Registration Dossier

Administrative data

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:
October 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- The substance in the test item is dissolved in approx. 30 % solvent (Substance content approx. 70 %).
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Test system epiCS has been validated for skin corrosion in a multicenter validation study and is commercially available.
Vehicle:
unchanged (no vehicle)
Details on test system:
DETAILS OF THE TEST PROCEDURE USED
The corrosive potential of the test item is assessed by determination of its cytotoxic effect on an in vitro reconstructed human epidermis. The test principle is based on the MTT assay reflecting the cell viability after exposure to the topically applied test item. All tests were performed in triplets for each time point. The test item was applied unchanged, i.e. 50 µl per insert for 3 min. (room temperature) and 60 min (incubator conditions). Cell viability was measured by the amount of MTT reduction (calculated on the basis of optical density compared to negative control).

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany).

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: RT (room temperature) for 3 min.-exposure and 37 ± 2° C (incubator conditions) for 60 min.-exposure

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Incubation conditions: 37 ± 2° C, 5 % CO2, maximum humidity
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES:
3 for test substance and negative control (for determination of cell viability the absorption of the isopropanol-extracts were measured in duplicates = 6 OD values for each)

DECISION CRITERIA:
- The mean optical density (OD) value obtained with the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100 %.
- Classification according to UN GHS: The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50% (Category 1A), or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15% (Category 1B/1C). The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Control samples:
yes, concurrent negative control
Amount/concentration applied:
50 μL per insert
Duration of treatment / exposure:
3 min. (room temperature) and 60 min. (37 °C)
Duration of post-treatment incubation (if applicable):
post-exposure incubation: none
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of exposure period 3 min.
Value:
76.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
other: no indication of corrosive potential
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of exposure period 60 min.
Value:
79.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
other: no indication of corrosive potential
Other effects / acceptance of results:
- DEMONSTRATION OF TECHNICAL PROFICIENCY:
The reliability of the test conduction was previously confirmed in an interlaboratory validation study.

Summary of results

 Sample No.  Test item  Time [min.]   OD mean*  StdDev    % Viability
 1 -3  Negative control (NaCl 0.9 %)  60  2.51  0.02 100 
 4 -6 Test item, undiluted  60  2.00  0.09  79.8
 10 -12  Negative control (NaCl 0.9 %)  3  2.612  0.14  100
 13 -15  Test item, undiluted  3  2.00  0.15  76.5

*: 6 values

Interpretation of results:
other: negative
Executive summary:

An in vitro study was performed for the assessment of skin corrosion. The experiment was carried out in accordance to OECD TG 431 using a commercially available reconstructed human epidermis test (RhE; epiCS®). In that study a test item with the substance at approx. 70 % (= "active ingredient") in approx. 30 % solvent was used. 50 µL of this test item was applied topically on the RhE. After an exposure period of 3 minutes (room temperature) and 60 minutes (37 °C; incubator conditions), the cell viability was 76.5 % and 79.8 %, respectively, as measured by a MTT conversion assay. The test item was thus not considered to have a corrosive potential to the skin.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2014
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)
Version / remarks:
(2013)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- The substance in the test item is dissolved in approx. 30 % solvent (Substance content approx. 70 %).
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Test system epiCS has been validated for skin irritation in a multicenter validation study and is commercially available.
Vehicle:
unchanged (no vehicle)
Details on test system:
DETAILS OF THE TEST PROCEDURE USED
The irritant potential of the test item is assessed by determination of its cytotoxic effect on an in vitro reconstructed human epidermis. The test principle is based on the MTT assay reflecting the cell viability after exposure to the topically applied test item. All tests were performed in triplets for each time point. The test item was applied unchanged, i.e. 30 µl per insert for 20 min. (room temperature). Cell viability was measured by the amount of MTT reduction (calculated on the basis of optical density compared to negative control).

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany).

