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Administrative data

Description of key information

Skin irritation: irritating (OECD 439; GLP compliant)
Eye irritation: not irritating (OECD 437 (2013); GLP compliant)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-07-03 to 2013-07-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 2010-07-22
Deviations:
no
Qualifier:
according to
Guideline:
other: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) for use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200), Rev. 1/19/2010.
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
, 2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2013-04-11
Test material information:
Composition 1
Details on test animals and environmental conditions:
Not applicable - Since this is an in vitro study there is no information on test animals.
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL of the undiluted test item were applied to each of triplicate tissues
Duration of treatment / exposure:
60 minutes
Observation period:
not applicable
Number of animals:
not applicable
Details on study design:
CELL CULTURE:
- Epi-200 SIT kits (Lot no.: 18346) and MTT-100 assays diluent were purchased from MatTek Corporation (82105 Bratislava, Slovakia).
- EpiDerm™ tissue (surface 0.6 cm²) consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.
Historical data and the quality certificate of the supplier of the test kit demonstrated the robustness of the test system or rather of the test kit.

TEST FOR DIRECT MTT REDUCTION BY THE TEST ITEM
- approximately 30 μL of the test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at room temperature for 60 minutes.
- untreated MTT medium was used as control.
- if the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.

TREATMENT:
- one day prior to the performance, the tissues were placed in the sterile 6-well plate.
- prior to the exposure of the test item and of the controls the EpiDerm™ tissues were inspected for quality.
- 0.9 mL of the assay medium was pipetted into each well of 6-well plates and the inserts with the EpiDerm™ tissues were pre-incubated for a total duration of about 19 hours (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH).
- after the pre-incubation of EpiDerm™ tissues, the negative control (Dulbecco's Phosphate Buffered Saline (DPBS) (30 μL)) and positive control (5% sodium lauryl sulphate (SLS) solution in deionised water (30 μL)) and the test item were added into the insert atop the corresponding EpiDerm™ triplicate tissues.
- the plates were placed into the incubator for 35 minutes at 37 ± 1.5 °C, 5 ± 0.5 % CO2, 95 ± 5% RH. Thereafter, plates were removed from the incubator and placed into the sterile hood until the period of 60 minutes was completed.
- after the end of the treatment interval the inserts were removed from the plate and tissues were rinsed with DPBS in order to remove any residual test material.
- tissues were transferred into new 6-well plates with 0.9 mL of fresh assay medium and tissues were incubated for nearly 24 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2.
- after incubation the inserts were transferred into new 6-wells plates containing fresh medium. Thereafter tissues were incubated for another about 17 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2.
- complete incubation time was approximately 43.5 hours.

CELL VIABILITY TEST:
- cell viability is measured by dehydrogenase conversion of MTT, in cell mitochondria, into a blue formazan salt.
- after the 43.5-hours incubation period, culture inserts were transferred from the holding plates plates containing 300 µL of MTT solution.
- after a 3-hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2), the MTT solution was aspirated from the wells, and the wells were rinsed with DPBS. Inserts were transferred onto new 24-well plates.
- the inserts were immersed into extractant solution (isopropanol) in each insert.
- the formazan salt was extracted for about 69.5 hours at room temperature.
- after the extraction period, the inserts were pierced to allow the extract to run into the well from which the insert was taken.
- per each tissue, 3 × 200 μL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate
- optical density was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany) with a 570 ± 1 nm filter.
- mean values were calculated from the 3 wells per tissue.

EVALUATION OF RESULTS:
- mean OD of the three negative control tissues was calculated.
- for each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula: Relative viability (%) = [OD test item / OD mean of negative control] x 100
- for the test item and the positive control the mean relative viability ± standard deviation of the three individual tissues was calculated and used for classification.
- an irritation potential of a test item according to EU classification R38 (according to directive 67/548/EEC), H315 (according to regulation (EC) 1272/2008) is recommended if the mean relative tissue viability of three individual tissues is reduced below 50% of the negative control.

Irritation / corrosion parameter:
other:
Value:
15.4
Remarks on result:
other:
Remarks:
Basis: mean. Time point: after 60 minutes incubation. Reversibility: no data. (migrated information)
Irritant / corrosive response data:
Compared to the relative absorbance value of the negative control the mean relative absorbance value decreased to 15.4% after exposure of the test item to the skin tissues. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

HISTORICAL DATA

Positive control

Negative control

Mean viability

5.7%

Mean absorption

1.858

Standard deviation

2.2%

Standard deviation

0.309

Range of viabilities

2.6 – 9.4%

Range of viabilities

1.423 – 2.651

Data of 102 studies performed from November 2011 until July 2013.

Result after treatment with the test item and the controls

Dose Group

 

Treatment Interval

Absorbance 570 nm Tissue 1*

Absorbance 570 nm Tissue 2*

Absorbance 570 nm Tissue 3*

Mean Absorbance of 3 Tissues

Mean Rel. Absorbance

[% of Negative Control]**

Negative control

60 minutes

 1.821

 1.815

 1.890

 1.842

 100.0

Positive control

60 minutes

 0.122

 0.107

 0.128

 0.119

 6.4

Test item

60 minutes

 0.267

 0.274

 0.313

 0.284

 15.4

* Mean of three replicate wells after blank correction

** Relative absorbance per treatment group [rounded values]: [100 X (mean absorbance test item / positive control)] / (mean absorbance negative control)

- Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

- After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 1.0 and ≤ 2.5 for the 60 minutes treatment interval thus showing the quality of the tissues.

- Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 6.4% thus ensuring the validity of the test system.

- The standard deviations between the % variabilities of the test item, the positive and negative controls were below 10% (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: 18%), thus ensuring the validity of the study.

