Registration Dossier

Administrative data

Description of key information

Skin irritation (in-vitro): Key study. Test method OECD Guideline 439. GLP study. The test item is considered as being non-irritant to skin under the test conditions.

Eye irritation (in vitro): Key study according to the OECD 438 Guideline with GLP. The test item is not classified as a severe irritant and not classified as non-irritant.

Eye irritation (in-vivo): Key study. Test method OECD Guideline 405. GLP study. The test item does not require classification as an eye irritant.


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:
From 27 April 2016 to 29 April 2016
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:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Source strain:
other: adult donors
Details on animal used as source of test system:
Not applicable
Justification for test system used:
The EPISKINTM (SM) model has been validated for irritation testing in an international validation study [9] and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE- Model used: EPISKINTM(SM) Model- Tissue batch number(s): 15-EKIN-047 and 16-EKIN-017- Expiration date: 15-EKIN-047 November 30, 2015, 16-EKIN-017 May 2, 2016- Date of initiation of testing: 27 April 2016TEMPERATURE USED FOR TEST SYSTEM- Temperature used during treatment / exposure: 23.8-27.5°C- Temperature of post-treatment incubation (if applicable): 37°CREMOVAL OF TEST MATERIAL AND CONTROLS- Number of washing steps: 3 steps two with PBS thoroughly and one with pipette- Observable damage in the tissue due to washing: washing was conducted without touching the epidermis in order to avoid damage- Modifications to validated SOP: noMTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE- MTT concentration: 0.3 mg/mL MTT- Incubation time: 3 hours (± 5 min)- Spectrophotometer: hermo Fisher Scientific, Catalogue Number: 240 72800, Serial Number: 0920-14. Date of calibration: 02 September 2014, calibration is valid until September 2016- Wavelength: 570 nmFUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA- Viability: Historical data for negative control is an OD of 0.827 with a range of 0.573-1.362. - Barrier function: historical data IC 50: specification 1.5mg/ml <= IC50 <= 3 mg/ml, result 2.8 mg/ml (16-EKIN-017) and 2.1 mg/ml (15-EKIN-017)- Morphology: well- differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum- Contamination: on blood, it was verified the absence of HIV1 and 2 antibodies, the absence of hepatitis C antibodies, the absence of hepatitis B antigen HBs, on epidermal cells of the donors it was verified the absence of bacteria, fungus and mycoplasma.- Reproducibility: historical control dataNegative control:Mean optical density (OD): 0.827, SD 0.182, range 0.573- 1.362, n= 67Positive control: Mean optical density (OD): 0.109 , SD 0.054, range 0.032-0.354, n= 63NUMBER OF REPLICATE TISSUES: 3CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE- Killed tissues- Procedure used to prepare the killed tissues (if applicable): For killed epidermis, living epidermis units (Manufacturer: SkinEthic, France, Batch No.:15-EKIN-047, Expiry Date: 30 November 2015) were placed in a 12 well plate with 2 mL of distilled water, then incubated at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere for 48 hours (±1 hour). At the end of the incubation the water was discarded and the dead epidermis units were frozen on 27 November 2015 (the frozen units can be used up to 6 months). Before use, the killed tissues were thawed at room temperature (at least 30 minutes in 2 mL of Assay Medium). Further use of killed tissues was similar to living tissues.- N. of replicates : 2- Method of calculation used: These tissues followed the same test item application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test item that may be present in the test disks. OD readings were conducted following the same conditions as for the other tissues.- Check-method for possible direct MTT reduction with test item: 10 mg of test item was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37°C in a shaking water bath for 3 hours.Purple colour of the mixture was detected in the test tube Thus, the test item reacted with MTT and therefore the use of additional controls was necessary.PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)- The test item considered to be irritant to skin (Category 2), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.- The test substance is considered to be non-corrosive to skin if the mean relative viability after 15 minutes exposure and 42 hours post incubation is more than (>) 50% of the negative control.OTHER:After receipt of the test system, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.Additional controls: As the test item was showed being an MTT-interacting substance and the test item had an intrinsic colour, two additional test item-treated killed tissues were used for the non specific OD evaluation. As colour change (purple) was observed after three hours of incubation of the test item in MTT working solution, thus the test material might interact with MTT. Therefore, additional controls and data calculations were necessary to exclude the false estimation of viability.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL- Amount(s) applied (volume or weight with unit): 20 mg of the test itemNEGATIVE CONTROL- Amount(s) applied (volume or weight): 50 μL of PBS- Concentration (if solution): not diluted, at the comercial concentration, Sigma-Aldrich Co. Batch number: BCBQ2925V, Expiry date: January 2020POSITIVE CONTROL- Amount(s) applied (volume or weight): 50 μL- Concentration (if solution): (5% (w/v) SDS solution
Duration of treatment / exposure:
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (23.8-27.5°C).
Duration of post-treatment incubation (if applicable):
The rinsed units were incubated for 42 hours (± 1h) at 37°C in an incubator with 5% CO2.
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
ca. 84.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
ca. 91.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
ca. 94.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
ca. 90.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:- Visible damage on test system: No damage was observed.- Direct-MTT reduction: Yes, the test item was showed being an MTT-interacting substance (see Table 2 on Any other information on results inc. tables”field)- Colour interference with MTT: Yes, colour change (purple) was observed after three hours of incubation of the test item(See Table 3 and 4 on “Any other information on results inc. tables” field).ACCEPTANCE OF RESULTS: - Acceptance criteria met for negative control: yes. The mean OD value of the three negative control tissues was in the recommended range (0.831). Standard deviation of the viability results for negative control samples was 4.3. The mean OD value of the blank samples (acidified isopropanol) was 0.046.- Acceptance criteria met for positive control: yes. The positive control treated tissues showed 6.7% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 2.0.- Acceptance criteria met for variability between replicate measurements: yes. The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 4.9.

