Registration Dossier

Administrative data

Description of key information

KGD 1409 is neither corrosive nor irritant to the skin in tests using reconstructed human epidermis (Wingenroth, 2015a+b). In vitro studies with KGD 1409 reveal no potential to serious eye damage (BCOP; Gmelin, 2015) or to irritant effects to the eyes (HET-CAM; Wingenroth, 2015d), whereas KGD 1409 is predicted as ocular irritant in a HCE test with human corneal epithelium (Wingenroth, 2015c).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Nov 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: P2D5000133
- Purity: 100 %
- Expiration date of the lot/batch: 2015-01-17
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
commercially available test method
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany).
- Cat.-No: CS-1001

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: RT (room temperature)
- Temperature of post-treatment incubation (if applicable): Incubator temperature: 37 ± 2° C (CO2 gas concentration: 5 %; Humidity: maximum)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/ml
- Incubation time: 3 hours
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

PREDICTION MODEL / DECISION CRITERIA:
- Corrosivity potential of test materials is predicted from the cell viabilities obtained after 3 min and 60 min treatment compared to the negative control. A chemical is classified "corrosive" (sub-category 1A) if the cell viability after 3 min treatment is decreased by more than 50 %. If cell viability after 3 min exposure is ≥ 50 %, while it is below 15 % after 60 min exposure the substance is also classified as corrosive, but sub-category 1 B/1 C. If cell viability after 3 min exposure is ≥ 50 % and after 60 min exposure ≥ 15 %, the substance is classified as non-corrosive.
Control samples:
yes, concurrent negative control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 0.9% NaCl

Duration of treatment / exposure:
3 and 60 minutes
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min
Value:
79.18
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min
Value:
106.21
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable

Table 1: Tabular summary of the results

 

  Sample No.    Test item  Time (min)   OD mean *    Std Dev    % Viability
1 - 3 Negative control NaCl 0.9 %  60  2.14  0.14  100.00 
10 - 12   KGD 1409  60

2.27

0.16

106.21 

16 - 18

Negative control NaCl 0.9 % 

2.46

0.14 

100.00 

25 - 27

KGD 1409 

3

1.94 

0.14

79.18

 

* 6 values

Interpretation of results:
other: no corrosive property
Executive summary:

A study for predicting a non-specific, corrosive potential of the test item by using reconstructed human epidermis (test method epiCS®) was performed according to OECD TG 431. For the determination of time related cytotoxic effects the incubation periods were 3 min. and 60 min. The MTT (Methylthiazoletetrazolium) viability test results (3 min.: 79.18 % viability; 60 min.: 106.21 % viability) showed, that KGD 1409 has no corrosive property under the conditions of the assay used.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Nov 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)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: P2D5000133
- Purity: 100 %
- Expiration date of the lot/batch: 2015-01-17
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
commercially available test method
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany).
- Cat.-No: CS-1001

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: RT (room temperature)
- Temperature of post-treatment incubation (if applicable): Incubator temperature: 37 ± 2° C (CO2 gas concentration: 5 %; Humidity: maximum)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/ml
- Incubation time: 3 hours
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

NUMBER OF TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
- The optical density of the isopropanol-extracts of 3 insert was determined by duplicate per insert = 6 OD values.

PREDICTION MODEL / DECISION CRITERIA
- The mean optical density (OD) values obtained with the test item were used to calculate the percentage of viability relative to the negative control, which is set at 100 %.
- According to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post treatment incubation is less than or equal (≤ ) to 50 %.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 0.9% NaCl in water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% SDS in physiological saline
Duration of treatment / exposure:
20 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
20 min
Value:
100.58
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- DEMONSTRATION OF TECHNICAL PROFICIENCY:

Reliability of the test was previously confirmed by interlaboratory validation

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: Tabular summary of the results

 Sample No.  Test item  OD mean *  Std Dev  % Viability
1 - 3  Negative control NaCl 0.9 %

 2.25

0.10

100.00

4 - 6

 Positive control SDS 5 %

 0.02

0.00

0.73

13 - 15

 Metalink U

 2.26

0.14

100.58

 

* 6 values

Interpretation of results:
other: no irritant property
Executive summary:

A study for predicting a skin irritation potential of the test item by using reconstructed human epidermis (test method epiCS®) was performed according to OECD TG 439. After an exposure period of 20 minutes, followed by a 42 hours post-treatment incubation period, the mean value of cell viability was measured to be 100.58 % in the MTT (Methylthiazoletetrazolium) conversion assay. Thus, KGD 1409 is considered to have no skin irritation category as defined in the UN GHS.

