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other: GLP compliant non-guideline study, available as unpublished report, minor restrictions, but adequate for assessment

Data source

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

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
according to:
- Lüpke N.P. (1985): Hen’s Egg Chorio allantoic Membrane Test for Irritation Potential. Fd. Chem. Toxic. 23, pp. 287 – 291.
- Spielmann, H. (1995): HET-CAM Test. In: Methods in Molecular Biology, 43 (eds.: O’Hare, S. and Atterwill, C. K.) pp. 199 – 204.
- Spielmann, H. et al. (1996): Results of a Validation Study in Germany on Two In Vitro Alternatives to the Draize Eye Irritation Test the HET-CAM Test and the 3T3 NRU Cytotoxicity Test. ATLA 24, pp. 741 – 858.
In addition the study follows the testing strategy for determination of eye irritation/corrosion as given in the following guideline: OECD Guideline for Testing of Chemicals No. 405, April 24, 2002 (“Acute Eye Irritation/Corrosion”).
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of the test substance used in the study report: 2-Chlor-1-(3-hydroxyphenyl)-ethanol
- pH-value: Ca. 4.5 (undiluted test substance, moistened with water)
- Homogeneity: The test substance was homogeneous by visual inspection.

Test animals / tissue source

Species:
other: fresh, fertilized hen eggs
Details on test animals or tissues and environmental conditions:
TEST system hen eggs
- Type of eggs/strain/quality: Fresh, fertilized hen eggs produced under controlled SPF conditions. White Leghorn, SPAFAS Inc., USA, SPF Premium
- Source: Charles River Deutschland GmbH, Extertal
- Weight range at start of incubation period: 50.8 g – 53.6 g

INCUBATION:
Breeding in an incubator at constant temperature of 37.5°C (± 0.5°C) and a relative humidity of 62.5% (± 7.5%). 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 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)
Amount / concentration applied:
single topical application of 25 µL bulk volume (about 19 mg)
Duration of treatment / exposure:
100 seconds
Number of animals or in vitro replicates:
3 eggs
Details on study design:
EXPERIMENTAL PROCEDURE
- 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 19 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 100 seconds. A final assessment of the CAM was performed immediately after washing.
- 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.
- 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 100 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 evaluation of the reactions was performed according to the following grading: 0 No visible change, 1 Slight reaction, 2 Moderate reaction, 3 Severe reaction.
- Positive controls: On each study day, before and/or after 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).

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: time until appearance of coagulation
Basis:
mean
Time point:
other: >100 seconds
Score:
0
Max. score:
3
Irritation parameter:
other: time until appearance of haemorrhagia
Basis:
mean
Time point:
other: >100 seconds
Score:
0
Max. score:
3
Irritant / corrosive response data:
The chorionallantoic membrane of the eggs did not show any irritation effects.

Applicant's summary and conclusion

Interpretation of results:
other: does not produce serious eye damage
Conclusions:
Under the conditions of the test the substance is considered to be of no rlsk of serious damage to the eyes.
Executive summary:

In a GLP-compliant study, the potential of the test substance to cause serious damage to the eyes/mucous membranes was assessed by a single topical application of 25μL bulk volume (about 19 mg) of the undiluted test substance to the chorionallantoic membrane (CAM) of fertilized and incubated hen eggs. Three eggs were observed for 100 seconds and then the test substance was removed by washing. The occurrence of vascular injury or intravascular coagulation in response to the test substance was recorded. The chorionallantoic membrane of the eggs did not show any irritation effects. Based on the results of this study it is concluded, that the test substance does not produce changes indicative for serious eye damage in the HET-CAM Test under the test conditions chosen.