Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 Nov - 13 Dec 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

adopted in 2003

GLP compliance:

yes (incl. QA statement)

Remarks:

(BAYERISCHES LANDESAMT FÜR GESUNDHEIT UND LEBENSMITTELSICHERHEIT, LANDESINSTITUT FÜR ARBEITSSCHUTZ UND PRODUKTSICHERHEIT, München, Germany)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

6-O-α-D-glucopyranosyl-D-fructose

EC Number:

237-282-1

EC Name:

6-O-α-D-glucopyranosyl-D-fructose

Cas Number:

13718-94-0

Molecular formula:

C12H22O11

IUPAC Name:

6-O-alpha-D-glucopyranosyl-D-fructose

Test material form:

solid: crystalline

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsD

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann, Borchen, Germany
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 16 - 23 g
- Housing: 5 animals per cage in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding (pre-screen test: lot no. 010812, main study: lot no. 160812)
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water: tap water, sulphur acidified to a pH value of approx. 2.8, ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

6.25, 12.5 and 25% (w/v)

No. of animals per dose:

5 (main study), 3 (pre-screen test including 2 test and 1 control animal)

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The solubility test revealed a maximum technically applicable concentration of 25% (w/v) of the test item in the vehicle DMSO
- Irritation:
2 mice were treated with 25% test item solution. 1 animal treated with the vehicle DMSO served as negative control. All animals were observed for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded prior to the first treatment (Day 1) and prior to termination (Day 6). Both ears of each mouse were observed for erythema/eschar formation and scored. Measurement of ear thickness was performed on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6.
No mortality or any signs of systemic toxicity were observed. No significant signs of irritation were observed as indicated by an erythema score ≥ 3 and/or an increased ear thickness of ≥ 25% on any day of measurement (25%: 0.19 mm on day 1, 0.17 mm on day 3, 0.18 on day 6; vehicle: 0.18 mm on day 1, 0.185 on day 3 and 0.19 0n day 6). According to this, the maximum technically applicable concentration of 25% (w/v) was selected as the highest test concentration in the main test.
- Lymph node proliferation response: Lymph node proliferation was not assessed in the range-finding test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by ß-scintillation
- Criteria used to consider a positive response: The test item was considered as a skin sensitizer, if exposure to at least one concentration of the test item resulted in an at least 3-fold or greater stimulation index in test animals compared to control mice (SI ≥ 3).
- Validation criteria: The positive control substance phenylenediamine (1%) should produce a positive LLNA response shown by a SI > 3 over the negative control group without causing excessive skin irritation (i.e. SI > 20) or systemic toxicity.

TREATMENT PREPARATION AND ADMINISTRATION:
25 µL of the test item was applied on the entire dorsal surface of each ear of each dose. The application was repeated once daily over three consecutive days. Local irritation reactions were assessed. On Day 6, an injection of 250 µL phosphate buffered saline containing 20 µCi of 3H-methyl thymidine was made into the tail vein of each experimental mouse. Approx. 5 h later, animals were sacrificed and the draining auricular lymph node of each ear was excised. A single cell suspension of lymph node cells, individually pooled for each animal, was prepared. After repeated washings, cells were precipitated with 5% trichloracetic acid at 4 °C overnight. The 3H-methyl thymidine-incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM) individually for each animal.

CLINICAL OBSERVATIONS:
All animals were observed prior to the application and once a day thereafter to detect signs of toxicity. Irritation at the site of application was monitored by erythema scoring according to the scoring scale defined in OECD GL 429 and recorded for each animal individually.

Positive control substance(s):

other: phenylenediamine (1% in DMSO)

Results and discussion

Positive control results:

The positive control substance induced a significant lymphoproliferative response as shown by a stimulation index value of 13.7.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

test substance at 6.25%

Results 2

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

test substance at 12.5%

Results 3

Key result

Parameter:

SI

Value:

0.9

Test group / Remarks:

test substance at 25%

Results 4

Parameter:

SI

Value:

13.7

Test group / Remarks:

positve control

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
No significant or dose-dependent increase in the DPM values was observed in the test groups compared to the controls. The determined mean DPM values per lymph node were 858.5 ± 261.9, 860.4 ± 305.6, 895.9 ± 92.9 and 744.1 ± 216.5 for the control, low- mid- and high-dose animals, respectively.

DETAILS ON STIMULATION INDEX CALCULATION
The proliferative response of lymphnode cells is expressed as the number of radioactive disintegrations per lymph node (DPM/NODE) and as the ratio of 3H-methyl-thymidine incorporation into lymph node cells of test group animals relative to that recorded for control group animals (stimulation index, SI). Before DPM/NODE values were determined, background values were substracted.

EC3 CALCULATION
The EC3 value could not be calculated as the SI of all concentrations was below 3.

CLINICAL OBSERVATIONS:
All animals survived throughout the test period without showing any clinical signs or signs of dermal irritation at the application site.

BODY WEIGHTS
All animals showed the expected body weight development.

Applicant's summary and conclusion

Interpretation of results:

other: not sensitising according to Regulation (EC) No 1272/2008

Conclusions:

CLP: not classified