3-phenyl-L-alanine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-05-08 to 2014-06-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study. Test procedure in accordance with generally accepted scientific standards and described in sufficient detail.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Principles of method if other than guideline:

The study was carried out according to:
ICCVAM-Recommended Test Method Protocol: Hen’s Egg Test – Chorioallantoic Membrane (HET-CAM) Test Method, published 2010.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-phenyl-L-alanine

EC Number:

200-568-1

EC Name:

3-phenyl-L-alanine

Cas Number:

63-91-2

Molecular formula:

C9H11NO2

IUPAC Name:

phenylalanine

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals / tissue source

Species:

other: CAM (chorioallantoic membrane) of fertile chicken eggs.

Strain:

other: CAM of chicken eggs

Details on test animals or tissues and environmental conditions:

Biological materials
Test system: Fertilized white Leghorn chicken eggs
- Source: Charles River Laboratories, Avian Products and Services, Germany GmbH, Sulzfeld, Germany
- Weight: 53 - 59 g
- Old: 6 days (at study initiation, before incubation)

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: Negative control item: 3 eggs treated with physiological saline. Positive control items: 3 eggs treated with NaOH (0.1 N), 3 eggs treated with SDS (1%).

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 300 mg/egg (used undiluted, grounded in a mortar to a fine dust)

Observation period (in vivo):

Starting 20 seconds after the application the reactions on the CAM were observed over a period of 300 seconds.

Number of animals or in vitro replicates:

3 eggs treated with the test item.

Details on study design:

1. Principle of the test
This method is increasingly used for the detection of eye mucosa membrane irritants. Eye irritation caused by external contact with chemical substances is characterized by corneal damage and/or conjunctival injury and/or iris defects.
The CAM of fertile eggs incubated for 9 days is a vital vascular membrane with a blood vessel complex. Effects which might produce irritancy in the conjunctiva are assessed by exposing the CAM to liquid or solid test items.

2. CAM preparation
a. Fresh, clean, fertile chicken eggs were selected. The eggs were candled and any eggs that were nonviable or defective were discarded. Excessively misshapen eggs or eggs with cracked or thin shells were not used. Shaking, unnecessary tilting, knocking, and all other mechanical irritation of the eggs were avoided when preparing.
b. The eggs were placed in an incubator with a rotating tray. The eggs were incubated at 38.3 ± 0.2°C and 58 ± 2% relative humidity in a Grumbach Compact S 84 incubator. The eggs were rotated five times per day until the day 8.
c. The eggs were candled on incubation day 8 and any nonviable or defective eggs were removed. Eggs were returned to the incubator (without hand rotation) with the large end of the eggs upwards for an additional day.
d. Eggs were removed from the incubator on day 9 for use in the assay. The eggs were again candled and any nonviable or defective eggs were discarded.
e. The air cell of the egg was marked. The section marked as the air cell was cut and then pared off. Care was taken when removing the eggshell to ensure that the inner membrane was not injured.
f. The inner membrane was moistened with 0.9% NaCl. A disposable pipette was used to apply the solution. The egg was placed into the incubator for a maximum of 30 minutes.
g. The egg was removed from the incubator, prior to its use in the assay, and the 0.9% NaCl solution was decanted. The inner membrane was carefully removed with forceps.

3. Observations
The chorioallantoic membrane (CAM) was carefully rinsed with 0.9% NaCl solution immediately before evaluation. Starting 20 seconds after the application the reactions on the CAM were observed over a period of 300 seconds. The time for the appearance of each of the noted endpoints was monitored and recorded, in seconds.
The following endpoints were observed:
- haemorrhage (bleeding from the vessels)
- vascular lysis (blood vessel disintegration)
- coagulation (intra- and extra-vascular protein denaturation)
Whereby,
Haemorrhage time = observed start (in seconds) of haemorrhage reactions on CAM
Lysis time = observed start (in seconds) of vessel lysis on CAM
Coagulation time = observed start (in seconds) of coagulation formation on CAM.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

mean - haemorrhage

Value:

0

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: All 3 individual values were 0 at each time interval of 0.5, 2 and 5 min. Max. score 7 after 0.5 min, 5 after 2 min, 3 after 5 min.

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

mean - lysis

Value:

0

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: All 3 individual values were 0 at each time interval of 0.5, 2 and 5 min. Max. score 5 after 0.5 min, 3 after 2 min, 1 after 5 min.

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

mean - coagulation

Value:

0

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: All 3 individual values were 0 at each time interval of 0.5, 2 and 5 min. Max. score 9 after 0.5 min, 7 after 2 min, 5 after 5 min.

Results 4

Irritation parameter:

in vitro irritation score

Run / experiment:

cumulative irritation acore

Value:

0

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Max. Score: 21

Any other information on results incl. tables

Evaluation

The current ICCVAM recommended protocol for which HET-CAM is recommended as a screening test to identify non-labelled ocular irritants uses an analysis method (i.e., the Irritation Score IS(A)) which is based on development of each of the three

HET-CAM endpoints at fixed time intervals of 0.5, 2, and 5 minutes (Table 1; Lüpke 1985).

The numerical time-dependent scores for lysis, haemorrhage, and coagulation (Table 2) are summed to give a single numerical value indicating the irritation potential of the test substance on a scale with a maximum value of 21.

Table 1: Scoring scheme for irritation testing

 

Effect	Score
	0.5 min	2 min	5 min
Lysis	5	3	1
Haemorrhage	7	5	3
Coagulation	9	7	5

 

Table 2: Classification of cumulative scores

Cumulative Score	Irritation Assessment
0 - 0.9	Practically none
1 - 4.9	Slight
5 - 8.9	Moderate
9 - 21	Strong

 

Criteria for an acceptable test

The HET-CAM assay is considered acceptable if the negative and positive controls each induce a response that falls within the classification of nonirritating and severely irritating, respectively. Historical control studies indicate that using 0.9% NaCl, as a negative control, the IS value was 0.0 (35 studies in the time period between 2010 and 2012). Historical control studies demonstrate that using 1% SDS and 0.1 N NaOH as positive controls results in IS values of approximately 8 to 12 or 17 to 19, respectively.

References

Lüpke, N. P., 1985: Hen's egg chorioallantoic membrane test for irritation potential Fd. Chem. Toxic. Vol. 23, No. 2, pp. 287 - 291

ICCVAM. 2010: ICCVAM-Recommended Test Method Protocol: Hen’s Egg Test - Chorioallantoic Membrane (HET-CAM) Test Method. NIH Publication No: 10-7553, published 2010.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

L-Phenylalanine did not show any eye irritancy potential in an in vitro GLP study using fertile chicken eggs (HET-CAM test).