Registration Dossier

Diss Factsheets

Administrative data

Description of key information

The results obtained in an in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilized testing conditions.

In an in vitro eye corrosive and severe irritant study, using the Isolated Chicken Eye model with the test item, no ocular corrosion or severe irritation potential was observed.

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:
25 June 2018 - 06 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
06 July 2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EpiSkin™ SOP, Version 1.8
Version / remarks:
February 2009
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: adult human donors
Justification for test system used:
The EPISKIN model has been validated for irritation testing in an international trial. It showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin(TM)SM, EPISKIN SNC Lyon, France, is a three-dimensional human epidermis model

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: approximately 25 mL PBS 1 x solution; rest of the PBS was removed from epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging to the epidermis)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 3 mg/mL stock solution; 2 mL of 0.3 mg/mL per well
- Incubation time: 3 hours
- Spectrophotometer: Thermo Scientific; Multiscan FC
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and 42 hours post incubation is less than or equal to 50 % of the negative control.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and 42 hours post incubation is greater than 50 % of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 10 µL

VEHICLE
- Amount applied: 10 μL

POSITIVE CONTROL
- Amount applied: 10 μL
- Concentration: 5 % aq. solution
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1st run
Value:
87
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the method the laboratory demonstrated the technical proficiency in a separate study using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Table 1: OD values and viability percentages of the negative control

 Substance    Optical Density (OD)  Viability (%)
 Negative Control (1xPBS)              1 1.319  101 
 2 1.431  110 
 3  1.169 89 
 mean 1.306  100 
 Standard deviation (SD)    10.08

Table 2: OD values and viability percentages of the positive control

 Substance    Optical Density (OD)  Viability (%)
 Positive Control (SDS, 5% aq.)  1 0.091  7
 2 0.082
 3  0.058
 mean 0.077 6
 Standard deviation (SD)   1.28

Table 3: OD values and viability percentages of the test item

 Substance    Optical Density (OD)  Viability (%)
 Test item  1 1.035  79
 2 1.185 91 
 3 1.197 92
 mean 1.139 87
 Standard deviation (SD)   6.89

Table 4: OD values and NSC % of additional control

 Substance    Optical Density (OD)  Non Specific Colour % (NSC %)
 Test item (test item treated tissues without MTT incubation)  1 0.063 3.1
 2 0.017
 mean 0.040

Table 5: Historical Control Data (Period of 2011 - 2018 July)

   Negative Control Data  Positive Control Data   Positive Control Data
   Phosphate Buffered Saline (1xPBS)  Sodium Dodecyl Sulphate (SDS) 5% aq. solution Viability (% control) 
 Mean OD  0.858  0.110 13 
Minimum OD  0.555  0.015
 Maximum OD  1.431  0.299  39
Interpretation of results:
GHS criteria not met
Conclusions:
The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilized testing conditions.
Executive summary:

A study according OECD TG 439 to determine the skin irritation potential of the test on reconstituted human epidermis in the EPISKIN model in vitro. Disks of epidermal units (three units) were treated with the test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. Epidermis units were then incubated at 37 ± 1 °C for 42 hours (± 1h) in an incubator with 5 ± 1 % CO2, ≥ 95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37 ± 1 °C in 5 ± 1 % CO2, ≥ 95 % humidified atmosphere and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically.

The test item has an intrinsic colour (light yellow), therefore two additional test item treated tissues were used for the non-specific OD evaluation. SDS 5 % aq. and 1 x PBS treated (three units / positive and negative control) epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control. In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 87 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

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:
13 June 2018 - 20 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye 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:
25 June 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Version / remarks:
14 February 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
chicken
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary
- Storage, temperature and transport conditions of ocular tissue: Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was happened by electric current). The heads were transported at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (19.3 ºC to 20.4 ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day.
- Time interval prior to initiating testing: 2 h
- Indication of any existing defects or lesions in ocular tissue samples: After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box).

