Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented and acceptable for assessment and in compliance with GLP regulations.

Data source

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

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
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. 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.
GLP compliance:
yes (incl. certificate)
Remarks:
BASF AG, Department of Experimental Toxicology and Ecology

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 4-Aminoazobenzol
- Analytical purity: 97% (analytical report No.: 07L00179)
- Test-substance No.: 07/0429-1

Test animals / tissue source

Species:
other: hen
Strain:
other: white leghorn
Details on test animals or tissues and environmental conditions:
- Type of eggs: Fresh, fertilized hen eggs produced under controlled SPF conditions. White Leghorn, SPAFAS Inc., USA, SPF Premium
- Source: Charles River Deutschland GmbH, Extertal
- Weight at study initiation: 56.6–58.2 g

ENVIRONMENTAL CONDITIONS
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 5 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:
unchanged (no vehicle)
Controls:
other: Positive control substances (aqueous solution of 0.1N NaOH and 1% Sodium dodecyl sulfate), which are known to cause moderate to severe irritation were tested, in order to demonstrate the sensitivity of the method.
Amount / concentration applied:
25 μL (about 18 mg)
Duration of treatment / exposure:
3.5 minutes
Observation period (in vivo):
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 3.5 minutes, respectively.
Number of animals or in vitro replicates:
3 eggs
Details on study design:
EXPERIMENTAL PROCEDURE:
A bulk volume of 25 μL (about 18 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 3.5 minutes. A final assessment of the CAM was performed immediately after washing.
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 3.5 minutes, respectively.
The time of appearance (in seconds after application) of intravascular resp. extravascular coagulation and, if applicable, other reactions (haemorrhagia, vessel lysis) were determined.


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).

- Oil-soluble test substances
mtc(100%) < 90: risk of serious damage to the eyes
or
mtc(100%) x 0.019 + mtc(10%) x 0.017 < 4.404: risk of serious damage to the eyes
mtc(100%) ≥ 90: no risk of serious damage to the eyes

- Water- and oil-insoluble test substances
mtc(100%) < 210: risk of serious damage to the eyes
mtc(100%) ≥ 210: no risk of serious damage to the eyes

- Water-soluble test substances
mtc(10%) < 90: risk of serious damage to the eyes
mtc(10%) ≥ 90: no risk of serious damage to the eyes


SCORING SYSTEM:
The evaluation of the reactions was performed according to the following grading:
0 No visible change
1 Slight reaction
2 Moderate reaction
3 Severe reaction

Results and discussion

In vivo

Results
Irritation parameter:
other: HET-CAM (Haemorrhagia and Coagulation)
Basis:
mean
Time point:
other: 210 s
Score:
0
Reversibility:
other: no effects observed
Other effects:
no other effects

Any other information on results incl. tables

Concentration

Egg no.:

Observation period (seconds)

Grading of effects observed after the test substanve removal by washing

Additional findings 

Haemorrhagia

Coagulation

Haemorrhagia

Coagulation

 

4-Aminoazobenzol

1

210

210

0

0

none

2

210

210

0

0

none

3

210

210

0

0

none

Concentration

Egg no.:

Observation period (seconds)

Grading of effects observed after the test substanve removal by washing

Additional findings 

Haemorrhagia

Coagulation

Haemorrhagia

Coagulation

 

Positive control (0.1N NaOH)

1

24

48

2

2

iv

2

24

42

2

2

iv

iv: intravascular coagulation

Based on the results of this study and applying the evaluation criteria it is concluded, that 4-Aminoazobenzol, CAS 60-09-3 does not produce changes indicative for serious eye damage in the HET-CAM Test under the test conditions chosen.

Applicant's summary and conclusion