Table 1: Optical Density (OD) and calculated relative viability % of the samples

 

Optical Density (OD)

Viability

Substance

 

Measured

Blank corrected

(% RV)

Negative Control:

1

0.858

0.812

97.6

Phosphate buffered saline

2

0.919

0.873

105.0

 

3

0.856

0.810

97.4

 

mean

0.831

100.0

Positive Control:

1

0.118

0.072

8.6

5% (w/v) SDS solution

2

0.084

0.038

4.6

 

3

0.103

0.057

6.9

 

mean

--

0.056

6.7

Test Item:

1

0.750

0.704

84.7

Nopcote 1661

2

0.809

0.763

91.8

 

3

0.828

0.782

94.1

 

mean

--

0.750

90.2

Table 2: Optical Density (OD) and the calculated Non-Specific MTT Reduction (NSMTT) of the Additional Control Tissues (Killed Epidermis)

Additional control

Optical Density (OD)

NSMTT

NSMTT%

(killed epidermis)

 

Measured

Blank corrected

T reated with

1

0.084

0.038

-0.013

 

Nopcote 1661

2

0.085

0.039

-0.012

-1.5

 

mean

0.038

*-0.012

 

T reated with

1

0.118

0.072

 

 

Physiological saline

2

0.075

0.029

 

 

(0.9% (w/v) NaCl)

mean

--

0.050

 

 

Table 3: Optical Density (OD) and the calculated Non Specific Colour % (NSCliving%) of the Additional Control Tissue

Additional control

Optical Density (OD)

NSC%

(living)

 

Measured

Blank corrected

T reated with

1

0.053

0.007

 

Nopcote 1661

2

0.055

0.009

0.9

 

mean

-

0.008

 

Mean blank value was 0.046

Table 4: Optical Density (OD) and the calculated Non Specific Colour % (NSCkilled%) of the Additional Control Tissues (Killed Epidermis)

Additional control

(killed epidermis)

Optical Density (OD)

NSC%

(killed)

 

Measured

ank corrected

T reated with

1

0.056

0.010

 

Nopcote 1661

2

0.066

0.020

1.7

 

mean

--

0.015

 


Interpretation of results:
GHS criteria not met
Remarks:
based on UN GHS/ CLP classification
Conclusions:
The test item was considered as being non-irritant to skin under the test conditions.
Executive summary:

