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Administrative data

Description of key information

The substance is not irritating to the skin and eye.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Expiry Date: 20 December 2020
Storage Conditions: at room temperature, protection from light
Description: Colourless to yellow liquid
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
This test uses the EpiDermTM reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
- Pre-Experiments: To check the non-specific MTT-reducing capability of the test item 30 μL of the test item were mixed per 1 mL MTT medium and incubated for 60 min at 37 ± 1 °C in the incubator. If the mixture turns blue/purple, the test item is presumed to have reduced MTT and the part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using killed tissues. For quantitative correction of results, two killed tissues were treated with 30 μL of the test item (KT) and two killed tissues were left untreated as a control (KU), respectively. All steps were performed exactly as described in the chapter below. NSMTT was then calculated relative to the negative control of living tissues (NK) according to the following formula: NSMTT [%] = [(ODKT - ODKU)/ODNK] * 100. If the test item is classified as non-irritant and if non-specific MTT reduction is ≤ 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) of the test item treated living tissues TM was corrected according to the following formula: TODTT = ODTM – (ODKT – ODKU). If non-specific MTT reduction is > 30% relative to the negative control of living epidermis, the test item is considered as incompatible with the test method. To check the colouring potential of the test item 30 μL of the test item were mixed per 300 μL aqua dest. and per 300 μL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for 60 min. If the test item is classified as non-irritant and colouring is detected by unaided eye-assessment, and the chemical in water and/or isopropanol absorbs light in the range of 570 ± 30 nm, the test item was checked for its tissue-colouring potential. For quantitative correction of results, the test was performed using two additional living tissues treated with 30 μL of the test item (TVT). All steps were performed exactly as described in the chapter below, except for the MTT-staining of the test item treated with tissues, which were incubated in medium without MTT. The non-specific colour of additional viable tissues (NSCliving) was then calculated according to the following formula: NSCliving [%] = [ODTVT/ODNK]*100. If NSCliving is ≤ 5% relative to the negative control of living epidermis, no correction of the results is necessary. If NSCliving is > 5% and ≤ 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) was corrected according to the following formula: TODTT = ODTM – ODTVT. If NSCliving is > 30% relative to the negative control of living epidermis, the test item is considered as incompatible with the test method. For test items which are classified as non-irritant and which act as non-specific MTT-reducers and show non-specific colouring of living tissues, a third control for non-specific colour in killed tissues (NSCkilled) was performed to avoid a possible double-correction for colour interference. Therefore, two killed tissues were treated with 30 μL of the test item (TKT). All steps were then performed exactly as described in the chapter below, except for the MTT-staining of the test item treated with tissues, which was incubated in medium without MTT. The non-specific colour of additional killed tissues (NSCkilled) was then calculated according to the following formula: NSCkilled [%] = [ODTKT/ODNK]*100. The true tissue viability was then calculated as the percent tissue viability obtained with living tissues minus NSMTT minus NSCliving plus NSCkilled. If the coloured test material or MTT reducing substance is classified as “irritant” i.e. mean tissue viability is < 50%, the correction procedures are not necessary.
- Experimental Procedure: Upon receipt of the EpiDermTM, the tissues were inspected visually and transferred into 6-well plates containing 0.9 mL assay medium per well. The surface was dried using a sterile cotton tip and the plates were incubated in a humidified incubator at 37 ± 1 °C, 5.0% CO2 for 60 ± 5 min. Subsequently the tissues were transferred into new wells containing 0.9 mL pre-warmed assay medium per well and were incubated for 18 ± 3 h in a humidified incubator at 37 ± 1 °C, 5.0% CO2. After this pre-incubation the tissues were treated with each dose group in triplicate, starting with the negative control. Start time was recorded with dosing of the first tissue and occurred sequentially for the other tissues, e.g. in one-minute intervals. After dosing of all tissues, all plates were transferred to the incubator for 35 ± 1 min. Afterwards all plates were removed from the incubator and placed under the sterile flow for the remaining time until the 60 ± 1 min incubation time of the first dosed tissue was over. Then the tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface, this process also occurred sequentially, e.g. in one-minute intervals. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper. The inserts were placed in prepared new 6-well plates containing 0.9 mL pre-warmed fresh assay medium per well and the tissue surface was dried using a sterile cotton tip. The plates were post-incubated at 37 ± 1 C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing 0.9 mL fresh assay medium and incubated for additional 18 ± 2 h. After this post-incubation period the bottom of the inserts were blotted on sterile blotting paper and the inserts were transferred in a prepared 24-well plate containing 300 μL pre-warmed MTT medium. This plate was incubated for 3 h ± 5 min at 37 ± 1 °C, 5.0% CO2, humidified to 95%. After the MTT incubation period, the tissues were rinsed three times with DPBS and afterwards placed on blotting paper to dry. The tissues were transferred into 12-well plates and immersed in 2 mL isopropanol, sealed to inhibit evaporation. Extraction was carried out protected from light at room temperature at least for 2 h with gentle shaking on a plate shaker. Before using the extracts, the plate had been shaken for at least 15 min on a plate shaker and the inserts were pierced with an injection needle. The extract was pipetted up and down 3 times before 2 x 200 μL aliquots per each tissue were transferred into a 96-well plate. OD was measured at 570 nm with a filter band pass of maximum ± 30 nm without reference wavelength in a plate spectrophotometer using isopropanol as a blank.