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:
Jan 2015
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: P2D5000133
- Purity: 100 %
- Expiration date of the lot/batch: 2015-06-12
Species:
other: isolated cornea from eyes of slaughtered cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Slaughterhouse Laame, Buchenhofen 26, 42329 Wuppertal, Germany
- Extraction: Staff of the slaughterhouse
- Transport: 1L containers with 500 mL HSS and 1 % penicillin/streptomycin solution; transport of the containers in coolers on ice
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 minutes
Duration of post- treatment incubation (in vitro):
90 minutes
Number of animals or in vitro replicates:
3
Details on study design:
- PREPARATION OF CORNEAS: Eyes were examined after delivery to the laboratory for any damage (like opacity, scratches or neovascularization) on the day of slaughter (1 day before the experiment). Eyes without any visible defects were transferred into new containers with fresh HSS solution supplemented with 1 % penicillin I streptomycin solution and 1 % FBS and stored overnight at refrigerator temperature (2-8 °C). Eyes with defects were discarded. On the next day (day of experiment) the containers with the eyes were transferred in an incubator at 32 °C (± 1 °C) for about 2 hours. For the preparation of the cornea the sclera of each eye was incised with a scalpel and cut by scissors. A 2-3 mm scleral edge was left around the cornea for further handling. The isolated corneas were placed with the epithelium side down into a prepared beaker filled with MEM medium supplemented with 1 %penicillin I streptomycin solution and 1 % FBS. Each cornea was placed in 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. The holders so prepared were transferred for at least 1 hour into the incubator at 32 °C (± 1 °C).
- SELECTION OF CORNEAS FOR APPLICATION: Following 1 hour in the incubator, the MEM medium was aspirated and the chambers were refilled with fresh MEM medium. For each cornea the reference opacity value was measured then. The mean and standard deviation of the measured values were calculated by using Microsoft Excel. The corneas with values within the range of mean ± standard deviation were selected for the actual test and assigned to the test groups. The numbers of the corneas selected were documented in an appropriate protocol. The holders were labeled with a new serial number for the further testing.
- APPLICATION OF THE TEST MATERIAL AND INCUBATION: Immediately before application, the medium was aspirated from the anterior chamber. 750 µL of the test material formulations were applied to the corneas, each through the holes of the anterior chamber. The holes of both chambers were sealed with adhesive tape and the holders were kept with the front side up, so that the formulations covered the cornea sufficiently. The holders were transferred into the incubator at 32 °C (± 1 °C) for the exposure time of 10 minutes. After the exposure, the formulations were aspirated from the anterior chamber. The corneas treated with the vehicle control, the negative and the positive control were rinsed at least 3 times with phenol red containing MEM to show effectiveness of test substance removal. The corneas treated with the test item were rinsed at first with com oil and then also at least 3 times with phenol red containing MEM. During the final rinse cycle the corneas were rinsed again with pure MEM medium in order to remove residues of the dye. The anterior chamber was then filled again with MEM medium to avoid drying out of the cornea. Before measuring opacity, fresh MEM medium was filled in the chambers.
- DETERMINATION OF 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.
- DETERMINATION OF PERMEABILITY: The medium in anterior chamber of each holder was replaced by 1 ml of fluorescein sodium solution (concentration 4 mg/mL). Afterwards the holders were incubated at 32 oc (± 1 °C) for about 90 minutes. After the incubation period, the medium of the posterior chamber was aspirated by a syringe and filled into a 10 mL tube. Three wells of a 96 well plate were filled with 300 )lL of each tube (triplicate determination). In addition, a standard series of 4 mg/mL sodium fluorescein solution was prepared and also filled into the 96-well plate, in triplicates. The permeability was determined by measuring the amount of fluorescein sodium which diffused through all cell layers of the cornea. The measurement was carried out at a wavelength of 490 nm (OD490) by an ELISA- Reader (Bio-Tek EL 808, Software Gen5).
- CALCULATION AND EVALUATION OF IN VITRO IRRITANCY SCORE (IVIS): All parameters and the IVIS values were calculated by using Microsoft Excel.
The opacity values were calculated by applying the following formulae:
1) Opacity= (Io/I-0.9894)/0.0251
2) Opacity change = opacity after application - opacity before application
3) Corrected opacity change= opacity change- mean opacity change NC
4) Mean opacity = 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 change + (15 x corrected OD490 change)
2) IVIS per group = mean of IVIS values per cornea in a group