The skin irritation potential of the test substance was determined in accordance with the OECD nº 439 with GLP. This experiment was conducted in a reconstructed human epidermis model; EPISKIN (SM). Disks of EPISKINTM (SM) (three units) were treated with the test item (20 mg) and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically. PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSC living) from the test item. The possible MTT interaction potential of the test item was examined using two additional test item treated and two negative control treated killed epidermis units. Furthermore to avoid a possible double correction for colour interference, a third set of controls for non-specific colour in killed tissues (NSC killed), two additional disks were used. Following exposure with the test item, the mean cell viability was 90.2% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid. In conclusion, in this in vitro EPISKIN model test with freeze-dried test item, the results indicate that the test item is non-irritant to skin.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 11 May 2016 to 17 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS- Source: S&K-LAP Kft. 2173 Kartal, Császár út 135, Hungary- Age at study initiation: 14 weeks old (male young adult)- Weight at study initiation: 3222 g – 3929 g- Housing: Rabbits were individually housed in AAALAC approved metal wire rabbit cages. Cages were of an open wire structure and cages were placed together to allow some social interaction with rabbit(s) in adjoining cages.- Diet: ad libitum- Water: ad libitum- Acclimation period: at least 28 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 20.0 – 24.0°C- Humidity (%): 28 – 68 %- Air changes (per hr): 15-20 air exchanges/hour- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.IN-LIFE DATES: From: 11 May 2016 To: 14 May 2016
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL - Amount(s) applied (volume or weight with unit): 0.1 g of test item
Duration of treatment / exposure:
The test substance was placed in the conjunctival sac. The treated eye of test animals was not rinsed with physiological saline solution, test item remained in the eye sac at the one hour observation time point
Observation period (in vivo):
The eyes were examined at 1, 24, 48 and 72 hours after treatment.
Number of animals or in vitro replicates:
3 animals
Details on study design:
REMOVAL OF TEST SUBSTANCE - Washing (if done): no washing was conducted. SCORING SYSTEM: The eye irritation scores were evaluated according to the scoring system by Draize (1977) and OECD 405 (02 October 2012) TOOL USED TO ASSESS SCORE: hand-slit lamp
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
ca. 0
Max. score:
4
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
ca. 0
Max. score:
4
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
ca. 0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
ca. 0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
ca. 0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
ca. 0
Max. score:
2
Irritation parameter:
conjunctivae score
Remarks:
Discharge
Basis:
animal #1
Time point:
24/48/72 h
Score:
ca. 0
Max. score:
3
Irritation parameter:
conjunctivae score
Remarks:
Discharge
Basis:
animal #2
Time point:
24/48/72 h
Score:
ca. 0
Max. score:
3
Irritation parameter:
conjunctivae score
Remarks:
Discharge
Basis:
animal #3
Time point:
24/48/72 h
Score:
ca. 0
Max. score:
3
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #1
Time point:
24/48/72 h
Score:
ca. 0.33
Max. score:
3
Reversibility:
fully reversible within: 48 h
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #2
Time point:
24/48/72 h
Score:
ca. 0.33
Max. score:
3
Reversibility:
fully reversible within: 48 h
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #3
Time point:
24/48/72 h
Score:
ca. 0.33
Max. score:
3
Reversibility:
fully reversible within: 48 h
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
ca. 0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
ca. 0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
ca. 0
Max. score:
4
Irritant / corrosive response data:
The test item, applied to rabbit eye mucosa, caused conjunctival effects at one hour after application which were fully reversible within 48 hours.
Other effects:
- Lesions and clinical observations:No Initial Pain Reaction (IPR) or any Pain Reaction (PR) was observed during the experimental period.Animal 1 clinical observations:At one hour after the application, conjunctival redness (score 1), chemosis (score 1) and discharge (score 1) were noted in the rabbit. Test item did not remain in the eye sac.At 24 hours after the application, conjunctival redness (score 1) was noted in the rabbit.At 48 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.At 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.Animal 2 clinical observations:At one hour after the application, conjunctival redness (score 1), chemosis (score 1) and discharge (score 1) were noted in the rabbit. Test item did not remain in the eye sac.At 24 hours after the application, conjunctival redness (score 1) was noted in the rabbit.At 48 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.At 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.Animal 3 clinical observations:At one hour after the application, conjunctival redness (score 1), chemosis (score 1) and discharge (score 1) were noted in the rabbit. Test item did not remain in the eye sac.At 24 hours after the application, conjunctival redness (score 1) was noted in the rabbit.At 48 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.At 72 hours after the application, no clinical signs, and no conjunctival or corneal effects were observed.- Other observations: The general state and behaviour of animals were normal throughout the study period. No mortality occurred during the study. The body weights of all rabbits were considered to be within the normal range of variability.