- Data Analysis: Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with DPBS. The test item is considered to be irritant to skin if the tissue viability after exposure and post-incubation is less or equal to 50%. Further testing is required to resolve between UN GHS categories 1 and 2 and decide on the final classification of the test substance. The test substance may be considered as non-irritant to skin in accordance if the tissue viability after exposure and post-treatment incubation is more than 50%.
- Test Acceptance Criteria: The test meets acceptance criteria if: mean absolute OD570 nm of the three negative control tissues is ≥ 0.8 and ≤ 2.8; mean relative tissue viability of the three positive control tissues is ≤ 20%; standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Irritation / corrosion parameter:
% tissue viability
Value:
77.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The mixture of 30 μL test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%. The mixture of 30 μL of the test item per 300 μl aqua dest. and/or per 300 μL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%. The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (77.3%) after 60 min treatment and 42 h post-incubation. The controls confirmed the validity of the study. The mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (1.385). The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (2.5%). Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (1.4% - 15.4%).
Interpretation of results:
GHS criteria not met
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:
04 Apr 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
adopted: 09 Oct 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Expiry Date: 20 December 2020
Storage Conditions: at room temperature, protection from light
Description: Colourless to yellow liquid
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Abattoir A. Moksel AG, Buchloe, Germany
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for defects and any defective eyes were discarded.
- Time interval prior to initiating testing: The corneas were incubated for one hour at 32 ± 1 °C
- Indication of any antibiotics used: Pen/Strep
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 750 μL of the test substance or the control substance was introduced into the anterior chamber.
Duration of treatment / exposure:
10 minutes incubation at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
- Illuminance measurement was performed after 2 hours incubation at 32 ± 1 °C.
- Optical density at 490 nm (OD490) was determined, 90 minutes after the illuminance measurement was performed.
Number of animals or in vitro replicates:
Triplicates
Details on study design:
- Preparation of the Corneas: The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1°C.
- Calibration of the Opacitometer: The opacitometer (BASF-OP3.0, Duratec GmbH) was switched on at least 15 min before starting the calibration procedure. The filter holder was placed into the opacitometer and the readout was adjusted to 1000 ± 10 lux using the “Calibrate”-turning knob. For calibration the glass filter F2 was introduced into the filter holder. The readout lay in the range between 540-560 lux. To test the linearity of the measurement, two additional calibration filters, glass filter F3 and glass filter F4, were measured. For these glass filters, the opacitometer displayed values between 300-310 lux and between 95-105 lux.
- Treatment of the Corneas: After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI. An initial measurement was performed on each of the corneas using the opacitometer. Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0 /1.1651 lux were used for the assay. The medium was removed from the anterior chamber and replaced with the test item or control. 750 µL of the test substance or the control substance was introduced into the anterior chamber. After 10 minutes incubation at 32 ± 1°C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed after 2 hours incubation at 32 ± 1°C. Also, each cornea was observed visually and pertinent observations were recorded. After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 4 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).
- Evaluation of Results: The following formula was used to calculate the opacity, whereas the values a and b are equipment-specific variables empirically determined by the manufacturer: Opacity=(I0/I-b)/a with a = 0.025 and b = 0.9894. The value I0 is the illuminance through a holder without cornea, but with windows and liquid. This value is determined by taking the mean for a set of cornea holders and is reevaluated periodically. The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment. The mean OD490 for the blank cuvettes was calculated. The mean blank OD490 was subtracted from the OD490 of each cuvette (corrected OD490). Any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor. The final-corrected OD490 of the test article and the positive control were calculated by subtracting the average-corrected OD490 of the negative-control corneas from the corrected OD490 value of each treated cornea: Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490. The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition. The following formula was used to determine the in vitro irritation score (IVIS): IVIS = mean opacity value + (15 x mean permeability OD490 value). The IVIS cut-off values for identifying test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are: ≤ 3: No Category; > 3 - ≤ 55: No prediction can be made; > 55: Category 1.
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean based on #1, #2, #3
Value:
1.41
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Mean based on #1, #2, #3
Value:
1.81
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein leakage
Run / experiment:
Mean based on #1, #2, #3
Value:
-0.014
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.
The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Table 1: Opacity

Cornea
No.