Interpretation of results:
Category 2 (irritant)
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item is an irritant to the skin.
According to the Directive 67/548/EEC and its subsequent amendments, the test substance is irritating to the skin (Xi; R38).
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item is classified as irritating to the skin (Category 2, H315).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
2013-08-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
adopted 2009-09-07
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
, 2010
Deviations:
no
Qualifier:
according to
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2013-04-11
Test material information:
Composition 1
Details on test animals or tissues and environmental conditions:
Not applicable - Since this is an in vitro study there is no information on test animals.
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL of the test item
Duration of treatment / exposure:
10 minutes
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
not applicable
Details on study design:
COLLECTION OF BOVINE EYES
- isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir and were transported in Hank’s BSS supplemented with streptomycin / penicillin at ambient temperature.

PREPARATION OF CORNEAE
- all eyes were carefully examined macroscopically for defects.
- the cornea was carefully removed from the eye.
- each cornea was mounted in a specially designed cornea holder according to the description given in OEDC guideline 437, annex III, that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. Both compartments of the holder were filled with incubation medium.
- for equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.
- at the end of the incubation period, the basal opacity was determined (t0) of all cornea.
- each corneae with a value of the basal opacity > 7 was discarded.

EXPOSURE OF THE TEST GROUPS TO THE CORNEAE
- the anterior compartment received the undiluted test item or negative control (saline) or positive control (2-Ethoxyethanol; tested neat) at a volume of 0.75 mL on the surface of the corneae.
- the corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath (incubation time: 10 minutes).
- the test item, positive control and negatice control were tested in triplicate.
- after the test item or control items were rinsed off with saline.
- the corneae were then incubated at 32 ± 1 °C for further two hours in a vertical position, followed by a second opacity reading (t130).
- permeability of the corneae was determined.

PERMEABILITY MEASUREMENT
- after the final opacity measurement, the complete medium was removed from the anterior compartment and replaced by 0.5% (w/v) sodium fluorescein solution in HBSS.
- corneae were incubated in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C.
- complete medium from the posterior compartment was removed, well mixed and the optical density at 490 nm was determined with a spectrophotometer (software SoftMax Pro Enterprise, version 4.7.1).

EVALUATION OF RESULTS
- Opacity: the change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t130 – t0), for each individual cornea.
The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
- Permeability: the corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IN VITRO IRRITATION SCORE CALCULATION
The following formula was used to determine the in vitro irritation score (IVIS) of the negative control:
In vitro Irritation Score = opacity value + (15 x OD490 value)
The following formula was used to determine the in vitro irritation score of the positive control and the test item:
In vitro Irritation Score = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group was calculated from the IVIS values.
Depending on the score obtained, the test item was classified into the followingc ategory according to OECD 437 (table 1 in the field "Any other information on materials and methods incl. tables" below).

CRITERIA FOR DETERMINATION OF A VALID TEST
The test is acceptable if the IVIS of the positive control is ≥ 30 and the IVIS of the negative control is ≤ 3.
Irritation parameter:
in vitro irritation score
Value:
0.08
Vehicle controls valid:
not examined
Negative controls valid:
no
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Relative to the negative control, the test item did not cause any increase of the corneal opacity or permeability. The calculated mean IVIS was 0.08.

Table 1: Results after 10 minutes incubation time

Test group

Opacity value = Difference (t130 – t0) of opacity

Permeability at 490 nm (OD490)

In vitro irritancy score

Mean in vitro irritancy score

 

 

Mean

 

Mean

 

 

Negative control

-1

 

-0.33

0.049

 

0.048

-0.27

 

0.38

0

0.048

0.72

0

0.046

0.69

Positive control

57.33

0.811

69.50

 

63.65

53.33

0.561

61.75

51.33

0.557

59.69

Test item

0.33

0.012

0.52

 

0.08

-0.67

0.007

-0.56

0.33

-0.003

0.29

* corrected values

- With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS= 0.38).

- The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae (mean IVIS =63.65).

Table 2: Historical data

 

Positive Control

Negative Control

Mean IVIS

72.53

1.15

Standard Deviation

19.02

0.78

Range of IVIS

33.59 – 153.20

0.00 – 2.84

Values of 190 studies with solid test items performed from February 2007 until July 2013

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In conclusion, according to the current study and under the experimental conditions reported, the test item is not irritating to the eye (IVIS = 0.08).
According to OECD 437 (adopted July, 26th 2013) a chemical that induces an IVIS≤ 3 is considered as not requiring classification for eye irritation or serious eye damage.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item not causes serious eye damage.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

An in vitro skin irritation study according to OECD439 (key_in vitro skin irritation_2013_Rel1) is considered to be reliable without restrictions

and the results indicate that the test item is irritant to skin.

Eye irritation:

An in vitro eye irritation study according to OECD 437 (key_in vitro eye irritation_2013_Rel1) is considered to be reliable without restrictions.

The test item is not irritating to the eye.


Justification for selection of skin irritation / corrosion endpoint:
GLP guideline study conducted with the test item

Justification for selection of eye irritation endpoint:
GLP guideline study conducted with the test item

Effects on skin irritation/corrosion: irritating

Justification for classification or non-classification

Skin irritation:

The test item does possess a skin irritation potential and does require classification as skin irritant according to Directive 67/548/EEC and its subsequent amendments (Xi; R38 Irritant to skin) and Regulation (EC) No 1272/2008 and subsequent regulations (Skin Irrit. Cat. 2; H315 Causes skin irritation).

 

Eye irritation:

The test item does not require classification for eye irritation and serious eye damage according to OECD 437 (adopted July, 26th 2013) and the test item not causes serious eye damage according to the Regulation (EC) No 1272/2008 and subsequent regulations.