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.

- DECISION CRITERIA: The IVIS cut-off values for identifying test chemicals as inducing serious eye damage (UN GHS Category 1) and test chemicals not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
IVIS <= 3 (No category), IVIS > 3 - <= 55 (No prediction can be made / No Category 1) or IVIS > 55 (Category 1)

Irritation parameter:
other: in vitro irritancy score (IVIS)
Run / experiment:
4 hrs
Value:
-5.03
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid

Table 1: Tabular in vitro irritancy scores (IVIS)

   Cornea No. Opacity per cornea  Permeability per cornea IVIS per cornea  IVIS mean  SD

 Negative control (0.9 % NaCl)

 1 2.33 0.010 2.49    
   2 3.18 0.007 3.29 2.32 1.06
   3 1.08 0.008 1.19    

 Positive control (1 % NaOH)

 4 69.12 1.639 93.70    
   5 104.45

0.547

112.65

 112.84

19.23

 

 6

111.16

1.401

132.17

 

 

 Test item (KGD 1409)

 13

- 5.81

0.005

- 5.74

 

 

 

 14

- 3.91 

- 0.003

- 3.96

- 5.03

0.94

 

 15

- 5.37

- 0.001 

- 5.38

 

 

No potential for serious eye damage was concluded from the study, as the IVIS was below 55 for the test item.

Interpretation of results:
other: no severe eye damage
Executive summary:

KGD 1409 was investigated in the Bovine Corneal Opacity and Permeability (BCOP) test according to OECD TG 437. The epithelial surface of the corneas was exposed to 750 µL of the undiluted test substance. Measurement of corneal opacity and permeability after a 10 minutes exposure followed by a post-treatment incubation of 90 minutes revealed an in vitro irritation score (IVIS) of - 5.03, well below the threshold for classification of serious eye damage (IVIS <=55). The positive (1 % NaOH) and negative (saline solution) controls confirmed the validity of the test. Thus, under the conditions of this test KGD 1409 was characterized by having no potential to seriously damage the eye.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Feb 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
no guideline available
Principles of method if other than guideline:
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. This is currently being addressed outside the validation study.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: P2D5000133
- Purity: 100 %
- Expiration date of the lot/batch: 2015-06-12
Species:
other: human corneal epithelium (HCE)
Strain:
other: not applicable
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 from the cell line HCE 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 minutes
Duration of post- treatment incubation (in vitro):
16 hours
Number of animals or in vitro replicates:
3
Details on study design:
The irritation potential of the test item is assessed by determination of its cytotoxic effect on an reconstructed human ocular epithelia. The test principle is based on the MTT assay reflecting the cell viability after exposure of the cornea equivalent to topically applied test item. After the exposure period the inserts were washed carefully with PBS. After a post-exposure incubation of 16 hours in the incubator (37 +/- 2 °C, 5 % CO2, maximum humidity) MTT reduction was performed. 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. 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:
60 min
Value:
1.97
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Table 1: Tabular summary of the results

 Sample No.  Test item  OD mean *  Std Dev  % Viability
 1 - 3  Negative control PBS  0.88  0.18  100.00
 4 - 6  Positive control SDS 0.3 %  0.03  0.02  3.05
 10 - 12  KGD 1409  0.02  0.02

