Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: HET-CAM, in accordance with the protocol proposed in the Official Journal of the Republic of France (#300) dated 26 December 1996
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals / tissue source

Strain:
other: Hen's eegs (White Leghorn)
Details on test animals or tissues and environmental conditions:
Temperature: 37 ºC

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Test system:
Fertilized hen egg (White Leghorn strain) weight from 50 to 65 g at receipt.

Reference items:
-Negative control: Lauryl Sulfo BetaYne (LSB) - CAS number: 14933-08-5 solution at 0.05 % in physiological serum
- Positive control: Lauryl Sulfo BetaYne (LSB) - CAS number: 14933-08-5 solution at 3.2 and 0.4 % in physiological serum

Series definition:
The test item diluted at 5 % is tested on four eggs (300 microL per egg). The reference items are tested in each analysis on two eggs (300 microL).
Duration of treatment / exposure:
300 microL of the warmed test item (undiluted or diluted) are carefully placed on the CAM with a pipette and the time clock is started. After contact for 20 seconds the membrane is rinsed with 5 ml of physiological saline at 37°C avoiding any violent splashing. The rinse liquid is removed by tilting the egg.
Duration of post- treatment incubation (in vitro):
Any irritant effects are observed for 5 minutes. The time interval when each effect appears (hyperaemia, haemorrhage, coagulation) is recorded by the all or nothing law. The presence and not the severity of the effect is recorded.
Number of animals or in vitro replicates:
Test item tested on four eggs, the reference items are tested in each analysis on two eggs.
Details on study design:
The principle of the method is based on the observation of the irritant effects (hyperaemia, haemorrhage, coagulation) which may occur within five minutes after placing a test item onto the chorio-allantoic
membrane (CAM) of an embryonic hen egg on the tenth day of incubation.
A score is established from the presence (or absence) of these effects and the time when they appear.
The mean score obtained from four eggs is used to classify the substance into four categories of irritant potential.

Test protocol:
The different stages are performed rapidly under suffic ient lighting not emitting heat. Drying of the CAM must be avoided (if drying occurs maintain the humidity level by spraying).
The egg is placed veltically on a SUppOlt (air pocket upwards).
The shell is cracked at the level of the air pocket and then removed to the level of the shell membrane.
Eggs which do not have a live hen embryo are removed.
The released surface is moistened with physiological saline solution warmed to 37°C and the solution is removed by tilting the egg.
The shell membrane is delicately detached with tweezers and then removed in order to uncover the underlying CAM. Any egg with a defective CAM or traces of haemorrhage is removed. The MCA
integrity is recorded for every egg on scores sheet at the time of the test.
300 !ll of the warmed test item (undiluted or diluted) are carefully placed on the CAM with a pipette and the time clock is started. After contact for 20 seconds the membrane is rinsed with 5 ml of
physiological saline at 37°C avoiding any violent splashing. The rinse liquid is removed by tilting the egg.
At the end of the test, eggs receive a lethal injection of 0.2 ml20 mglml pentobarbital.

Reading procedure:
Any irritant effects are observed for 5 minutes. The time interval when each effect appears (hyperaemia, haemorrhage, coagulation) is recorded by the all or nothing law. The presence and not
the severity of the effect is recorded.

Hyperaemia or injection: Capillaries which were invis ible before adding the substance become visible whereas capillaries which were already visible dilate and become redder. This phenomenon
may affect large diameter vessels.

Haemorrhage: Release of blood escaping from the vessels and or capillaries may take on different appearances, particularly « cauliflower» patches, diffuse sheet, or punctate (the blood escapes
intermittently at di fferent places in the membrane).
It must be noted that:
Haemorrhage may take on a transient appearance: this must nevertheless by counted.
Masked hyperaemia must be counted if massive haemorrhage occurs during the first 30 seconds.

Coagulation, opacity andlor thrombosis:
Opacity: opalescent sheet or direct opacification of part or all of the membrane (take care that this effect is not due to the physico-chemical properties of the substance in aqueous medium, formation of a colloid or precipitate etc.).
Thrombosis: discontinuation of blood flow producing a segmented appearance of the vessels (alternating areas of strangulation more or less dark turgescent areas). Changes occurring in the capillaries are not counted.

Results and discussion

In vitro

Results
Irritation parameter:
other: evaluation score
Run / experiment:
Mean score
Value:
ca. 0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Same results found for each egg (total number of eggs: 4), details:

Hyperanemia: Notation: 0 , Score: 0
Haemorrhage: Notation: 0, Score:0
Opacity: Notation 0 , Score 0

Subsequently, Score for each egg is 0, and Mean score (4 eggs) is 0.

Control: Lauryl sulfobetaïne
At 3.2% (positive control), score on 2 eggs: 17 (Irritant)
At 0.4% (positive control), score on 2 eggs: 10 (Moderately irritant or Irritant)
At 0.05% (negative control), score on 2 eggs: 0 (Practically non-irritant or Slightly irritant)
Results found for the reference items allow to validate the test.

Any other information on results incl. tables

An evaluation score is assigned to each phenomenon according to its occurrence time:

Scores according to the time:

Time

Effect                                                             T < 30s                       30s<T<2min              2<1'<5min

Hyperaemia                                                        5                                   3                               1

Haemorrhage                                                     7                                   5                             3

Coagulation, opacity and/or thrombosis              9                                   7                                5

The score for each egg is the sum of each phenomenon scores. The test item rating is the arithmetic mean, rounded to two decimal points, of the scores obtained for all eggs (maximum rating 21).

The test item irritation potential (pure or dilute) is given by the following scale:

Rating (N)              Classification

N < 1              Practically non irritant

1 =< N<5        Slightly irritant

5 =< N <9       Moderately irritant

N >9               Irritant

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the retained experimental conditions, the irritant potential of the test item Acetylglycyl-beta-alanine tested diluted at 5 %, may be classified as practically non irritant according to the adopted scale.