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: RT (room temperature)
- Temperature of post-treatment incubation (if applicable): 37 ± 2° C (incubator temperature)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/ml
- Incubation time: 3 hours
- Incubation conditions: 37 ± 2° C, 5 % CO2, maximum humidity
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3 for test substance. positive and negative control (for determination of cell viability the absorption of the isopropanol-extracts were measured in duplicates = 6 OD values for each)

DECISION CRITERIA:
- The mean optical density (OD) value obtained with the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100 %.
- Classification according to UN GHS: The test substance is considered to be at least irritant to skin if the viability after 20 minutes exposure is less than or equal to 50% (Category 2 or 1; depending on outcome of corosivity test). The test substance is considered to be non-irritant to skin if the viability after 20 minutes exposure is greater than 50% .
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
30 μL per insert
Duration of treatment / exposure:
20 min. (room temperature)
Duration of post-treatment incubation (if applicable):
42 hours (37 °C)
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
82.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- DEMONSTRATION OF TECHNICAL PROFICIENCY:
The reliability of the test conduction was previously confirmed in an interlaboratory validation study.

Summary of results

 Sample No.  Test item  OD mean*  StdDev  % Viability
 1 -3  Negative control (NaCl 0.9 %)  2.49  0.05  100.00
 4 -6  Positive control (SDS 5 %)  0.04  0.03  1.53
 7 -9  Test item, undiluted  2.05  0.11  82.3

*: 6 values

Interpretation of results:
other: negative
Executive summary:

An in vitro study was performed for the assessment of skin irritation on a reconstructed human epidermis (RhE; epiCS®). The experiment was carried out in accordance to OECD TG 439 using a test item with the substance at approx. 70 % (= "active ingredient") in approx. 30 % solvent. 30 µL of this test item was applied topically on the RhE. After an exposure period of 20 minutes (room temperature) and a post-exposure incubation of 42 hours (37 °C, 5 % CO2, maximum humidity), the cell viability was 82.3 %, as measured by a MTT conversion assay. The test item was thus not considered to have an irritant potential to the skin.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
November 2014
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:
(2013)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- The substance in the test item is dissolved in approx. 30 % solvent (Substance content approx. 70 %).
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Eyes of slaughtered cattle were isolated and transferred in Hank’s balanced salt solution (HBSS) supplemented with penicillin/streptomycin. During transportation the containers with the eyes were ice-cooled.

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse Laame, Buchenhofen 26, 42329 Wuppertal, Germany
- indication of any existing defects or lesions in eyes: Eyes were examined for any damage (like opacity, scratches or neovascularization) on the day of slaughter (1 day before the experiment). Eyes with defects were sorted out and disposed of.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
750 µl per cornea
Duration of treatment / exposure:
10 min. (32 °C)
Duration of post- treatment incubation (in vitro):
2 hours (32 °C)
Number of animals or in vitro replicates:
3 corneas
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Eyes without defects were transferred into containers with fresh medium (Hank's salt solution supplemented with 1 % penicillin/streptomycin solution and 1 % FBS) and stored overnight in the refrigerator (2-8 °C). On the next day (day of experiment) the containers with the eyes were placed in an incubator at 32 °C (± 1 °C) for about 2 hours. For the cornea preparation the sclera was then incised with a scalpel and cut by scissors. The isolated corneas were placed with the epithelium side down into a prepared cup filled with MEM medium supplemented with 1 % penicillin/streptomycin solution and 1 % FBS. Each cornea was brought into a cornea holder with the endothelial side on the sealing ring of the posterior chamber. The anterior chamber was then fixed by screws on the other side. The chambers were filled with MEM medium, avoiding air bubbles, and were placed for at least 1 hour in the incubator at 32 °C (± 1 °C).

QUALITY CHECK OF THE ISOLATED CORNEAS
For each cornea the reference opacity value was determined before testing. The mean and the standard deviation of opacity was calculated. Only the corneas with values within the range of the standard deviation were selected for the study and assigned to the groups.

NUMBER OF REPLICATES
Three corneas were used for each test item, positive and negative control.

APPLICATION DOSE AND EXPOSURE TIME / TREATMENT METHOD / POST-INCUBATION PERIOD / REMOVAL OF TEST SUBSTANCE
The medium was aspirated from the anterior chamber of the cornea holder immediately before application of substances. In each case 750 μL of the test item, positive or negative control was applied to the corneas. For application of the viscous test item the open chamber method was used. The corneas were then transferred into the incubator at 32 °C (± 1 °C) for the exposure time of 10 minutes. After exposure, the substances were aspirated from the anterior chamber and the corneas were rinsed at least 3 times with MEM containing phenol red to show effectiveness of test substance removal. During the final rinse cycle the corneas were rinsed again with pure MEM medium in order to remove residues of the dye. After fresh medium was added the holders were incubated for about 2 hours at 32 °C (± 1 °C). Before measuring opacity the medium was again exchanged .

