Fatty acids, C8-18 and C18-unsatd., reaction products with diethanolamine and propylene oxide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well-documented GLP study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Principles of method if other than guideline:

There are no official guidelines for the HET-CAM Test; however, the study was conducted according to the following publications:
- 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.

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C8-18 and C18-unsatd., reaction products with diethanolamine and propylene oxide

EC Number:

600-026-8

Cas Number:

1000817-22-0

Molecular formula:

Unspecified

IUPAC Name:

Fatty acids, C8-18 and C18-unsatd., reaction products with diethanolamine and propylene oxide

Details on test material:

Name of test substance: Kerocom FM 38
Batch identification: 2008250
Purity: The test substance was characterized analytically.
Homogeneity: The test substance was homogeneous by visual inspection.
Storage stability: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
pH-value: Ca. 5 (undiluted test substance); ca. 5 (10% preparation in olive oil)

Test animals / tissue source

Species:

other: hen eggs

Strain:

other: Fresh, fertilized hen eggs (White Leghorn) produced under controlled SPF conditions.

Details on test animals or tissues and environmental conditions:

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: 55.5 g – 58.8 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.
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: olive oil

Amount / concentration applied:

Single topical application of 0.3 mL of the undiluted test substance and 0.3 mL of a 10% test-substance solution in olive oil.
The test-substance preparation was produced on a weight by weight (w/w) basis shortly before application by stirring with a magnetic stirrer.

Duration of treatment / exposure:

up to 64 seconds

Number of animals or in vitro replicates:

3 eggs (undiluted)
3 eggs (test substance 10% in olive oil)

Details on study design:

Randomization: A randomization was not 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: The application procedure was chosen based on the chemical and physical properties of the test substance. A volume of 0.3 mL per egg of the liquid test substance / test substance solution was applied with a syringe and the CAM was observed until unambiguous irritation reactions were detected. It was possible to observe the reactions through the test substance/test-substance solution. The time until appearance of reactions was recorded.
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.

Results and discussion

In vitro

Results

Irritation parameter:

other: serious eye damage

Run / experiment:

HETCAM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: According to the results of the HET-CAM test, the test substance causes serious eye damage

In vivo

Irritant / corrosive response data:

The undiluted test substance caused moderate intravascular coagulation and slight haemorrhagia in all eggs after 51 – 64 seconds.
The 10% test-substance solution in olive oil caused moderate intravascular coagulation and slight haemorrhagia in all eggs after 30 – 41 seconds.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye)

Remarks:

Migrated information Criteria used for interpretation of results: EU