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: The test item was applied in such a way that the approximately 2 cm^2 entire surface of a plastic film was covered with test substance.
Duration of treatment / exposure:
10 sec
Duration of post- treatment incubation (in vitro):
The control and test item treated eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ± 5 minutes were considered acceptable.
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 % (w/v) was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL saline solution. Then the fluorescein-treated cornea was examined with a slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged (i.e., fluorescein retention ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit. The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper. The nictitating membrane and other connective tissue were cut away. The prepared eyes were kept on wet papers in a closed box to maintain an appropriate humidity. The treatment group and the concurrent positive control consisted of three eyes. The negative control group consisted of one eye. The enucleated eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again, too much pressure on the eye by the clamp was avoided. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with saline solution dripping from a stainless steel tube, at a rate of approximately 3 or 4 drops/minute. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity. The appropriate number of eyes was selected and, after being placed in the superfusion apparatus, the eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the saline solution which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or a high corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured using the depth measuring device. Any eye with cornea thickness deviating more than 10 % from the mean value for the eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatisation started and was conducted for approximately 45 to 60 minutes. The temperature of the circulating water was verified to ensure that the temperature in all chambers was in the range of 32 ± 1.5 °C during the acclimatisation and treatment periods.

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than ±5-7 % within approximately 45 to 60 minutes before the start of application. Slight changes in thickness (0 % to 2 %) were observed in the eyes, finding considered as normal when maintaining enucleated eyes. Following the equilibration period, the fluoresce in retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. The location of any minor findings was marked on the record sheet as a drawing, if applicable. If any eye was considered to be unsuitable following baseline assessment, it was discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: NaCl (9 g/L saline)

POSITIVE CONTROL USED: Acetic acid 10 % (v/v)

APPLICATION DOSE AND EXPOSURE TIME: 2 cm^2 for 10 sec

OBSERVATION PERIOD: up to 240 min

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The time of application was monitored.After an exposure period of 10 seconds the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove the entire residual test item, if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to a minimum.

SCORING SYSTEM:
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment

DECISION CRITERIA: decision criteria as indicated in the TG was used.
Irritation parameter:
percent corneal swelling
Remarks:
at up to 240 min
Run / experiment:
1st run
Value:
3
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
1st run
Value:
0.8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
1st run
Value:
1.2
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method the laboratory demonstrated the technical proficiency in a separate study using the ten Proficiency Chemicals according to OECD Test Guideline No. 438.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Table 1: Results for the test item

 Observation   Value   ICE Class
 Mean maximum corneal swelling at up to 75 min 3% 
 Mean maximum corneal swelling at up to 240 min  4%
 Mean maximum corneal opacity  0.8 II 
  Mean fluoresin retention  1.2  II
 Other Observations None    
Overall ICE class 1xI, 2xII    

Table 2: Results for positive control Acitic acid 10% (v/v) solution

 Observation   Value   ICE Class
 Mean maximum corneal swelling at up to 75 min 24%  III 
 Mean maximum corneal swelling at up to 240 min 33% IV
 Mean maximum corneal opacity 4.0 IV
  Mean fluoresin retention 0.5  I
 Other Observations Corneal opacity score 3 was observed in three eyes at 30 minutes after the post-treatment rinse.
Overall ICE class 1xI, 2xIV  

Based on the overall ICE Class the positive control Acetic acid 10 % (v/v) solution was classed as corrosive/severely irritating, UN GHS Classification: Category 1.

Table 3: Results for the negative control NaCl (9 g/L saline)

 Observation   Value   ICE Class
 Mean maximum corneal swelling at up to 75 min 0% 
 Mean maximum corneal swelling at up to 240 min  0%
 Mean maximum corneal opacity  0.0 I
  Mean fluoresin retention  0.0  I
 Other Observations None    
Overall ICE class 3xI  

Based on the overall ICE Class the negative control NaCl (9 g/L saline) had no significant effects on the chicken eye in this study.

Positive and negative control values were within the corresponding historical control data ranges.