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The measurements of opacity were carried out using an opacitometer BASF OP3.0 (with integrated light meter testo 545 and Comfort 3.4 SP6 software from Testo AG, Lenzkirch). Before each measurement the opacitometer was adjusted to about 1000 LUX and a filter calibration measurement was carried out by using 3 different filters.
- Corneal permeability: The medium in the anterior chamber of each holder was replaced by 1 mL of fluorescein sodium solution (concentration 4 mg/mL). Then the holders were incubated at 32 °C (± 1 °C) for about 90 minutes. After the incubation period, the medium of the posterior chamber was aspirated and measured for the amount of fluorescein. The permeability was determined by the amount of fluorescein which diffused through all cell layers of the cornea. The measurement was carried out with an ELISA - Reader (Bio-Tek EL 808, Software Gen5, OD 490 nm).

SCORING SYSTEM:
In Vitro Irritancy Score (IVIS)
The opacity values were calculated by applying the following formulae:
1) Opacity = (Io/I-0.9894)/0.0251
[Io =single value of the measurement of empty holder with medium but without cornea, measured 1-2 days before the experiment.
I = individual value of each opacity measurement before and after application 0.9894 / 0.0251 is a constant, which is required for calculation.]
2) Opacity change = opacity after application - opacity before application
3) Corrected opacity value = opacity change - mean opacity change NC
4) Mean opacity value = mean of all corrected opacity changes per group

The permeability values were calculated by applying the following formulae:
1) OD490 change = OD490 value - mean blank value OD490
2) Corrected OD490 change = OD490 change - mean OD490 change NC
3) Mean OD490= mean of all corrected OD490 changes per group

Calculation of In Vitro Irritancy Score (IVIS):
1) IVIS per cornea = corrected opacity value + (15 x corrected OD490 change)
2) IVIS per group = mean of IVIS values per cornea in a group

DECISION CRITERIA:
- Classification according to UN GHS: A test substance is considered seriously damaging the eye if an IVIS value > 55 is obtained (UN GHS Cat 1). Test substances that cause IVIS values ≤ 55 are considered not to seriously damage the eye (no UN GHS Cat 1).
Irritation parameter:
in vitro irritation score
Remarks:
(IVIS)
Run / experiment:
mean of 3 corneas
Value:
27.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: no serious damage to the eye indicated

Summary of results:

 Cornea No. Opacity change  Permeability per cornea IVIS per cornea  IVIS mean  SD
 neg. control (NaCl 0.9 %)  1  2.6  0.006  2.7    
   2  1.4  0.005  1.4  1.8  0.7
   3  1.3  0.005  1.4    
 pos. control (NaOH 1 %)  4  99.9  1.03 115    
   5  105  1.09  121  117 3.0
   6  95.8  1.34  116    
 test item  7  2.1  0.002  2.1    
 8  4.9  0.005  5.0  27.6  41.7
   9  75.7*  0.003  75.8*    

* visible residues of the test item on the cornea after 2-hour incubation

Interpretation of results:
other: negative
Executive summary:

The substance was investigated for its potential to seriously damage the eye in the Bovine Corneal Opacity and Permeability (BCOP) test according to OECD TG 437. In this study a test item that contained approx. 70 % of the pure substance (= "active ingredient") in approx. 30 % solvent was used. 750 µL of this test item was applied onto the epithelial surface of an isolated bovine cornea. Measurement of corneal opacity and permeability after a 10-minute exposure time and a 2-hour post-exposure incubation revealed a mean IVIS well below the cut-off value of 55, that is indicating a classification for serious eye damage according to OECD TG 437. The individual IVIS (three replicates tested) were 2.1, 5.0, and 75.8; the latter is considered to be an artefact, since visible residues of the test item on the cornea diminished the opacity measured. The positive (1 % NaOH-solution) and negative (0.9 % NaCl-solution) controls confirmed the validity of the test.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Test system HET-CAM: Assessment of the eye irritation potential of the test item by observing its effect on the extra-embryonal blood vessel system (e.g. haemorrhage, vessel lysis, coagulation) of the fertile 8 days incubated chicken egg.

