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Administrative data

Description of key information

Skin irritation: irritating (OECD 439; GLP compliant)
Skin corrosion: not corrosive (OECD 431; 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:
2013-11-27 to 2013-12-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 2013-07-26
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
adopted 2009
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
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)
Controls:
other: Control (DPBS) skin cultures were used.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL of the undiluted test item


Duration of treatment / exposure:
60 minutes
Observation period:
not applicable
Number of animals:
Number of skin tissue replicates per dose:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates
Details on study design:
CELL CULTURE:
- Epi-200 SIT kits (Lot no.: 18399 Kit B) 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.

TEST FOR DIRECT MTT REDUCTION
- 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 nearly24 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 about 23 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 18.5 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2.
- complete incubation time was about 41.5 hours.

CELL VIABILITY TEST:
- cell viability is measured by dehydrogenase conversion of MTT, in cell mitochondria, into a blue formazan salt.
- after the 41.5-hours incubation period, culture inserts were transferred from the holding plates to 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).
- the formazan salt was extracted for 70 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 (OD) 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 (%) = [(meanOD test item/positive control)/ ODmean of negative control] x 100
- for the test item and the positive control the mean relative viability ± rel. 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:
3.8
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 3.8% 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

Rel. standard deviation

2.2%

Rel. 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.

Results 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

 1.301

 1.480

 1.410

 1.397

 100.0

Positive control

60 minutes

 0.068

 0.071

 0.057

 0.065

 4.7

Test item

60 minutes

 0.056

 0.047

 0.054

 0.053

 3.8

* 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 ≥ 0.8 and ≤ 2.8 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 4.7% thus ensuring the validity of the test system.

- The rel. standard deviations between the % variabilities of the test item, the positive and negative controls were below 12% (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).
Executive summary:

In this in vitro study the test substance was tested for its skin irritation potential according to OECD guideline 439 (2013) by the means of the human skin model test using EpiDerm™. Triplicates of EpiDerm™ tissues were exposed to either the undiluted test item (30 µL), the positive control (5% sodium lauryl sulphate solution; 30 µL), or the negative control (DPBS; 30 µL) for 60 minutes. The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 41.5 hours the tissues were treated with the MTT solution for 3 hours following 70 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

Compared to the relative absorbance value of the negative control the mean relative absorbance value decreased to 3.8% 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. The controls confirmed the validity of the study.

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:
2013-12-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
adopted 2013-07-26
Deviations:
yes
Remarks:
please refer to the field "Rationale for reliability incl. deficiencies" above
Qualifier:
according to
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997
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)
Controls:
other: Control (saline) bovine corneae were used.
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:
Number of bovine corneae per dose:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates
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 buffered salt solution (HBSS) 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.
- 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 following category 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 was acceptable if
- the positive control gives an IVIS that falls within two standard deviations of the current historical mean, and if
- the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
Irritation parameter:
other: in vitro irritancy score
Basis:
mean
Time point:
other: 10 minutes
Score:
2.84
Irritant / corrosive response data:
Relative to the negative control, the test item caused slight opacity but no permeability on the corneae. The calculated mean IVIS was 2.84.

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

0

 

0.00

0.044

 

0.043

0.66

 

0.65

0

0.043

0.65

0

0.042

0.63

Positive control

58.00

0.600*

67.00

 

58.49

45.00

0.432*

51.48

48.00

0.599*

56.99

Test item

1.00

0.001*

1.02

 

2.84

4.00

0.025*

4.38

2.00

0.075*

3.13

* corrected values

- With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS= 0.65).

- The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae (mean IVIS =58.49).

Table 2: Historical data

 

Positive control

Negative control

Mean IVIS

72.74

1.13

Standard deviation

18.79

0.77

Range of IVIS

33.59 – 153.20

0.00 – 2.84

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

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Relative to the negative control, the test item caused a very slight increase of the corneal opacity. Permeability effects did not occur. In conclusion, according to the current study and under the experimental conditions reported, the test item is not irritating to the eye (the calculated IVIS = 2.84). 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.
Executive summary:

In this in vitro study the test substance was tested for its eye irritation potential according to the OECD guideline 437 (2013) using fresh bovine corneae. Three corneae were exposed to either the undiluted test item (0.75 mL), the positive control (2 -Ethoxyethanol (neat); 0.75 mL), or the negative control (saline; 0.75 mL) for 10 minutes (incubation temperature: 32 ± 1 °C). After the incubation phase the test item, the positive control, and the negative control were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in incubation medium. Next, the corneal opacity and permeability were measured and the In vitro Irritancy Score (IVIS) was calculated for the test item and the controls.

Relative to the negative control, the test item caused slight opacity but no permeability on the corneae. The calculated mean IVIS was 2.84 (threshold for serious eye damage: IVIS≥ 55. 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.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation / corrosion:

Two in vitro studies, one on skin corrosion and one on skin irritation, were conducted with the test item. The in vitro skin irritation test according to OECD 439 (WoE_in vitro skin irritation_2014_RL1) showed that the test substance is irritating to the skin. According to testing and assessment strategy for skin corrosion/irritation in Figure R.7.2–3 (ECHA, 2015: Guidance on Information Requirements and CSA, Chapter R.7a) following a bottom-up approach an in vitro skin corrosion test according to OECD 431 (WoE_in vitro skin corrosion_2014_RL1) was conducted and indicated that the test item is not corrosive to the skin. Based on these studies and Figure R.7.2–3 (ECHA, 2015: Guidance on Information Requirements and CSA, Chapter R.7a) and Section 3.2.2.6 (ECHA, 2015: Guidance on the Application of the CLP Criteria), the test item is classified as irritating to skin Cat. 2.

Eye irritation:

An in vitro eye irritation study according to OECD 437 (key_in vitro eye irritation_2014_RL1) is considered to be reliable without restrictions.

The test item is not irritating to the eye.


Justification for selection of skin irritation / corrosion endpoint:
Two in vitro studies, one on skin corrosion and one on skin irritation, were conducted with the test item. The in vitro skin irritation test (OECD 439) showed that the test substance is irritating to the skin. According to testing and assessment strategy for skin corrosion/irritation in Figure R.7.2–3 (ECHA, 2015: Guidance on Information Requirements and CSA, Chapter R.7a) following a bottom-up approach an in vitro skin corrosion test (OECD 431) was conducted and indicated that the test item is not corrosive to the skin.

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 corrosion:

The test item does not possess a skin corrosive potential and does not require classification as corrosive to skin according to Directive 67/548/EEC and its subsequent amendments and Regulation (EC) No 1272/2008 and subsequent regulations.

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 its 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.