Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-05-28 to 2008-06-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: ECVAM international validation study on In Vitro tests for acute skin irritation (Altern Lab Anim. 2007 Dec; 35 (6):559-601)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2007-01-19

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

Test animals

Details on test animals and environmental conditions:
not applicable

Test system

Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approx. 12 mg of the neat solid test item were applied to each of triplicate tissues

- no further significant information stated
Duration of treatment / exposure:
15 ± 1 minutes
Observation period:
not applicable
Number of animals:
not applicable
Details on study design:
Three tissues of the human skin model EPISKIN were treated with either the test item (12 mg), the negative or the positive control (15 mL) for 15 minutes.

NEGATIVE CONTROL
- deionised water: 15 µL were applied to each of triplicate tissues for 15 ± 1 minutes

POSITIVE CONTROL
- a 5 % SLS (Sodium lauryl sulphate) solution in deionised water, freshly prepared; 15 µL were applied to each of triplicate tissues for 15 ± 1 minutes

CELL CULTURE
- EPISKIN kits were purchased from Skinethic LAboratories and consisted of normal, human-derived epidermal keratinocytes, cultered to form a multilayered, highly differentiated model of the human epidermis
- it consistes of organised basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers, analogous to those found in-vivo
- surface: 0.38 cm2
- cultured on specially prepared cell culture inserts

EXPERIMENTAL PERFORMANCE
- after 23.5 hours incubation of EPISKIN tissues at 37±1 °C, they were treated with the test item; the inserts were transferred into 12-well plates containing pre-warmed maintenacne medium
- negative and positive control and the test item were added into the insert atop the concerning EPISKIN triplicate tissues and the test item tissue is wetted with 15 µL deionised water
- th eplates were placed into the incubator for 15 ± 1 min at 37 ± 1 °B, 5 ± 0.5 % CO2

REMOVAL OF TEST SUBSTANCE
- Washing: with PBS after removing of the inserts from the plate
- Time after start of exposure: approx. 15 min
- then the inserts were placed with 2 mL maintenace mdeium and were incubated for 42 ± 1 h at 37 ± 1 °C, 5 ± 0.5 % CO2

IMMUNOASSAY
- after 42 h incubation, samples of all treatment groups were shaken for 15 min to homogenise the released mediator before sampling
- at least 1.6 mL of each well were taken and stored in the freezer until analysis
- the amount of released IL-1α was determined according to the instruction from the "Quantikine kit" (Quantikine Human IL -1α Immunoassay kit was purchased from R & D Systems)

MTT-REDUCTION TEST
-MTT solution: 0.3 mg MTT Formazan salt were dissolved in 1 mL PBS
- 12 mg solid test item were added to 1 mL MTT solution and incubated in the dark at room temperature for 60 minutes (+ untreated MTT control)
- if the solution colour turns blue/purple, the test item was able to reduce MTT and a negative functional test should be done with freezed killed tissue

MTT ASSAY
- after completion of treatment procedure, cell culture inserts for all time points were transferred into MTT-plates (0.3 mg/mL MTT per well) and incubated for 3 hours (at 37 ± 1 °C, 5 ± 0.5 % CO2) and then rinsed with PBS
- colour changed was measured

- no further significant information stated

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other:
Remarks on result:
other:
Remarks:
Basis: mean 15.24 pg/mL. Time point: after 15 min incubation. Remarks: in-vitro test on human skin tissues. (migrated information)

In vivo

Irritant / corrosive response data:
see table 1
Other effects:
none

Any other information on results incl. tables

Table 1: Results after treatment with Naringin DHC

Dose

Group

Treatment

interval

Absorbance

570 nm

Tissue 1*

Absorbance

570 nm

Tissue 2*

Absorbance

570 nm

Tissue 3*

Mean

Absorbance of 3 Tissues

Rel.

Absorbance

[% of neg.

Control]

Negative

Control

15 min

1.070

1.038

0.954

1.021

100.0

Positive control

15 min

0.317

0.264

0.323

0.301

29.5

Naringin

DHC

15 min

0.795

1.077

1.088

0.987

96.7

* mean of three replicate wells after blank correction

- the absorbance values after treatment with the negative control were well above the required criterion of mean OD ≥ 0.8 showing the quality of the tissues
- treatment with the positive control induced a decrease in relative absorbance as compared to the negative control to 29.5% thus ensuring the validity of the test system
- after treatment with the test item the relative absorbance values were not decreased

- the optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour

- the mean concentration of released IL-1α did not show an increase; it was was 15.24 pg/mL and therefore below the threshold for an irritant potential of 60 pg/mL

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study Naringin DHC is not irritant to skin and therefore must not be classified.