INDIVIDUAL SCORES FOR OCULAR IRRITATION

Animal Number

Sex

Cornea Opacity

Iris

Conjunctivae

Redness

Chemosis

Discharge

811
Animal 1

male

0.00

0.00

0.33

0.00

0.00

813
Animal 2

male

0.00

0.00

0.33

0.00

0.00

828
Animal 3

male

0.00

0.00

0.33

0.00

0.00

Animal 1

Time

Score of irritation

IPR/ PR

Other sign

Conjunctivae

Cornea

Iris


Control eye

R

CH

D

OD

OE

R

Pre- treatment

0

0

0

0

0

0

0

0

0

Post-treatment
(h =hour)

1 h

1

1

1

0

0

0

0

0

0

24 h

1

0

0

0

0

0

0

0

0

48 h

0

0

0

0

0

0

0

0

0

72 h

0

0

0

0

0

0

0

0

0

Animal 2

Time

Score of irritation

IPR/ PR

Other sign

Conjunctivae

Cornea

Iris


Control eye

R

CH

D

OD

OE

R

Pre- treatment

0

0

0

0

0

0

0

0

0

Post-treatment
(h =hour)

1 h

1

1

1

0

0

0

0

0

0

24 h

1

0

0

0

0

0

0

0

0

48 h

0

0

0

0

0

0

0

0

0

72 h

0

0

0

0

0

0

0

0

0

Animal 3

Time

Score of irritation

IPR/ PR

Other sign

Conjunctivae

Cornea

Iris


Control eye

R

CH

D

OD

OE

R

Pre- treatment

0

0

0

0

0

0

0

0

0

Post-treatment
(h =hour)

1 h

1

1

1

0

0

0

0

0

0

24 h

1

0

0

0

0

0

0

0

0

48 h

0

0

0

0

0

0

0

0

0

72 h

0

0

0

0

0

0

0

0

0

Abbreviations:  R = Redness                OD = Opacity degree of density

CH = Chemosis               OE        = Extent of opaque area

D = Discharge                 IPR/PR = Initial or any pain reaction

0    = Normal (in case of control eye and other lesions)

Interpretation of results:
GHS criteria not met
Conclusions:
Under these experimental conditions, the test item does not require classification as an eye irritant.
Executive summary:

The eye irritation potential of the test item was determined in accordance with the OECD Nº 405 with GLP. The test item was applied on three New Zealand male White rabbits (0.1g/eye). Animals were observed at 1h, 24h, 48 and 72h after the test item was applied. The corneal opacity score, the iris score and the conjunctive score (Discharge, Redness and Chemosis) were evaluated. No Initial Pain Reaction (IPR) or any Pain Reaction (PR) was observed during the experimental period. The test item, applied to rabbit eye mucosa, caused conjunctival effects at one hour after application which were fully reversible within 48 hours.The mean chemosis score, mean discharge score, mean cornea score and mean iris score were all 0.0, whereas the mean redness score was 0.33.According to the UN Globally Harmonised System of Classification and Labelling of Chemicals, the test item does not require classification as an eye irritant.

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

Additional information

In- vitro skin irritation: Key study: An in-vitro skin irritation study was performed according to OECD 439 Guideline and EU B.46 Method (Reconstructed Human Epidermis Model Test) with GLP. A single dose of 20 mg of test item was applied to the epidermis model EPISKIN (SM). Under the conditions of this study, the test item was considered as being non-irritant to skin.

In-vitro eye irritation: Key study: An in-vitro eye irritation study was performed according to OECD 438 Guideline and EU B.48 Method (Isolated chicken eye test method for identifying ocular corrosives and sever irritants) with GLP. A single dose of 30 mg of test item was applied on chicken eyes. Under the conditions of this study, the test item is not classified as a severe irritant and not classified as non-irritant since the overall ICE Class was 1xI 2xII.

In-vivo eye irritation: Key study: An in-vivo eye irritation study was performed according to OECD 405 Guideline with GLP. A single dose of 0.1 g f test item was applied to the eye mucosa of three New Zealand White rabbits. Under the conditions of this study, the test item does not require classification as an eye irritant.

Justification for classification or non-classification

Based on the available data, the substance is not classified for skin irritant and eye irritant according to CLP Regulation no. 1272/2008.