 1.97

 

* 6 values

 

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Executive summary:

An in vitro study for assessing ocular irritation was conducted in a human corneal epithelial (HCE) cell model. 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: 523-537, 2010), and is routinely used by cosmetic and pharmaceutical companies. Undiluted test item was applied topically to the reconstructed HCE tissue (30 µL). After an exposure period of 60 minutes, followed by a 16 hours post-treatment incubation period, the cell viability was 1.97 % as measured by a MTT conversion assay. As the cut-off for a non-irritant to the eye is 50 % (a test substance is predicted to be an ocular irritant if the relative tissue viability (%) exposed to the test substance is ≤ 50), KGD 1409 was considered to be irritating to eyes.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Feb 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
no guideline available
Principles of method if other than guideline:
The protocol for this study is executed according to the ICCVAM Test Method Evaluation Report: Appendix G: ICCVAM Recommended Protocol for Future Studies Using the Hen’s Egg Test-Chorioallantoic Membrane (HET-CAM) Test method, November 2006.

These tests are also related to OECD 405, with inclusion of the ‘Manual of decisions for implementation of the sixth and seventh amendments to Directive 67/548/EEC on dangerous substances (Directives 79/831/EEC and 92/32/EEC).
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: P2D5000133
- Purity: 100 %
- Expiration date of the lot/batch: 2015-06-12
Species:
other: hen's egg chorioallantoic membrane (HET-CAM)
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
The test system used for this study is the fertile Lohmann Brown hen egg (Brinkschulte Josef GmbH & Co.KG, 48308 Senden, Germany), which is incubated for 8 days before test item application.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
300 µL per egg
Duration of treatment / exposure:
single application
Observation period (in vivo):
300 seconds after substance application
Number of animals or in vitro replicates:
4 eggs per group (negative control, positive control, test substance)
Details on study design:
TEST SYSTEM:
The fertile Lohmann Brown hen eggs (Brinkschulte Josef GmbH & Co.KG, 48308 Senden) were incubated in an incubator with an automatic rotating device (e.g. Ehret GmbH), optimum temperature : 37.5 °C, relative humidity 63 %. After 7 days of incubation, 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 NaCI 0.9 % and carefully removed with forceps. Only eggs with normally developed embryos and blood vessel systems were used for testing.

METHODS:
This study is used to evaluate the potential ocular irritancy or corrosion of a test item as measured by its ability to induce toxicity in the chorioallantoic membrane of a chicken egg. A 100 % concentration is tested on the chorioallantoic membrane (HET-CAM) of a chicken embryo. Effects are measured by the onset of haemorrhage, vessel lysis or coagulation during the first 300 seconds after application. Times till appearance of each of these endpoints are used to calculate an irritation score. However, there is no clear discrimination between strong irritation and corrosion. The protocol for this study is executed according to the ICCVAM Test Method Evaluation Report: Appendix G: ICCVAM Recommended Protocol for Future Studies Using the Hen's Egg Test-Chorioallantoic Membrane (HET-CAM) Test method, November 2006. These tests are also related to OECD 405, with inclusion of the 'Manual of decisions for implementation of the sixth and seventh amendments to Directive 67/548/EEC on dangerous substances (Directives 79/831/EEC and 92/32/EEC).

POSITIVE AND NEGATIVE CONTROL:
Physiological saline solution (0.9 % NaCl, 300 µL) was used as negative control. A SDS (sodium dodecyl sulfate) solution (1 %, 300 µL) was used as positive control (Sigma- Aldrich; Cat.No.: L5750-100G).

APPLICATION OF TEST MATERIAL:
300 µL of the test item was applied to the CAM (4 eggs each). NaCI 0.9 % treated eggs were used as negative contrals, SDS 1 % as positive contral (in
quadruplicate each). 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.

OBSERVATIONS:
0 = no effect
1 = vasodilatation, slight haemorrhage (H)
2 = vessel lysis, strong haemorrhage (L)
3 = blood-coagulation, albumen-coagulation (C)

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 IS.