The protocol for this study is executed according to the ICCVAM Test Method Evaluation Report Appendix B3: ICCVAM-Recommended Protocol for Future Studies Using the Hen's Egg Test–Chorioallantoic Membrane (HET-CAM) Test Method, September 2010.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- The substance in the test item is dissolved in approx. 30 % solvent (Substance content approx. 70 %).
Details on test animals or tissues and environmental conditions:
The test system used for this study is the fertile Lohmann Brown hen egg, incubated for 8 days before application of test item.

SOURCE OF FERTILIZED CHICKEN EGGS
- Source: Brinkschulte Josef GmbH & Co.KG, 48308 Senden, Germany
- Indication of any existing defects or lesions in eggs: After 7 days of incubation, all eggs were candled in order to discard those that were defect and to mark the air bubble.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
300 µL per chicken egg
Duration of treatment / exposure:
5 min.
Duration of post- treatment incubation (in vitro):
not applicable
Number of animals or in vitro replicates:
4 eggs
Details on study design:
SELECTION AND PREPARATION OF EGGS
After 7 days in an incubator (37.5 °C, relative humidity 63%) with an automatic rotating device, all eggs were candled in order to discard those that were defect and to mark the air bubble. The eggs were replaced into the incubator with the large end upward but not rotated, thus ensuring accessibility to the CAM region. At day 8 of incubation the sections marked for the air bubble were sawed out of the shell. The inner membrane was moistened with NaCl 0.9 % and carefully removed with forceps. Only eggs with normally developed embryos and blood vessel systems were used for testing.

NUMBER OF REPLICATES
4 eggs each were used for the test substance, negative and positive controls.

APPLICATION DOSE AND EXPOSURE TIME / TREATMENT METHOD
For the positive (1% sodium dodecyl sulfate) and negative control 300 µl were applied per egg. Due to the viscosity of the test item a different kind of application has been selected. A piece of mesh was immersed in 300 µl of the unchanged test item and placed on the CAM. The size of mesh corresponding to approximately half of the CAM surface.

METHODS FOR MEASURED ENDPOINTS:
Observations of effects to the blood vessels, albumen or embryo over a period of 300 seconds after substance application are determined for each single egg. The time to the appearance of each of the observations mentioned above has been monitored and recorded. If no effect appeared during the observation period of 300 seconds (observation = 0) the result was assigned as negative for the related endpoint, and the factor set to 0 for this endpoint when calculating the Irritation Score (IS).

Formula for calculation of IS: IS = 5 x (301 - H [sec]/300) + 7 x (301 - L [sec]/300) + 9 x (301 - C [sec]/ 300)

where H= observed start of haemorrhage reactions in seconds, L= observed start of vessel lysis, strong haemorrhage in seconds and C= observed start of blood - coagulation, albumen - coagulation in seconds

Assessment of Irritation Scores (IS) according to Luepke, N.P., Fd. Chem. Toxic 23, 287-291, 1985:
Score                
0 - 0.9               no irritation
1 - 4.9               slight irritation
5 - 8.9               moderate irritation
9 - 21                strong irritation

DECISION CRITERIA:
A test substance is considered to cause severe eye irritation when the Irritation Score is equal to or above 9.
Irritation parameter:
other: Irritation Score
Run / experiment:
Maximum Value 21; mean of 4 eggs
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Results with the test item:

 Egg  Effect

 Effects detected after

[sec]

 IS

1

H > 300 

L > 300  

C > 300 0

2

H > 300  
   L > 300  
   C > 300 0
3  H > 300  
 L > 300  
   C > 300 0
 4  H > 300  
   L > 300  
   C > 300 0

H= observed start of haemorrhage reactions in seconds, L= observed start of vessel lysis, strong haemorrhage in seconds and C= observed start of blood - coagulation, albumen - coagulation in seconds

Interpretation of results:
other: negative
Executive summary:

The HET-CAM (Hen's Egg Test on the Chorioallantoic Membrane) was conducted according to a protocol of the ICCVAM Test Method Evaluation Report 2010 in order to assess the eye irritation potential of the substance. In this test eight days incubated fertile hen eggs were opened and 300 µL of the test item was applied onto the chorioallantoic membrane. The test item contained the substance at 70 % (= "active ingredient") in 30 % solvent. Due to the viscosity of the test item, instead of pipetting the test item directly, a piece of mesh soaked with 300 µL was applied onto the chorioallantoic membrane. The effects occuring during the first 300 seconds after application were measured and used to calculate the irritation score. Based on this irritation score (0 of maximal 21) the test item was identified as non irritant to the chorioallantoic membrane.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
December 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Test system HCE: Assessment of the ocular irritation potential by determination of the cytotoxic effect of a test item on the human corneal epithelium model (exposure 60 min./rt followed by 16 hours incubation at 37 °C, subsequently MTT).

