Hesperidin

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study without significant deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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:

Hesperidin

EC Number:

208-288-1

EC Name:

Hesperidin

Cas Number:

520-26-3

Molecular formula:

C28H34O15

IUPAC Name:

5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-3,4-dihydro-2H-chromen-7-yl 6-O-(6-deoxy-alpha-L-mannopyranosyl)-beta-D-glucopyranoside

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

orange brown powder
Batch# 120323
Purity: 91.15% based on dry mass

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non-pregnant. After an acclimatisation period of at least five days the animals were selected at random and given a number unique within the study by indelible ink-marking on the tail and a number written on a cage card. At the start of the
study the animals were in the weight range of 15 to 23 g, and were eight to twelve weeks old.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

For the purpose of the study, the test item was freshly prepared as a suspension in dimethyl formamide. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. The concentrations used were 50%, 25%, 10% and 0% (vehicle control).

No. of animals per dose:

4 animlas per dose including vehicle control.

Details on study design:

Test Item Administration
Groups of four mice were treated with the test item at concentrations of 50%, 25% or 10% w/w in dimethyl formamide. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 µl of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item 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 item 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.
3.2.2.4 Terminal Procedures
Termination: 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 190 g) 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 Incorporation: After approximately eighteen hours incubation at approximately 4°C, the precipitates were recover ed by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by β-scintillation counting. The "Poly Q" 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 Inc, 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 item will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item 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

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.85

Test group / Remarks:

10% (%w/w) concentration in DMF

Results 2

Parameter:

SI

Value:

2.09

Test group / Remarks:

25% (%w/w) concentration in DMF

Results 3

Parameter:

SI

Value:

2.73

Test group / Remarks:

50% (%w/w) concentration in DMF

Results 4

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: vehicle: 6024.73 DPM (753.09 DPM/node) 10 weight% in DMSO: 11135.87 DPM (1391.98 DPM/node) 25 weight% in DMSO: 12568.12 DPM (1571.02 DPM/node) 50 weight% in DMSO: 16447.30 DPM (2055.91 DPM/node)

Any other information on results incl. tables

Local skin irritation was recorded on both ears once daily according to Draize scores. All scores showed zero (i.e. no erythema).

Measurements of ear thickness and mean ear thickness changes were recorded at 50% concentration resulting in less than 25% increase thus indicative of absence of irritation.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item was considered to be a non-sensitiser under the conditions of the LLNA-test.

Executive summary:

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group and were 1.85 at 10% by weight in DMF, 2.09 at 25% by weight in DMF and 2.73 at 50% by weight in DMF (all <3 and thus negative).

Light brown coloured residual test item on the ears was noted in all test animals. There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Bodyweight 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.

As a result the test item was considered to be a non-sensitiser under the conditions of the test.