IRRITATION SCORE:
Following formula was used to generate an irritation score (IS):
IS = 5 x (301-sec H)/300 + 7 x (301- sec L)/300 + 9 x (301- sec C)/300

H= observed start in seconds of haemorrhage reactions
L= observed start in seconds of vessel lysis, strong haemorrhage
C= observed start in seconds of blood-coagulation, albumen-coagulation
Irritation parameter:
in vitro irritation score
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
The test substance was identified as "non-irritant" to the chorioallantoic membrane (irritation score (IS) = 0) under the conditions of this assay.

Table 1: Summary of results from HET-CAM test

 Compound

Irritation Score (IS)

Assessment

 Negative control (0.9 % NaCl)

0

non-irritant

 Positive control (1 % SDS)

12

strong irritant

Test substance (KGD 1409)

0

non-irritant

 

Interpretation of results:
not irritating
Remarks:
Migrated information
Executive summary:

KGD 1409 was identified as "non-irritant" to the chorioallantoic membrane (irritation score (IS) = 0) in the HET-CAM test according to a protocol recommended by ICCVAM, November 2006.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A study for predicting a non-specific, corrosive potential of the test item by using reconstructed human epidermis (test method epiCS®) was performed according to OECD TG 431 (Wingenroth, 2015a). For the determination of time related cytotoxic effects the incubation periods were 3 min. and 60 min. The MTT (Methylthiazoletetrazolium) viability test results (3 min.: 79.18 % viability; 60 min.: 106.21 % viability) showed, that KGD 1409 has no corrosive property under the conditions of the assay used.

A study for predicting a skin irritation potential of the test item by using reconstructed human epidermis (test method epiCS®) was performed according to OECD TG 439 (Wingenroth, 2015b). After an exposure period of 20 minutes, followed by a 42 hours post-treatment incubation period, the mean value of cell viability was measured to be 100.58 % in the MTT (Methylthiazoletetrazolium) conversion assay. Thus, KGD 1409 is considered to have no skin irritation category as defined in the UN GHS.

KGD 1409 was investigated in the Bovine Corneal Opacity and Permeability (BCOP) test according to OECD TG 437 (Gmelin, 2015). The epithelial surface of the corneas was exposed to 750 µL of the undiluted test substance. Measurement of corneal opacity and permeability after a 10 minutes exposure followed by a post-treatment incubation of 90 minutes revealed an in vitro irritation score (IVIS) of - 5.03, well below the threshold for classification of serious eye damage (IVIS <=55). The positive (1 % NaOH) and negative (saline solution) controls confirmed the validity of the test. Thus, under the conditions of this test KGD 1409 was characterized by having no potential to seriously damage the eye.

KGD 1409 was identified as "non-irritant" to the chorioallantoic membrane (irritation score (IS) = 0) in the HET-CAM test according to a protocol recommended by ICCVAM, November 2006 (Wingenroth, 2015d).

An in vitro study for assessing ocular irritation was conducted in a human corneal epithelial (HCE) cell model (Wingenroth, 2015c). 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: 523-537, 2010), and is routinely used by cosmetic and pharmaceutical companies. Undiluted test item was applied topically to the reconstructed HCE tissue (30 µL). After an exposure period of 60 minutes, followed by a 16 hours post-treatment incubation period, the cell viability was 1.97 % as measured by a MTT conversion assay. As the cut-off for a non-irritant to the eye is 50 % (a test substance is predicted to be an ocular irritant if the relative tissue viability (%) exposed to the test substance is ≤ 50), KGD 1409 was considered to be irritating to eyes.

Justification for classification or non-classification

Skin irritation

Based on the study results of in vitro skin irritation/corrosion tests a classification according to Regulation (EC) No. 1272/2008 (CLP) is not required.

Eye irritation

Based on the study results of in vitro eye irritation/corrosion tests a classification according to Regulation (EC) No.1272/2008 (CLP) with Eye Irrit. 2 ( H319: Causes serious eye irritation) is recommended.