Reaction mass of 1,4:3,6-dianhydro-D-glucitol monooleate and 1,4:3,6-dianhydro-D-glucitol dioleate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Harlan Laboratories Ltd, Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1,4:3,6-dianhydro-D-glucitol

EC Number:

211-492-3

EC Name:

1,4:3,6-dianhydro-D-glucitol

Cas Number:

652-67-5

Molecular formula:

C6H10O4

IUPAC Name:

1,4:3,6-dianhydro-D-glucitol

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Bicester, Oxon, UK
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: individually in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet: ad libitum, 2014 Teklad Global Rodent diet supplied by Harlan Teklad, Lackthorn, Bicester, Oxon, UK
- Water: ad libitum, tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0, 10, 25, 50 % (w/w)

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Irritation/systemic toxicity: Using available information regarding the systemic toxicity potential of the test material, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the test material at a concentration of 50 % w/w in dimethyl formamide, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Any signs of toxicity or excessive local irritation noted during this period were recorded. The body weight was recorded on Day 1 (prior to dosing) and on Day 6.
- Lymph node proliferation response: Not determined

MAIN STUDY
- Test item preparation: The test material was freshly prepared as a solution in dimethyl formamide. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. The test material was formulated within two hours of it being applied to the test system; it is assumed that the formulation was stable for this duration.
- Test material administration: The mice were treated by daily application of 25 μI of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of four mice received the vehicle alone in the same manner.
- 3H-Methyl Thymidine Administration: Five days following the first topical application of the test material or vehicle (Day 6) all mice were injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmol, ARC UK Ltd) giving a total of 20 μCi to each mouse.
- Clinical observations and body weights: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded. The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).
- Termination: Approximately five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.
- Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labelled with the project number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190g) for ten minutes. The pellet was resuspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 mL of 5 % Trichloroacetic acid (TCA).
- Determination of 3HTdR Incorperation: After approximately eighteen hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450g) for ten minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by ß-scintillation counting. The "Poly Q TM„ vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments lnc, Fullerton, CA, USA).
- Interpretation of results: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitiser".

Positive control substance(s):

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

Results and discussion

Positive control results:

A stimulation index of 5.16 was deterimined for 15 % hexyl cinnamic aldeyde in dimethylformamide.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

>= 0.55 - <= 0.71

Test group / Remarks:

The stimulation index was determined to be 0.71, 0.55 and 0.55, for the 10, 25 and 50 % (w/w) exposed animals, respectively. All these results were considered negative.

Results 2

Key result

Parameter:

other: disintegrations per minute (DPM)

Value:

>= 5 179.54 - <= 9 416.03

Test group / Remarks:

The DPM were determined to be 9416.03, 6655.76, 5216.33, and 5179.54 for the 0, 10, 25 and 50 % (w/w) exposed animals, respectively

Any other information on results incl. tables

RANGE FINDING TEST

No signs of systemic toxicity were noted.

 

MAIN TEST

- Viability/mortality: There were no deaths.

- Clinical signs: No signs of systemic toxicity were noted in the test or control animals during the test.

- Body weight: Body weight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met