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Description of key information

Skin Irritation:

The following classification criteria apply:

EU DSD & CLP Not classified for Irritation.

UN GHS Not classified for Irritation (category 3 can not be determined).

Eye irritation:

No category. Not requiring classification to UN GHS or EU CLP.

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:
21 October 2015 - 26 October 2015
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:
Variation between the three identically treated negative control tissues exceeded the ≤ 18% standard deviation limit as given in the study protocol. The standard deviation for the negative control group in this study was 20%. However as there was a high confidence in the results, ie there were no borderline results in the negative control, positive control or test item treated groups a repeat of the study was considered unnecessary.
Deviations:
yes
Remarks:
This deviation was considered not to affect the purpose or integrity of the study.
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Variation between the three identically treated negative control tissues exceeded the ≤ 18% standard deviation limit as given in the study protocol. The standard deviation for the negative control group in this study was 20%. However as there was a high confidence in the results, ie there were no borderline results in the negative control, positive control or test item treated groups a repeat of the study was considered unnecessary.
Deviations:
yes
Remarks:
This deviation was considered not to affect the purpose or integrity of the study.
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch: L002
Purity: 95.4%
Physical state / Appearance: Pale yellow liquid
Expiry date: 15 August 2016
Storage Conditions: Room temperature in the dark
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: The main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Source strain:
not specified
Justification for test system used:
Reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.
Vehicle:
unchanged (no vehicle)
Details on test system:
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation (if applicable): 37 °C
DYE BINDING METHOD
- Dye used in the dye-binding assay:MTT
- Spectrophotometer: microplate reader
- Wavelength: 562 nm
- Filter:without
- Filter bandwidth: na
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if relative mean tissue viability is ≤50%
- The test substance is considered to be non-irritant to skin if relative mean tissue viability is >50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
10 µL (26.3 µL/cm2): the test item
10 µL of DPBS: negative control
10 µL of SDS 5% w/v: positive control
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
Triplicate tissues
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main
Value:
82.6
Vehicle controls validity:
not applicable
Negative controls validity:
not valid
Remarks:
There were no borderline results in the negative control, positive control or test item treated groups a repeat of the study was considered unnecessary.
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Mean OD562 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item (Table 1).

Table 1: Mean OD562 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item

OD562of tissues

Mean OD562of triplicate tissues

±SD of OD562

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Item

0.871

0.702

0.146

124.1

100*

20.8

0.619

88.2

0.617

87.9

Positive Control Item

0.042

0.084

0.041

6.0

12.0

5.9

0.124

17.7

0.085

12.1

Test Item

0.642

0.580

0.056

91.5

82.6

8.0

0.564

80.3

0.534

76.1


OD=Optical Density

SD=       Standard deviation

*=        The mean viability of the negative control tissues is set at 100%

Interpretation of results:
GHS criteria not met
Remarks:
UN GHS Not classified for Irritation (category 3 can not be determined).
Conclusions:
The test item was classified as non-irritant.
The following classification criteria apply:
EU DSD & CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 November 2015 - 04 November 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
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch: L002
Purity: 95.4%
Physical state/Appearance: pale yellow liquid
Expiry Date: 15 August 2016
Storage Conditions: room temperature in the dark
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: local abattoir
- Age of animals: 12 to 60 months old
- Storage: refrigerated on arrival and used within 24 hours of receipt
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL of the test item or control items
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS: Only corneas free of damage were used.

QUALITY CHECK OF THE ISOLATED CORNEAS: macroscopically examined before and after dissection

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: sodium chloride solution

POSITIVE CONTROL USED: Ethanol

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL of the test item or control items for 10 minutes.

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: yes, 120 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 4
- POST-EXPOSURE INCUBATION: 120 minutes

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Each cornea was visually observed
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microplate reader (OD492)
- Histopathology: possible conduct of histopathology

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
IVIS CLASSIFICATION
≤ 3 No category. Not requiring classification to UN GHS or EU CLP
> 3; ≤55 No prediction of eye irritation can be made
> 55 Category 1. UN GHS or EU CLP Causes serious eye damage
Irritation parameter:
in vitro irritation score
Run / experiment:
Main
Value:
1.5
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:
Corneal Opacity and Permeability Measurement: Table 1
Corneal Epithelium Condition: Table 2

Table 1     Individual and Mean Corneal Opacity and Permeability Measurements

 

 

Treatment

 

CorneaNumber

 

Opacity

Permeability(OD)

InVitroIrritancyScore

Pre-Treatment

Post-Treatment

PostIncubation

Post-Incubation

-Pre-Treatment

CorrectedValue

 

CorrectedValue

 

Negative Control

19

4

5

5

1

 

0.006

 

 

22

4

4

6

2

 

0.004

 

 

23

4

4

6

2

 

0.001

 

 

 

 

 

 

1.7*

 

0.004¨

 

1.7

 

Positive Control

5

3

35

33

30

28.3

0.446

0.442

 

18

3

35

34

31

29.3

1.308

1.304

 

24

4

31

29

25

23.3

0.880

0.876

 

 

 

 

 

 

27.0·

 

0.874·

40.1

 

 

Test Item

26

4

4

4

0

0.0

0.065

0.061

 

27

3

4

4

1

0.0

0.006

0.002

 

28

4

3

9

5

3.3

0.008

0.004

 

 

 

 

 

 

1.1·

 

0.023·

1.5

 

 OD=Opticaldensity       *=Meanofthepost-incubation-pre-treatmentvalues      ¨=Meanpermeability                      ·=Meancorrectedvalue

Table 2     Corneal Epithelium Condition Post Treatment and Post Incubation

 

 

Treatment

 

CorneaNumber

Observation

PostTreatment

PostIncubation

 

Negative Control

19

clear

clear

22

clear

clear

23

clear

clear

 

Positive Control

5

cloudy

cloudy

18

cloudy

cloudy

24

cloudy

cloudy

 

Test Item

26

clear

clear

27

clear

clear

28

clear

clear

Interpretation of results:
GHS criteria not met
Conclusions:
No category. Not requiring classification to UN GHS or EU CLP.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Justification for classification or non-classification

Skin irritation:

According to the criteria in CLP Annex I, 3.2.2 and the OECD guideline In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method which states that 'test chemicals that produce cell viabilities above the defined threshold level, may be considered non-irritants (i.e. > 50%, No Category)', the test substance is classified as No Category.

Eye irritation:

According to the criteria in CLP Annex I 3.3. and 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, the test substance is classified as No Category.