Test Item

Initial
Opacity

Final
Opacity

Change of Opacity Value

Corrected Opacity Value

1

 

1.11

1.61

0.50

 

2

Negative

1.11

1.87

0.75

 

3

Control

1.11

2.16

1.05

 

MV

 

1.11

1.88

0.77

 

4

 

3.19

34.99

31.80

31.03

5

Positive

2.16

29.80

27.64

26.87

6

Control

2.27

29.49

27.22

26.45

MV

 

2.54

31.43

28.89

28.12

7

 

0.29

2.46

2.17

1.41

8

Test Item

-0.38

1.83

2.21

1.44

9

 

-0.73

2.01

2.75

1.98

MV

 

-0.28

2.10

2.38

1.61

MV = mean value

Table 2: Permeability

Cornea
No.

Test Item

OD490

Corrected OD490 Value

1

 

0.022

 

2

Negative

0.045

 

3

Control

0.015

 

MV

 

0.027

 

4

 

0.985

0.958

5

Positive

1.166

1.139

6

Control

0.905

0.878

MV

 

1.019

0.991

7

 

0.014

-0.013

8

Test Item

0.017

-0.010

9

 

0.010

-0.017

MV

 

0.014

-0.014

 MV = mean value

Table 3: In Vitro Irritation Score

Cornea
No.

Test Item

Corrected Opacity

Corrected
OD490 Value

IVIS

1

 

0.50

0.022

 

2

Negative

0.75

0.045

 

3

Control

1.05

0.015

 

MV

 

0.77

0.027

1.18

4

 

31.03

0.958

 

5

Positive

26.87

1.139

 

6

Control

26.45

0.878

 

MV

 

28.12

0.991

42.99

7

 

1.41

-0.013

 

8

Test Item

1.44

-0.010

 

9

 

1.98

-0.017

 

MV

 

1.61

-0.014

1.41

MV = mean value

 

Table 4: Historical Mean In Vitro Irritation Score of the Positive Control

 

IVIS Positive Control - Ethanol 100 %

Mean Value (MV)

48.57

Standard Deviation (SD)

9.69

MV- 2xSD

29.20

MV+2xSD

67.94

Number of Replicates providing Historical Mean: 50

Positive controls are updated after every single experiment or at
least every 3 months
 

Interpretation of results:
GHS criteria not met
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

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

Additional information

Skin irritation:

Skin irritation was investigated in an in vitro skin irritation test using the Reconstructed Human Epidermal Model - EpiDerm™ Standard Model (EPI-200 -TM) according to OECD Guideline 439 and following GLP. In the pre-experiment, a mixture of 30 μL test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent: the mixture did not turn blue/purple. Therefore, the non-specific reduction of MTT (NSMTT) equalled 0%. In the same pre-experiment, a mixture of 30 μL of the test item per 300 μl aqua dest. and/or per 300 μL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, the non-specific colour (NSC) equalled 0%. In the main experiment, the test item (undiluted), negative control (DPBS), and positive control (5% SDS solution) were applied on the tissues in triplicate for 60 minutes followed by a 42 h post-incubation period. The mean relative tissue viability (% negative control) was > 50% (77.3%) after 60 min treatment and 42 h post-incubation. In conclusion, in this study under the given conditions the test item showed no irritant effects. The relative mean tissue viability after 60 min of exposure and 42 h post-incubation was > 50%. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Eye irritation:

Eye irritation was investigated in a Bovine Corneal Opacity and Permeability Assay according to OECD 437 and following GLP. Eyes were collected from a slaughterhouse. The eyes were carefully examined for defects and any defective eyes were discarded. 750µL of the item (undiluted), negative control (physiological saline 0.9% NaCl), or positive control (ethanol 100%) were applied in triplicate on the corneas for 10 minutes. After a 2 -hour post-incubation period the illuminance measurement (opacity) was performed. After another 90 minutes, the optical density at 490 nm (OD490) was determined (permeability). Based on the results, the IVIS-score was determined according to OECD guideline 439. The IVIS-score was determined to be 1.41. The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid. The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control. In conclusion, according to the evaluation criteria the test item is classified into UN GHS No Category.

Justification for classification or non-classification

The test substance does not have to be classified as irritating to the skin or eye according to Regulation (EC) No 1272/2008.