Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-09-19 to 2008-10-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
, 2002
Deviations:
yes
Remarks:
modified EC B.42 ,method according to Ehlings et al. 2005
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
, 2004
Deviations:
yes
Remarks:
modified EC B.42 ,method according to Ehlings et al. 2005
Principles of method if other than guideline:
The test was performed in accordance with the method according to Ehlings et al (2005): An european inter-laboratory validation of alternative endpoints of the murine local lymph node assay: first round, Toxicology 212 (2005) 60-68 and Ehlings et al (2005): An european inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round, Toxicology 212 (2005) 69-79

Threshold values of the stimulation indices of lymph node cell count and ear weight were calculated by dividing the average values per group of the test item treated animals by the vehicle treated ones. Values above 1.4 (cell count) or 1.1 (ear weight) are considered positive
(these values were fixed empirically during the inter-laboratory validation of this method). In addition, the lymph node weights were determined for concentration related properties.
GLP compliance:
yes
Remarks:
signed 2007-04-20
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Naringin DHC; 1-[4-[[2-O-(6-Deoxy-L-mannopyranosyl)-D-glucopyranosyl]oxy]-2,6-dihdroxyphenyl]-3(4-hydroxyphenyl)-1-propanone
- Substance type: technical product
- Physical state: solid, whitish powder
- Analytical purity: 99 % [a/a]
- water content: 6.4 %
- Lot/batch No.: 2
- Expiration date of the lot/batch: January 2010
- Storage condition of test material: 15-25 °C, dark, don't freeze, keep away from strong light and heat

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Mice were supplied by Charles River Deutschland GmbH. The animals were nulliparous and non-pregnant and healthy.
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: 19-26 g
- Housing: The animals were kept singly in MAKROLON cages (type III) with a basal surface of approx. 39 cm x 23 cm and a height of approx. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. The cages were changed and cleaned once a week. Periodic analysis of the bedding material for contaminants based on EPA/USA is conducted at least once a year by LUFA-ITL
- Diet: Commercial diet ssniff® R/M-H V1534 served as food (ssniff Spezialdiäten GmbH, 59494 Soest, Germany; see Appendix 2: Composition of the diet). This food was offered ad libitum. Food residue was removed. Periodic analysis of the food for contaminants based on EPA/USA is conducted at least twice a year by LUFA-ITL. Certificates of analysis of the composition and for contaminants were provided by the manufacturer and are included in the raw data.

- Water: tap water (ad libitum)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: 12 - 18 per hour
- Photoperiod: 12 hours light/dark cycle

no more information given

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Remarks:
400
Concentration:
Animals were treated with the test material at concentrations of 10%, 25% or 50% (w/w) in PEG 400. The vehicle was
selected on the basis of maximising the test concentrations and solubility whilst producing a solution suitable for application of the test item. Particular care should be taken to ensure that hydrophilic materials are incorporated into a vehicle system, which wets the skin and does not immediately run off. Thus, wholly aqueous vehicles had to be avoided. PEG 400 is a clinically relevant solvent.
No. of animals per dose:
6 animals per group and dose
Details on study design:
RANGE FINDING TESTS: The doses were selected from the concentration series 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5% etc. according the recommendations of the guidelines. A 50% (w/w) solution of Naringin DHC was the maximum feasible concentration for an administration volume of 25 μL/ear. In a preliminary dose range finding experiment 2 female animals per dose level were treated with 0.5%, 1%, 2%, 5%, 10%, 25% and 50% (w/w) solutions of Naringin DHC. The high dosed animals revealed only marginally increased values for the cell count and lymph node weights compared to the control. Hence, a top concentration of 50% (w/w) Naringin DHC was employed for the main study.

Administration: The test item solution was administered to the dorsum of both animal's ears at an application volume of 25 μL/ear.