The HCE model is recognized in the scientific community as a highly valuable model for the identification of substances that do not require classification for severe eye damage/eye irritancy (e.g. Cotovio et. al., Tox. in Vitro, 24, 2010, 523-537), and is routinely used by cosmetic and pharmaceutical companies. It has been prevalidated (van Goethem et. al., Tox in Vitro 20, 2006, 1-17; Alépée et al., Tox in Vitro 27, 2013, 1476-1488) and has entered formal ECVAM validation in 2010. Although a high reproducibility was attested within the validation process, a further need for optimization was identified. A recently conducted multi-laboratory validation study again demonstrated that the assay in general is a promising tool that may be used in combination with others for a solid eye irritation risk assessment (Alépée et al., Toxicol in Vitro 31, 2016, 43–53).
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- The substance in the test item is dissolved in approx. 30 % solvent (Substance content approx. 70 %).
Details on test animals or tissues and environmental conditions:
The experiment was carried out on a Human Corneal Epithelial (HCE) Model, which is standardized and commercially available (SkinEthic, France). Inserts were of 0.5 cm² size. When cultivated at the air-liquid interface in a chemically defined medium, the immortalized human cornea epithelial cells reconstruct a corneal epithelial tissue (mucosa), without a stratum corneum, ultra-structurally (tissue morphology and thickness) similar to the corneal mucosa of the human eye.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
30 µL per insert
Duration of treatment / exposure:
60 min. (room temperature)
Duration of post- treatment incubation (in vitro):
16 hours (37°C, 5% CO2, maximum humidity)
Number of animals or in vitro replicates:
3 inserts
Details on study design:
DETAILS OF THE TEST PROCEDURE USED
The irritation potential of the test item is assessed by determination of its cytotoxic effect on a reconstructed human ocular epithelium. The test principle is based on the MTT assay reflecting the cell viability after exposure of the cornea equivalent to topically applied test item.
The test substance was applied unchanged, i.e. 30 µL per insert of the test item with a substance content of approx. 70 %, for 60 min at room temperature. After the exposure period the inserts were washed carefully with PBS. MTT reduction was performed after a post-exposure incubation of 16 hours in the incubator (37 +/- 2 °C, 5 % CO2, maximum humidity). Cell viability was measured by the amount of MTT reduction, i.e. an OD value following exposure to the negative or positive control substances or the test item.

NUMBER OF TISSUE REPLICATES
Tests were performed in triplets for test substance, positive and negative control.

DECISION CRITERIA:
A test substance is predicted to be an ocular irritant if the mean relative tissue viability (%) exposed to the test substance is ≤ 50 %.
Irritation parameter:
other: cell viability (%)
Run / experiment:
decision criteria: ocular irritant if cell viability
Value:
138
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Summary of results:

Sample No.

 Test item

 %Viability

1 - 3

Negative control (PBS)

100.00

4 - 6

Positive control (SDS 0.3%)

17.3

10 - 12

test substance

138

Interpretation of results:
other: negative
Executive summary:

An in vitro study for assessing ocular irritation was conducted in a human corneal epithelial cell model (HCE; SkinEthic TM). This model is recognized in the scientific community as a highly valuable model for the identification of substances that do not require classification for serious eye damage/eye irritancy (e.g. Cotovio et al., Toxicol. in Vitro, 24, 2010, 523 -537), and is routinely used by cosmetic and pharmaceutical companies.

In this study a test item containing approx. 70 % of the pure substance (= "active ingredient") in approx. 30 % solvent was used. 30 µL of this test item was applied topically to the reconstructed HCE tissue. After an exposure period of 60 minutes (room temperature), followed by a 16 hours post-treatment incubation period (37 °C, 5 % CO2, maximum humidity), the cell viability was fully remained (above 100 %) as measured by a MTT conversion assay. Based on this assay no potential for eye irritation was concluded.

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

Respiratory irritation

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

Additional information

No in vivo studies for irritation/corrosion are available for the substance.

