Registration Dossier

Administrative data

Endpoint:
eye irritation
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: HET-CAM in vitro system follows the testing strategy for determination of eye irritation/corrosion as given the OECD guideline 405

Data source

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

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
Due to animal welfare reasons, the potential of severe irritation is determined in the HET-CAM in vitro system before deciding on a possible study in the rabbit as suggested in OECD Guideline 405.
Deviations:
yes
Remarks:
The HET-CAM Test is an alternative in vitro method for testing of severe eye/mucous membrane damage using the chorionallantoic membrane of fertilized, incubated hen eggs.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Gelb LD 6259 (No 08/0646-1)
- Physical state: solid/ sun yellow
- Analytical purity: Main component Gelb LD 6259: HPLC fingerprint at 230 and 400 nm: 87.6% and 89.1 area% , respectively. Free acid content: about 61.3 g/100g
- batch No.: 0817 VP 01
- Stability under test conditions: the stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility
- Storage condition of test material: room temperature
- Other: the test substance was homogeneous by visual inspection, pH value: ca. 5 (undiluted test substance, moistened with water)

Test animals / tissue source

Species:
other: in vitro test system
Strain:
other: in vitro test system
Details on test animals or tissues and environmental conditions:
In vitro system using fresh unfertilized eggs produce under controlled SPF conditions
- Type of eggs: White Leghorn, SPAFAS Inc., USA, SPF Premium
- Reasons for the selection of the type of eggs: hen eggs are recommended as preferred type of eggs in the quoted references. Analogous to test animals, controlled SPF housing guarantees a defined microbiological status of the eggs.
- Supplier: Charles River Deutschland GmbH, Extertal
- Identification: At start of incubation period continuous numbering of the eggs with a felt pen.
- Weight range at start of incubation period: 55.6 g – 59.0 g
- Climate: breeding in an incubator at constant temperature of 37.5°C (± 0.5°C) and a relative humidity of 62.5% (± 7.5%).
Automatic rotating device: Until including incubation day 8 and/or day 9, the eggs were rotated automatically 6 times a day. On the day before application the eggs were placed with the blunt end upward and were not rotated until preparation. The incubation conditions were checked daily. Deviations were recorded.
- Candling of the eggs: the eggs were candled before the start of incubation and on the 9th and/or 10th day. Any defective or unfertilized eggs were discarded.

Test system

Vehicle:
other: undiluted test substance or diluted (10%) in highly deinized water
Amount / concentration applied:
TEST MATERIAL
- A bulk volume of 25 μl (about 15 mg) per egg of the ground solid test substance was applied on approximately the half of the membrane area, starting from the center. The visible parts of the membrane were observed. The test substance was removed by washing with 0.9% aqueous NaCl-solution after 50 seconds. A final assessment of the CAM was performed immediately after washing.
- A volume of 0.3 ml per egg of the test-substance solution was applied with a syringe and the CAM was observed up to a maximum time period of 50 seconds. It was possible to observe the reactions through the test-substance solution. The time until appearance of reactions was recorded.

TEST GROUPS
- Selection procedure: before the application, the CAM was investigated for signs of preexisting damage. Only eggs with intact and sufficient vessels were used.
- Number of eggs / egg weights for each testsubstance concentration at start of the incubation
period:
----------------------------------------------------------------------------------------
Test substance concentration /Egg /Identification No. /Weight day (-9) (g)
Undiluted test substance /01 /15 /59.0
/02 /16 /57.7
/03 /17 /55.6
Test substance 10% in highly deionized water /01 /12 /56.1
/02 /13 /58.3
/03 /14 /58.9
------------------------------------------------------------------------------------------

