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Description of key information

The substance is not classified for skin/eye irritation/corrosion, based on the results of an in vitro eye irritation/corrosion study and an in vitro skin irritation study.

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:
17 August 2015 - 24 August 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:
(2013)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
(2012)
Deviations:
no
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
other: epidermal keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 15-EKIN-033; source: SkinEthic Laboratories, Lyon, France). This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which resulted in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

TESTS FOR INTERFERENCE WITH MTT ENDPOINT
- The test substance was checked for possible colour interference before the study was started. Approximately 10 mg of the test substance was added to 90 µL Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 µL Milli-Q water was tested concurrently. At the end of the shaking period a colour check was performed.
- The test substance was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, approx. 10 mg of the test substance was added to 2 mL MTT solution (0.3 mg/mL in PBS). The mixture was incubated for 3 hour at 37°C. A negative control, sterile Milli-Q water was tested concurrently.

APPLICATION/TREATMENT
The test was performed on a total of 3 tissues per test substance together with negative and positive controls. The skin was moistened with 5 μL Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test substance to the tissue and the solid test substance (23.4 to 27.2 mg) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5% SDS in PBS (positive control) respectively. The positive control was re-spread after 7 minutes contact time.

REMOVAL OF TEST SUBSTANCE
- Washing: PBS
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
42 hours at 37°C

CELL VIABILITY MEASUREMENT
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-medium (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μL isopropanol. Tubes were stored refrigerated and protected from light for 69.5 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

SCORING SYSTEM
Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amounts applied: 23.4 to 27.2 mg per skin tissue
- The skin tissues were moistened with 5 µL Milli-Q water before application

NEGATIVE CONTOL:
- Amount applied: 25 µL Phosphate buffered saline (PBS)

POSITIVE CONTROL
- Amount applied: 25 µL
- Concentration: 5% (aq) Sodium dodecyl sulphate in PBS
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of 3 replicates
Value:
117
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Mean tissue viability for the test substance was > 50% (117%), therefore the test substance is considered not to be irritant to the skin.

TESTS FOR INTERFERENCE WITH MTT ENDPOINT

As no colour changes were observed in the tests, it was concluded that the test substance did not interact with the MTT endpoint.

Interpretation of results:
other: the substance does not need to be classified for skin irritation according to GHS and CLP
Conclusions:
An in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles. It is concluded that this test is valid and that the substance is not irritating in the in vitro skin irritation test.
Executive summary:

In an in vitro skin irritation test using a human skin model (EPISKIN Small Model), conducted according to OECD 439 and GLP principles, the influence of the substance on the viability of human skin was tested. Reliable positive and negative controls were included. The substance was applied directly to 0.38 cm2 cultured skin (23.4 to 27.2 mg, in presence of 5 μL Milli-Q water). After 15 minutes, the substance was removed and cells were cultured for 42 hours at 37°C. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 13% whereas the substance showed cell viability of 117%. Since the mean relative tissue viability after exposure to the substance was above 50%, it is concluded that the substance is not irritating in the in vitro skin irritation test. Based on the results of this study, the substance is not classified for skin irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) and Regulation (EC) No 1272/2008 (CLP Regulation).

Endpoint:
skin corrosion: in vitro / ex vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reason / purpose:
data waiving: supporting information
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:
August 3, 2015 - August 4, 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)
Version / remarks:
(2013)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
(2010)
Deviations:
no
Qualifier:
according to
Guideline:
other: The Ocular Toxicity Working Group (OTWG) of ICCVAM and NICEATM, Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The BCOP Test Method, March 2006
Deviations:
no
Qualifier:
according to
Guideline:
other: In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006
Deviations:
no
Qualifier:
according to
Guideline:
other: Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- pH (1% in water, indicative range): 6.5 - 6.2
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL of a 20% (w/v) suspension in the vehicle per cornea

NEGATIVE CONTROL
- Amount applied: 750 µL of the vehicle per cornea

POSITIVE CONTROL
- Amount applied: 750 µL of a 20% (w/v) Imidazole solution in the vehicle per cornea
Duration of treatment / exposure:
4 hours
Details on study design:
TEST SITE
- Isolated bovine cornea
- Corneas that had an initial opacity reading higher than 7 were not used
- Three corneas were used for each treatment group, at random.

