 2.5%, 5%, 10, and 25% on one ear each on three consecutive days. Clinical signs were recored 24 +/- 4 hours after each application. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity. The test item in the main study was assayed at 1, 10 and 25%. The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- 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 1, 10, and 25 % (w/w) in dimethylformamide. The application volume, 25 µl, was spread over the entire dorsal surface (diam. ca. 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).
- Criteria used to consider a positive response: A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 3-fold or greater increase in incorporation of 3 HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

ADMINISTRATION OF ³H-METHYL THYMIDINE (³HTdR)
- Five days after the first topical application, all mice were administered with 250 µl of 78.6 µCi/ml 3HTdR (corresponds to 19.7 µCi 3HTdR per mouse) by intravenous injection via a tail vein.

DETERMINATION OF INCORPORATED ³HTdR
- Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium (Release, WDT, D-30827 Garbsen).
- The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times 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 plastic scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid (Perkin Elmer (LAS) GmbH, D-63110 Rodgau) and thoroughly mixed.
- The level of 3HTdR incorporation was then measured on a beta-scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, D-63110 Rodgau). Similarly, background 3HTdR levels were also measured in two 1 ml-aliquots of 5 % trichloroacetic acid. The beta-scintillation counter expresses 3 HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Positive control substance(s):

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

Statistics:

Mean values and standard deviations were calculated for the body weights.

Results and discussion

Positive control results:

Background I: 43 dpm
Background II: 38 dpm
Control group 1: 2270 dpm, 2230 dpm-BG; 278.7 dpm per lymph node (8 lymph nodes)
Test group 2 (5%): 11733 dpm, 11693 dpm-BG, 1461.6 dpm per lymph node (8 lymph nodes); S.I. = 5.24
Test group 3 (10%): 16484 dpm; 16444 dpm-BG, 2055.4 dpm per lymph node (8 lymph nodes); S.I. = 7.38
Test group 4 (25%):20812 dpm; 20772 dpm-BG, 2596.4 dpm per lymph node (8 lymph nodes); S.I. = 12.45

The EC3 value was estimated to be < 5%. A precise calculation of the EC3 value was not possible since the S.I. was above the threshold of 3 even at the lowest tested concentration. The obtained values are within the expected range and the positive control experiment is considered fully valid.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

2.94

Test group / Remarks:

test group 4 (25%)

Remarks on result:

other:

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

- Background I: 21 dpm - Background II: 23 dpm - Control group 1: 2163 dpm, 2141 dpm-BG*; 267.6 dpm per lymph node** (8 lymph nodes) - Test group 2 (1%): 4335 dpm, 4313 dpm-BG*, 539.1 dpm per lymph node** (8 lymph nodes) - Test group 3 (10%): 6932 dpm; 6910 dpm-BG*, 863.8 dpm per lymph node** (8 lymph nodes) - Test group 4 (25%): 6310 dpm; 6288 dpm-BG*, 786.0 dpm per lymph node** (8 lymph nodes) * The mean value was taken from the figures of background (BG) I and BG II ** Since the lymph nodes of the animals of a dose group were pooled, DPM/nodewas determined by dividing the measured value by the number of lymph nodespooled

Results 3

Parameter:

SI

Value:

2.01

Test group / Remarks:

test group 2 (1%)

Results 4

Parameter:

SI

Value:

3.23

Test group / Remarks:

test groud 3 (10%)

Results 5

Parameter:

EC3

Value:

8.3

Any other information on results incl. tables

- Viability/mortality: no deaths occured during the study period.

- Clinical signs: no symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

- Body weights: the body weight of the animals, recorded prior to the first application and prior to the treatment with ³HTdR, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Conclusions:

The test item RT003066 was found to be a skin sensitiser under the described conditions and should be classified as skin sentitizer category 1B based on the calculated EC3 value, according to CLP Regulation.