Duration of treatment / exposure:
50 s
Observation period (in vivo):
50 s
Number of animals or in vitro replicates:
Number of eggs: 3 (undiluted test substance) and 3 (10% test-substance solution)
Details on study design:
1. Conduct of the study
- Randomization: no randomization was performed. Within a study eggs of comparable weight (± 10 g) were used.
- Route of application: after careful removal of the eggshell including the inner membrane directly onto the chorionallantoic membrane.
- Application procedure: a bulk volume of 25 μL (about 15 mg) per egg of the ground solid test substance was applied on approximately the half of the membrane area, starting from the center. The visible parts of the membrane were observed.
- Preparation and opening of the eggs: the eggs were candled on the day of application to ensure viability and in order to mark the location of the air chamber with a felt pen. The eggshell was cut along the marking line with an electric drill and removed exposing the egg membrane, which forms the inner barrier between egg content and air chamber. This membrane was moistened with warm physiological saline and the eggs were then placed in the incubator again until they were used for testing (maximum of 30 minutes between opening the eggs and application).
- Selection of the eggs: After removal of the egg membrane the CAM was investigated for signs of pre-existing damage, which would exclude the egg from the assay. Only eggs with an adequate vascular system and even CAM surface were used for the study.

2. Assessment of reactions
After application of the test substance the chorionallantoic membrane was observed by means of a stereomicroscope until unambiguous irritation reactions were detected or up to a maximum time period of 50 seconds, respectively.
The time of appearance (in seconds after application) of intravascular resp. extravascular coagulation, and if applicable other reactions (haemorrhagia, vessel lysis), were determined. The following grading was used: 0 (No visible change); 1 (Slight reaction); 2 (Moderate reaction); and 3 (Severe reaction). Any further findings not covered by this scale were described in the raw data.

3. Positive controls
On each study day, before application of test substances, positive control substances (aqueous solution of 0.1 M NaOH and 10% SDS (Sodium dodecyl sulfate)), which are known to cause moderate to severe irritation were tested, in order to demonstrate the sensitivity of the method.

EVALUATION OF RESULTS
The evaluation depends on the solubility of the test substance, the tested concentrations and the time until appearance of unambiguous intravascular coagulation of middle-sized vessels or extravascular coagulation of the treated CAM, respectively.
The mean time until appearance of reaction over the eggs of a treatment group was calculated (mean time to coagulation = mtc in seconds). For water-soluble test substances: mtc(10%) < 50 risk of serious damage to the eyes and mtc(10%) ≥ 50 no risk of serious damage to the eyes

Usually the result of a 10% aqueous solution is used for evaluation. If the undiluted test substance (mtc100%) is considerable more reactive than the 10% solution (mtc 100% considerably lower than 50 seconds), further information on the properties of the test substance or results from structurally related compounds will be taken into account for evaluation.

HISTORICAL CONTROL DATA
Historical control values of positive controls were gathered over an appropriate time period and demonstrate the reproducibility of results and robustness of the procedures.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: Haemorrhagia
Basis:
mean
Time point:
other: 50 s
Score:
0
Max. score:
3
Reversibility:
other: no findings
Irritation parameter:
other: coagulation
Basis:
mean
Time point:
other: 50 s
Remarks on result:
other: Coagulation was not observed
Irritant / corrosive response data:
The chorionallantoic membrane of the eggs treated with the undiluted test substance and with a 10% test-substance solution in highly deionized water did not show any irritation effects (see Table 1&2).
Other effects:
No other effects were observed (see Table 1&2).

Any other information on results incl. tables

Table: Test substance findings

 

Concentration:

Egg-No.:

Observation period (seconds):

Grading of effects observed after test substance removal by washing:

Additional findings

 

Haemorrhagia:

Coagulation:

Haemorrhagia:

Coagulation:

 

Undiluted

1

50

50

0

0

none

2

50

50

0

0

none

3

50

50

0

0

none

10% in highly deionized water

1

50

50

0

0

none

2

50

50

0

0

none

3

50

50

0

0

none

 

Table: Positive control findings

 

Concentration:

Egg-No.:

Observation period (seconds):

Grading of effects observed after test substance removal by washing:

Additional findings

 

Haemorrhagia:

Coagulation:

Haemorrhagia:

Coagulation:

0.1 M NaOH

1

20

40

2

2

iv

2

28

42

2

2

iv

Mean

n = 2

24

41

2

2

 

10% SDS

1

22

41

2

2

iv, ev

2

21

35

2

2

iv, ev

Mean

n = 2

22

38

2

2

iv, ev

Ev = extravascular coagulation; iv = intravascular coagulation

Applicant's summary and conclusion