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Description of key information

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 2012
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Official journal of the European union dated august, 24'h, 2009
Deviations:
no
Qualifier:
according to
Guideline:
other: ECVAM protocol
Version / remarks:
November 5th, 2008
Deviations:
no
GLP compliance:
yes
Test system:
human skin model
Details on test system:
Human reconstructed epidermis, SkinEthic model 0.5 cm', maintained according to the supplier instructions.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Series definition:
16 microL of the test item diluted at 5 %, as well as the reference items, are tested on three epidermis.
Test protocol:
16 microL ± 0.5 microL of the test item have been deposited with a positive displacement micropipette on the surface of the epidermis and a 7.5 mm diameter nylon mesh is gently applied on the surface of the
epidermis with tweezers.
16 microL ± 0.5 microL of reference item are deposited with a micropipette positive displacement on the surface of the tissue. A 7.5 mm diameter nylon mesh is gently applied on the surface of the epidermis in with
tweezers.
Duration of treatment / exposure:
The epidermis are incubated in 0.3 ml of maintenance medium (24 wells plate) at room temperature for 42 minutes ± 1 minute.
Duration of post-treatment incubation (if applicable):
Nylon meshes are removed and the epidermis are rinsed with 25 ml of PBS by epidemlis (25 times 1 ml using a dispenser). The residual PBS is eliminated on absorbent paper. If necessary the epidermis
can be gently swept with a cotton tip. The epidermis are incubated in 2 ml of growth medium in 6 wells plate at 37 °C, 5% CO, for 42 hours ± 1 hour.
All epidermis (living and dead) are incubated in 0.3 ml of maintenance medium at 1 mg/ml MIT in 24 well plate.
After 3 hours ± 5 minutes incubation at 37°C, 5% CO2 outside of inserts is rinsed with1I to 2 ml of PBS.
Extraction is performed by placing the epidermis into wells filled with 0.8 ml of isopropanol and covered with 0.7 ml isopropanol for 2 hours ± 5 minutes under gentle agitation protected from light.
The absorbance is measured in triplicat on 200 microL extract in 96-wells plates. Absorbances are measured at 540 nm against a blank consisting with isopropanol.
Number of replicates:
3 replicates for test item, 3 replicates for positive control and 3 replicates for negative control
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue Viability (%)
Value:
ca. 100
Negative controls validity:
valid
Positive controls validity:
valid
      ASSESMENT OF VIABILITY           CONCLUSION
      Abs.  mean  Standard deviation Viability %  

 Negative control

PBS

Ep.1

Ep.2

Ep.3 

1.904 1.894 1.892

1.951 1.946 1.905

1.778 2.124 1.777 

 1.908  0.103  100%  Non Irritant

 Positive control

SDS 5%

Ep.1

Ep.2

Ep.3 

0.020 0.020 0.018

0.021 0.021 0.019

0.022 0.022 0.020 

 0.020

 0.001

 1.1%

 Irritant

Test item 

Ep.1

Ep.2

Ep.3 

2.160 2.148 2.108

1.862 1.907 1.971

1.707 1.680 1.620 

 1.907

 0.207

 100%

 Non Irritant

Interpretation of results:
GHS criteria not met
Conclusions:
In accordance with the Regulation EC No. 127212008 on classification, labelling and packaging of substances and mixtures, the test item must not be classified.
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
Assess on 10 volunteers the irritant potential of the studied test item after its unique application, maintained for 48 hours in contact with the skin, with the help of a semi-occlusive patch.
GLP compliance:
no
Remarks:
Patch test
Species:
other: Human
Details on test animals and environmental conditions:
11 volunteers of the female or male sex from 18 to 65 years of age, with a normal skin, without any dermatological lesion on the experimental area, should be included in the study.
Type of coverage:
semiocclusive
Preparation of test site:
other: semi-occlusive patch
Vehicle:
not specified
Amount / concentration applied:
Single application of 160 mg of the studied test item diluted at 5 %, on the external face of the arm, maintained for 48 hours in contact with the skin, with the help of a semi-occlusive patch.
Duration of treatment / exposure:
48 hours in contact with the skin, with the help of a semi-occlusive patch.
Observation period:
22/04/2012 to 24/05/2012
Number of animals:
11 volunteers of the female or male sex from 18 to 65 years of age, with a normal skin, without any dermatological lesion on the experimental area, should be included in the study.
Details on study design:
The clinical quotation is made 30 minutes after the patch removal and takes in account the erythema, the papules, the vesicles and the blisters. According to their intensity, the quotation is spread out from 0 to 3. The total sum of the scores, divided by the number of volunteers, defines the mean irritation index (M.I.I.), which allows to classify arbitrarily the test item into “non irritant, slightly irritant, moderately irritant, very irritant and severely irritant”.
M.I.I. =< 0.20 Non irritant
0.20 < M.I.I. =< 0.50 Slightly irritant
0.50 < M.I.I. =< 2 Moderately irritant
2 < M.I.I. =< 3 Very irritant
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
48 h
Score:
0
Max. score:
0
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
11 volunteers have been included and analyzed. The individual scores of product are 0.00 for the 11 volunteers.
Interpretation of results:
GHS criteria not met
Conclusions:
The test item, applied diluted at 5 %, can be considered as non irritant after an application with the help of a semi-occlusive patch for 48 consecutive hours on 11 volunteers.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
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
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
Strain:
other: Hen's eegs (White Leghorn)
Details on test animals or tissues and environmental conditions:
Temperature: 37 ºC
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.
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.

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

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.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

An in vitro skin irritation study on human reconstructed epidermis (SkinEthic model, OECD guideline 439) and a 48 hours insult patch test were performed.

Under the retained experimental conditions and according to the criteria for classification, packaging and labelling of dangerous subsatances and preparations in accordance with EE derectives 657/548, 2001/59 and 99/45 /, the test item diluted at 5% can be considered as non irritant, and must not be classifed.

To evaluate the eye irritation potential of the substance a HET-CAM test (2009) has been done. According to the score for this test method the substance has been classified as " practically non irritant".