MAIN STUDY
Day 1:
The weight of each animal was individually identified and recorded. In addition, ear swelling measurements were carried out at the helical edge of both ears using an Oditest micrometer. Open application of 25 μL of the appropriate dilution of the test item, the vehicle alone or the positive control (as appropriate) were administered to the dorsum of each ear.
- Days 2 and 3:
The application procedure carried out on day 1 was repeated.
- Day 4 (24 hours after the last application the animals were sacrificed under ether anaesthesia by cutting the aorta abdominalis): Ear swelling measu ements were carried out at the helical edge of both ears using an Oditest micrometer.
Punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and immediately weighed on an analytical balance.
Lateral pairs of auricular lymph nodes draining the ear tissue were excised, carefully separated from remaining fatty tissue and weighed on an analytical balance immediately following preparation. The lymph nodes were then stored on ice in PBS5/0.5% BSA6 and subjected to the preparation of single cell suspensions by mechanical tissue disaggregation. The cells were counted automatically in a cell counter.

CLINICAL OBSERVATIONS: Animals were observed once daily for any clinical signs of local systemic irritation at the application site or of systemic toxicity. Observations were recorded for each individual animal. Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.
In addition, animals were checked regularly throughout the working day from 7:30 a.m. to 4:30 p.m. On Saturdays and Sundays animals were checked regularly from 8:00 a.m. to 12:00 noon with a final check performed at approximately 4:00 p.m.,
if applicable. Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.

BODYWEIGHTS: The weight of each mouse was recorded at the time of allocation of animals to groups (test day 1) and at the time of necropsy (test day 4). These values are presented in Table 6.

Analysis of results:
The so-called stimulation (or LLN-) indices were calculated by dividing the average lymph node cell counts or ear weights (punch biopsies) per group of the test item treated animals by the vehicle treated ones. Thus, in case of no stimulating effect the index for the lymph node cell count is always below 1.4, for the ear weights 1.1 (cut-off values). Indices above 1.4 or 1.1, respectively, are considered positive. For each dose group the values obtained for lymph node cell count, lymph node weight and weight of ear punch biopsies were compared to the respective values of the control group employing the U-Test according to MANN and WHITNEY (p≤ 0.01 with Bonferroni correction). In addition, the average ear thickness per group was compared to the vehicle control group.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
U-Test according to MANN and WHITNEY (p≤ 0.01 with Bonferroni correction)

Results and discussion

Positive control results:
50 % (w/w) Stimulation index (SI): 2.285 Result: positive (cell count) SI: 1.795 (lymph node weight)

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Threshold values of the stimulation indices of lymph node cell count and ear weight were calculated by dividing the average values per group of the test item treated animals by the vehicle treated ones. Values above 1.4 (cell count) or 1.1 (ear weight) are considered positive (these values were fixed empirically during the inter-laboratory validation of this method). In addition, the lymph node weights were determined for concentration related properties. 10 % (w/w) SI: 1.323 (cell count) SI: 1.565 (lymph node weight) 25 % (w/w) SI: 1.146 (cell count) SI: 1.477 (lymph node weight) 50 % (w/w) SI: 0.872 (cell count) SI: 1.159 (lymph node weight)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: not applicable

Any other information on results incl. tables

Treatment with Naringin DHC at concentrations of 10%, 25% or 50% (w/w) revealed marginally dose-related, though statistically significantly increased values (p ≤ 0.01) for the cell count and lymph node weights for concentrations of 25% and 50%. However, the stimulation index of the cell count did not exceed the threshold level of 1.4 and, further, the stimulation index of ear weights did not exceed the threshold level of 1.1, either. Hence, the test item is classified as not stimulating. The positive control group caused the expected increases in cell count and ear weights (statistically significant at p ≤ 0.01). The values for the stimulation index of cell count and ear weight were 2.3 and 1.2, respectively. No changes in behaviour were observed in any of the treated animals.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Under the present test conditions, Naringin DHC at concentrations of 10, 25 or 50% (w/w) in PEG 400 did not reveal any sensitising properties in the local lymph node assay in mice.