1,4:3,6-dianhydro-2,5-di-O-methyl-D-glucitol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 - 05 Apr 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

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)

Version / remarks:

24 April 2002

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Test material information

Constituent 1

Reference substance name:

1,4:3,6-dianhydro-2,5-di-O-methyl-D-glucitol

EC Number:

226-159-8

EC Name:

1,4:3,6-dianhydro-2,5-di-O-methyl-D-glucitol

Cas Number:

5306-85-4

Molecular formula:

C8H14O4

IUPAC Name:

1,4:3,6-dianhydro-2,5-di-O-methyl-D-glucitol

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J Rj.

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier S.A.S., Le Gnest st Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 20.8 - 24.0 g
- Housing: individually in cages of standard dimensions on sawdust bedding
- Diet: RM1(E)-SQC SDS/DIETEX feed (quality controlled / radiation sterilised)
- Water: ad libitum
- Acclimation period: minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 45 - 65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:

other: for 25, 50 and 75% solutions: sterile water

Concentration:

25, 50, 75% (v/v) and 100%

No. of animals per dose:

2 (preliminary study), 4 (main study)

Details on study design:

PRE-SCREEN TESTS:
A preliminary study was carried out to evaluate the maximum homogeneous concentration causing slight to moderate irritation by cutaneous application without causing toxic effects for the main study. Four concentrations of 25, 50 and 75% (v/v) in sterile water and 100% (undiluted) were tested, each in at least two different animals (two concentrations per mouse, one concentration per ear).
- Compound solubility: The test susbtance was soluble at 75% (v/v) in sterile water.
- Irritation: The application of the test substance at 25%, 50%, 75% (v/v) in sterile water and 100% did not induce colouring of the application site. Grading of all skin reactions was therefore possible. No sign of irritation was observed in animals.
- Systemic toxicity: No mortality was observed during the preliminary study.
- Ear thickness measurements: No increase of ear thickness was noted at all tested concentrations.

MAIN STUDY
Since undiluted test substance caused no irritation in preliminary study, same doses were chosen.
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: ELISA BrdU
- Criteria used to consider a positive response: The test substance can be regarded as a sensitiser if at least one test concentration produces a test/control ratio equal to or greater than 3.0 (Stimulation Index SI > 3) [1]. However, the magnitude of the stimulation index was not the only factor used in determining the biological significance of a skin sensitisation response. Other relevant criteria such as cellularity and ear thickness were taken into account for the interpretation of the results.

TREATMENT PREPARATION AND ADMINISTRATION: A volume of 25 μL of test substance, vehicle or positive control was applied to the dorsum of both ears of all animals daily for three consecutive days. Two days after the third application on Day 5, an intraperitoneal injection of 250 µL of 0.9% sodium chloride containing 2.5 mg of BrdU was made. Approximately 5 h after BrdU injection, the mice were sacrificed and draining auricular lymph nodes from each mouse ear were excised. A single cell suspension was prepared by separation through a 200 µm-mesh nylon filter. Cell suspensions were re-suspended in 2 mL of a solution containing phosphate buffer solution and 1% of antibiotics and antimycotics for counting of lymphocytes (cellularity). 25 μL of cell suspension was seeded in 96 well tissue culture plate. On the next day (Day 7) BrdU was measured by ELISA using a commercial kit.

Positive control substance(s):

other: Dinitrochlorbenzol (DNCB) 0.5% in acetone

Statistics:

Results of Stimulation Index and ear thickness were given as means ± SEM (Standard Error of the Mean). The effects of the test item on Stimulation Index and ear thickness were compared with that of the control group using an analysis of variance with a Dunnett´s test in case of significance (P ≤ 0.05). Body weight changes were given as mean ± SEM (Standard Error of the Mean) and were compared with the control group using an analysis of variance with Dunett´s test in case of significance (P ≤ 0.05).

Results and discussion

Positive control results:

In the positive control animals were treated with DNCB 0.5% discrete erythema was noted on both ears on Day 4 and Day 6. An increase in ear thickness (+10%) was noted compared to Day 1. This increase was statistically significant in comparison with ear thickness of animals treated with vehicle positive control (acetone). Cellularity index was 21.88 with a total amount of viable cells of 14.1x10E6 compared to 1.21x10E6 with the vehicle control group and the significant stimulation index was 4.3.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.1

Test group / Remarks:

50% (v/v)

Results 2

Parameter:

SI

Value:

0.9

Test group / Remarks:

75% (v/v)

Results 3

Parameter:

SI

Value:

1.1

Test group / Remarks:

100% (v/v)

Cellular proliferation data / Observations:

IRRITATION, EAR THICKNESS AND EAR WEIGHTS:
Erythema: The test substance at 50%, 75% and 100% did not provoked irritation.
Ear thickness: No significant increase was noted in animals treated with the test substance at 50%, 75% and 100% during the study except in animals treated with the test substacne at 75% on Day 2 in comparison with vehicle group but there were only an increase of 5% in comparison with Day 1. Therefore it was not a relevant significant increase.

CELLULAR PROLIFERATION DATA: Groups treated with vehicle, test substance at 50%, 75% and 100% , acetone and DNCB at 0.5% showed a cell viability of 73%, 68%, 73%, 64%, 71%, and 86% respectively. The total amount of viable cells for each groups were of 1.21x10E6 ; 1.24x10E6 ; 1.19x10E6 ; 0.86x10E6 , 0.58x10E6 and 14.1x10E6 cells per node, respectively. Cellularity index were of 1.15; 0.98 and 1.06 and stimulation index were 1.1, 0.9 and 1.1with the test substance at 50%, 75%, and 100%, respectively.

DETAILS ON STIMULATION INDEX CALCULATION: Stimulation Index (SI), expressed as the ratio of mean OD of the test nodes relative to that recorded for control (vehicle) nodes and Cellularity Index expressed as the ratio of mean amount of cells (x 10E6 cells) in the treated group and the mean amount of cells (x 10E6 cells) in the vehicle group.

EC3 CALCULATION: No EC3 value could be calculated.

CLINICAL OBSERVATIONS: No clinical signs or mortalities were observed during the main study.

BODY WEIGHTS: The body weight change of the treated animals with test substance was similar to that of the vehicle control animals.

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of the local lymph node assay, the test substance at concentrations of 50, 75 and 100% did not produce a stimulation index equal to or greater than 3 and no EC3 value was calculated. Therefore, the test substance was considered to be not sensitising.