Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 21, 2008 - June 13, 2008
Reliability:
1 (reliable without restriction)

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: HET-CAM
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
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 / tissue source

Details on test animals or tissues and environmental conditions:
in vitro eye irritation testing using chicken eye.

The Lohmann Selected Leghorn chicken was selected for several reasons: the ability to hatch the eggs of this breed is very consistent and reproducible. There did not appear to be any hereditary defects in this breed. The eggs were obtained from the LSL Rhein-Main Geflügelvermehrungsbetriebe (D-64807 Dieburg, Germany) on day one. Afterwards, they were incubated at 37.5 +- 0.5°C for eight days. While incubation the eggs were rotated to prevent an attachement of the embryo to one side of the egg. On day eight the eggs were candled and non-vialbe eggs were discarded. The rest of the eggs were placed upward in the incubator and were incubated for another day without rotation.

Test system

Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
0.1 g of the test item was applied to the membrane off the eggs so that at least 50% of the membrane was covered by the test item. The test item was applied to 6 eggs, each with 0.1 g test substance.
Duration of treatment / exposure:
20 seconds
Observation period (in vivo):
300 seconds
Number of animals or in vitro replicates:
not applicable, see under "amount/concentration applied"
Details on study design:
With the HET-Cam test an irritative or corrosive potential is determined by a detection of damages in blood vessels under the chorioallantoic membrane of 9 day incubated chicken eggs.

- Incubation of the eggs: Eggs were incubated at 37.5 ± 0.5 °C for eight days. While incubation the eggs were rotated to prevent an attachment of the embryo to one side of the egg. On day eight the eggs were candled and non-viable eggs were discarded. The rest of the eggs were placed upward in the incubator and were incubated for another day without rotation.

- Treatment: In the HET-Cam assay the test item was tested pure. Six eggs were treated with the test item. Therefore each an amount of approximately 0.1 g of the test item was applied to the membrane so that at least 50% of the membrane was covered by the test item. After 20 sec treatment the test item was washed of the membrane with warm water. Right after this the observation for 300 sec was started. During the observation for 300 seconds lesions in close proximity to the covered membrane were noted.

- Observations: The membrane of the eggs were observed for 300 sec. Lesions of the underlying blood vessels were noted. In general there were three endpoints which were observed and the time when that particular endpoint was observed was noted. The three endpoints were: haemorrhage, coagulation and lysis of the blood vessel

- Positive controls:
(A) Name: sodium dodecyl sulfate (purity > 99%)
Supplier: Merck (64293 Darmstadt, Germany)
Cat. No.: 1.13760
Solvent: deion. water; a 1% solution of SDS in water
(B) Name: sodium hydroxide solution c(NaOH) = 0.1 mol/L (0.1 N)
Supplier: Merck (64293 Darmstadt, Germany)
Cat. No.: 1.09141
The positive controls were tested in only one concentration (each 300 µL) and three times each (3 eggs were used for each positive control).

- Negative controls: eggs treated with each 300 µL physiological NaCl solution (0.9% (v/v)) without other substances) were used as negative controls. 3 eggs were tested.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: haemorrhage
Basis:
mean
Time point:
other: after 300 seconds
Score:
0
Max. score:
0
Remarks on result:
other: Mean irritancy index of 6 eggs
Irritation parameter:
other: coagulation
Basis:
mean
Time point:
other: after 300 seconds
Score:
0
Max. score:
0
Remarks on result:
other: Mean irritancy index of 6 eggs
Irritation parameter:
other: lysis of the blood vessel
Basis:
mean
Time point:
other: after 300 seconds
Score:
0
Max. score:
0
Remarks on result:
other: Mean irritancy index of 6 eggs
Irritant / corrosive response data:
In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item Naringin dihydrochalcone (DHC) does not possess any irritating potential.
Other effects:
During the observation for 300 seconds lesions in close proximity to the covered membrane were noted.

Any other information on results incl. tables

Table1: Results with DHC, as well as negative and positive controls:

Test group

Time until haemorrhage (seconds)

Time until lysis (seconds)

Time until coagulation (seconds)

Irritancy index

Mean irritancy index

Negative control (0.9% (v/v) NaCl in H2O)

301

301

301

0.00

0.00

Negative control

301

301

301

0.00

Negative control

8

301

301

0.00

Positive control 0.1N NaOH

7

59

16

19.08

19.04

Positive control 0.1N NaOH

8

50

14

19.37

Positive control 0.1N NaOH

13

73

19

18.66

Positive control 1%SDS

23

127

301

8.86

8.71

Positive control 1%SDS

24

145

301

8.27

Positive control 1%SDS

301

113

301

9.00

Test item

301

301

301

0.00

0.00

Test item

301

301

301

0.00

Test item

301

301

301

0.00

Test item

301

301

301

0.00

Test item

301

301

301

0.00

Test item

301

301

301

0.00

Mean irritancy index of 3 eggs: positive control: (sodium dodecyl sulfate) = 8.71 and positive control (sodium hydroxide solution) = 19.04

Mean irritancy index of 6 eggs: Test item = 0.00

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item Naringin dihydrochalcone (DHC) does not possess any irritating potential.