 

The in vitro studies for the endpoints skin irritation/corrosion and damage to the eye/eye irritation were performed with a test item that contained the substance (= "active ingredient") at approx. 70 % in approx. 30 % solvent. This is favourable in view of risk assessment, since it is the form the substance is produced and handled. Moreover, the pure substance is a solid, but the isolation is technically very complex and not manageable with reasonable efforts. For these reasons the dissolved substance was used in the in vitro assays, although an assessment of the irritant/corrosive properties of the substance at a 100 % concentration could not be achieved.

 

An in vitro study is available for the assessment of skin corrosion. The experiment was carried out in accordance to OECD TG 431 using a commercially available reconstructed human epidermis test (RhE; epiCS®). In that study 50 µL of the unchanged test item (see above) was applied topically on the RhE. After an exposure period of 3 minutes (room temperature) and 60 minutes (37 °C, 5 % CO2, maximum humidity), the cell viability was 76.5 % and 79.8 %, respectively, as measured by a MTT conversion assay. The test item was thus not considered to have a corrosive potential to the skin.

In vitro skin irritation was also assessed by using a commercially available RhE (epiCS®). The experiment was carried out in accordance to OECD TG 439. In this study 30 µL of the unchanged test item (see above) was applied topically on the RhE. After an exposure period of 20 minutes (room temperature) and a post-exposure incubation of 42 hours (37 °C, 5 % CO2, maximum humidity), the cell viability was 82.3 %, as measured by a MTT conversion assay. The test item was thus also not considered to have an irritant potential to the skin.

 

The substance was further investigated for its potential to seriously damage the eye in the Bovine Corneal Opacity and Permeability (BCOP) test according to OECD TG 437. In this study 750 µL of the unchanged test item (see above) was applied onto the epithelial surface of an isolated bovine cornea. Measurement of corneal opacity and permeability after a 10-minute exposure time and a 2-hour post-exposure incubation revealed a mean IVIS well below the cut-off value of 55, that is indicating a classification for serious eye damage according to OECD TG 437. The individual IVIS (three replicates tested) were 2.1, 5.0, and 75.8; the latter is considered to be an artefact, since visible residues of the test item on the cornea diminished the opacity measured. The positive (1 % NaOH-solution) and negative (0.9 % NaCl-solution) controls confirmed the validity of the test.

An in vitro study for assessing ocular irritation was conducted in a human corneal epithelial cell model (HCE; SkinEthic TM). This model is recognized in the scientific community as a highly valuable model for the identification of substances that do not require classification for serious eye damage/eye irritancy (e.g. Cotovio et al., Toxicol. in Vitro, 24, 2010, 523 -537), and is routinely used by cosmetic and pharmaceutical companies.

In this study 30 µL of the unchanged test item (see above) was applied topically to the reconstructed HCE tissue. After an exposure period of 60 minutes (room temperature), followed by a 16 hours post-treatment incubation period (37 °C, 5 % CO2, maximum humidity), the cell viability was fully remained (above 100 %) as measured by a MTT conversion assay. Based on this assay no potential for eye irritation was concluded.

As further test for the assessment of eye irritation the HET-CAM (Hen's Egg Test on the Chorioallantoic Membrane) was conducted according to a protocol of the ICCVAM Test Method Evaluation Report 2010. In this test eight days incubated fertile hen eggs were opened and 300 µL of the unchanged test item (see above) was applied onto the chorioallantoic membrane. Due to the viscosity of the test item, instead of pipetting the test item directly, a piece of mesh soaked with 300 µL was applied onto the chorioallantoic membrane. The effects occurring during the first 300 seconds after application were measured and used to calculate the irritation score. Based on this irritation score (0 of maximal 21) the test item was identified as non irritant to the chorioallantoic membrane.

 

Overall, no irritant or corrosive potential was concluded for the substance up to a 70 % concentration, neither in skin irritation/corrosion studies nor in studies investigating the irritant potential to the eye/potential for serious eye damage.

Justification for classification or non-classification

According to Regulation (EC) No 1272/2008, Annex I, no classification is warranted for skin irritation/corrosion or for serious damage to the eye/eye irritation up to a 70 % concentration.

According to Regulation (EC) No 1272/2008, Annex I, no classification is warranted for respiratory irritation (STOT SE3), since the available study on acute inhalation exposure does reveal only slight unspecific clinical signs up to the maximal technically attainable concentration of 3498 mg/m³. Although these signs may reflect a respiratory irritant potential, the low severity would not justify classification for respiratory irritation. For further details see chapter Acute Toxicity.