Table 4: Historical control data of Positive control (Period of 2012 - 2017)

ACETIC ACID 10% (V/V) SOLUTION HISTORICAL CONTROL Dose level: 30 µL / eye

n=270

Relative
obobservation time
(m(min)

Corneal thickness

Corneal opacity score

Fluorescein retention

30

 

75

120

180

240

 30

75

120

180

240

 

∆FR

 

Maximium

swelling (%):

42

47

52

54

54

Max. OS:

4.0

4.0

4.0

4.0

4.0

Max. FR:

2.8

Minimum swelling (%):

2

4

6

9

11

Min. OS:

2.5

2.8

3.3

3.5

3.5

Min. FR:

0.2

Average:

18

24

28

31

33

Average:

3.5

3.8

3.9

3.9

3.9

Average:

0.6

 

Table 5: Historical control data of Negative control (Period of 2011 - 2017)

NaCl (9 g/L saline) SOLUTION HISTORICAL CONTROL Dose level: 30 µL / eye

n=188

Relative
obobservation time
(m (min)

Corneal thickness

Corneal opacity score

Fluorescein retention

 30

 

75

120

180

240

30 

75

120

180

240

  

∆FR

Maximum

swelling (%):

3

5

5

5

5

Max. OS:

0.5

0.5

0.5

0.5

0.5

Max. FR:

0.5

Minimum swelling (%):

0

0

0

0

0

Min. OS:

0

0

0

0

0

Min. FR:

0.0

Average:

0.2

0.4

0.5

0.5

0.5

Average:

0.0

0.1

0.1

0.1

0.1

Average:

0.0

Remark:

n = number of examined eyes

∆FR = Difference between fluorescein retention and fluorescein retention reference value

OS = Opacity score

Interpretation of results:
GHS criteria not met
Conclusions:
In this in vitro eye corrosive and severe irritant study, using the Isolated Chicken Eye model with the test item, no ocular corrosion or severe irritation potential was observed. The overall ICE score was 1xI, 2xII.
Executive summary:

A study according OECD TG 438 was performed (Isolated Chicken Eye Test, ICET) to evaluate the potential ocular corrosivity or severe irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The test compound was applied in a single dose onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" [category 1 of the Globally Harmonised System for the Classification and Labelling of chemicals (GHS)], or 2: not requiring classification for eye irritation or serious eye damage according to the GHS.

The test item, Acetic acid 10 % (v/v) solution (positive control) and NaCl (9 g/L saline) (negative control) were applied in such a way that the entire surface of the cornea was uniformly covered with the test substance or positive or negative control. Three test item treated eyes and three positive control eyes and one negative control eye were used in this study.

After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye.

In this ICET, the test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE classes were once I (based on corneal swelling of 4 % within 240 min) and twice II (based on a fluorescein retention of 1.2 and opacity score of 0.8). Positive and negative controls showed the expected results. The experiments were considered to be valid.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

A study according OECD TG 439 to determine the skin irritation potential of the test on reconstituted human epidermis in the EPISKIN model in vitro. Disks of epidermal units (three units) were treated with the test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. Epidermis units were then incubated at 37 ± 1 °C for 42 hours (± 1h) in an incubator with 5 ± 1 % CO2, ≥ 95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37 ± 1 °C in 5 ± 1 % CO2, ≥ 95 % humidified atmosphere and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically.

The test item has an intrinsic colour (light yellow), therefore two additional test item treated tissues were used for the non-specific OD evaluation. SDS 5 % aq. and 1 x PBS treated (three units / positive and negative control) epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control. In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 87 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

Eye irritation

A study according OECD TG 438 was performed (Isolated Chicken Eye Test, ICET) to evaluate the potential ocular corrosivity or severe irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The test compound was applied in a single dose onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" [category 1 of the Globally Harmonised System for the Classification and Labelling of chemicals (GHS)], or 2: not requiring classification for eye irritation or serious eye damage according to the GHS.

The test item, Acetic acid 10 % (v/v) solution (positive control) and NaCl (9 g/L saline) (negative control) were applied in such a way that the entire surface of the cornea was uniformly covered with the test substance or positive or negative control. Three test item treated eyes and three positive control eyes and one negative control eye were used in this study.

After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye.

In this ICET, the test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE classes were once I (based on corneal swelling of 4 % within 240 min) and twice II (based on a fluorescein retention of 1.2 and opacity score of 0.8). Positive and negative controls showed the expected results. The experiments were considered to be valid.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data, the test item is not classified for skin or eye irritation according to Regulation (EC) No 1272/2008 (CLP), as amended for the 12th time in Regulation (EU) No 2019/521.

Categories Display