2-Propenoic acid, 2-methyl-, 2-(4-benzoyl-3-hydroxyphenoxy)ethyl ester

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: see 'Remark'

Remarks:

The study was conducted in compliance with GLP (incl. certificate). There are no official guidelines for the HET-CAM test; however, the study was conducted according to following publications: Lüpke NP, 1985, Fd. Chem. Toxic. 23, pp. 287 – 291; Spielmann H, 1995, Methods in Molecular Biology, 43, pp. 199 – 204; Spielmann H. et al, 1996, ATLA 24, pp. 741 – 858.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

no guideline followed

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 (CAM) of fertilized, incubated hen eggs. The test substance is applied to the CAM and the reactions of the membrane (e.g. haemorrhages, coagulation, alterations of blood vessels or albumen) are observed and evaluated.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-benzoyl-3-hydroxyphenoxy)ethyl 2-methylprop-2-enoate

Cas Number:

16613-04-0

Molecular formula:

C19 H18 O5

IUPAC Name:

2-(4-benzoyl-3-hydroxyphenoxy)ethyl 2-methylprop-2-enoate

Details on test material:

- Name of test material (as cited in study report): ULS-611, Test substance No. 08/0140-1
- Analytical purity: 99.2 % (99.2 g/100 g)
- Impurities (identity and concentrations): not specified
- Homogeneity: The test substance was homogenous by visual inspection
- Storage stability: the stability under storage conditions over the study period was guaranteed by the manufacturer

Test animals / tissue source

Species:

other: fresh fertilized hen eggs

Details on test animals or tissues and environmental conditions:

TEST SYSTEM HEN EGG
- 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, External
- Weight at study initiation: 54.7-58.6 g
- The eggs were candled before the start of incubation and on the 9th and/or 10th day. Any defective or unfertilized eggs were discarded.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37.5 °C in incubator
- Humidity (%): 62.5 % (+- 7.5 %)
- 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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: 0.1 M NaOH and 10 % sodium dodecyl sulfate (SDS) served as positive controls and were tested on each study day; historical control data were also documented.

Amount / concentration applied:

TEST MATERIAL
- Amount applied: ca. 10 mg test material, undiluted

Duration of treatment / exposure:

100 seconds

Observation period (in vivo):

100 seconds, followed by a final assessment after washing

Number of animals or in vitro replicates:

3 eggs

Details on study design:

Route of application:
After careful removal of the eggshell including the inner membrane, the test material is directly applied onto the chorionallantoic membrane (CAM).

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.

Application procedure:
A bulk volume of ca. 10 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.

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
Any further findings not covered by this scale were described.

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

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: Haemorrhagia

Basis:

mean

Remarks:

of all 3 eggs

Time point:

other: > 100 seconds

Score:

0

Results 2

Irritation parameter:

other: Coagulation

Basis:

mean

Remarks:

of all 3 eggs

Time point:

other: > 100 seconds

Score:

0

Irritant / corrosive response data:

After application of the test substance onto the CAM of three eggs, the effects were observed for 100 seconds and then the test substance was removed by washing. The occurrence of vascular injury (haemorrhagia)or intravascular coagulation in response to the test substance was recorded.
The chorionallantoic membrane of the eggs did not show any irritation effects.

Any other information on results incl. tables

Table 1: Summary of the effects observed on the treated CAM for the test substance ULS-611 and for the positive controls NaOH and SDS

Concentration	Egg-No.	Observation period (seconds)		Grading effects after washing
		Haemorrhagia	Coagulation	Haemorrhagia	Coagulation
undiluted test substance	1	> 100	> 100	0	0
	2	> 100	> 100	0	0
	3	> 100	> 100	0	0
0.1 M NaOH	1	23	37	2	2
	2	22	36	2	2
10 % SDS	1	16	28	2	2
	2	24	47	2	2
	3	26	49	2	2