REMOVAL OF TEST SUBSTANCE
- Washing: yes, with MEM+phenol red
- Time after start of exposure: 4 hours (at 32 °C)

SCORING SYSTEM:
- After exposure, the cornea was thoroughly rinsed to remove the test substance followed by immediate opacity measurement
- Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated, after another incubation period of 90 minutes at 32 °C. OD490 values of less than 1500 were used in the calculation.
- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate the mean in vitro score:
Mean in vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).
- Opacity and permeability values were also evaluated individually

TOOLS USED TO ASSESS SCORES:
- Opacitometer (OP-KIT, MC2, Clermont-Ferrand, France) and microplate reader (TECAN Infinite® M200 Pro Plate Reader)

DATA EVALUATION:
- A test substance that induces a mean IVIS >55 is classified with Eye Irr. Cat. 1 in accordance with the CLP Regulation
- A test substance that induces a mean IVIS ≤ 3 is not classified for Eye irritation in accordance with the CLP Regulation
- For a test substance that induces a mean IVIS of >3 - ≤55, it cannot be concluded whether the substance needs to be classified or not and for these substances, more (in vivo) information is needed
Irritation parameter:
in vitro irritation score
Run / experiment:
mean of 3 replicates
Value:
1
Vehicle controls validity:
valid
Positive controls validity:
valid

- Summary of opacity, permeability and in vitro scores:

 

Treatment

Mean

Opacity

Mean

Permeability

Mean in vitro

Irritation Score

Negative control

0.0

0.000

0.0

Positive control

105.0

1.939

134.1

L 125 PLUS

1.0

-0.003

1.0

- Individual in vitro irritancy scores:

 

Eye

In vitro Irritancy Score

Negative control

1

-0.6

2

0.3

3

0.4

Positive control

4

124.2

5

132.5

6

145.6

L 125 PLUS

7

2.3

8

-0.8

9

1.4

 

No pH effect of the test substance was observed on the rinsing medium.

Interpretation of results:
other: the substance does not need to be classified for eye irritation/corrosion according to GHS and CLP
Conclusions:
Based on the results of a Bovine Corneal Opacity and Permeability test in which the substance did not induce ocular irritation (mean in vitro irritancy score of 1.0), the substance is not classified for eye irritation/corrosion.
Executive summary:

Using the Bovine Corneal Opacity and Permeability test (BCOP test) the substance was screened for its eye irritancy potential in accordance with OECD 437 and according to GLP principles. The substance was applied as a 20% (w/v) suspension (750 µL) directly on top of the corneas. Adequate negative and positive controls were included. The substance did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score (IVIS) of 1.0 after 240 minutes of treatment. Since the substance induced a mean IVIS ≤ 3, the substance is not classified for eye irritation/corrosion according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) and Regulation (EC) No 1272/2008 (CLP Regulation).

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion

In an in vitro skin irritation test using a human skin model (EPISKIN Small Model), conducted according to OECD 439 and GLP principles, the influence of the substance on the viability of human skin was tested. Reliable positive and negative controls were included. The substance was applied directly to 0.38 cm2cultured skin (23.4 to 27.2 mg, in presence of 5 μL Milli-Q water). After 15 minutes, the substance was removed and cells were cultured for 42 hours at 37°C. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 13% whereas the substance showed cell viability of 117%. Since the mean relative tissue viability after exposure to the substance was above 50%, it is concluded that the substance is not irritating in the in vitro skin irritation test.

This inorganic substance does not have a rate of hydrolysis. Also metabolism is not considered relevant. Therefore the longer incubation period in the in vitro study for skin corrosion will not affect the substance as such and thus, as the substance is not irritating to skin, it is concluded that the substance is also not corrosive to skin.

Eye irritation/corrosion

Using the Bovine Corneal Opacity and Permeability test (BCOP test) the substance was screened for its eye irritancy potential in accordance with OECD 437 and according to GLP principles. The substance was applied as a 20% (w/v) suspension (750 µL) directly on top of the corneas. Adequate negative and positive controls were included. The substance did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score (IVIS) of 1.0 after 240 minutes of treatment. Since the substance induced a mean IVIS ≤ 3, the substance is not classified for eye irritation/corrosion.

Based on the results of this study, performing a second in vitro and/or an in vivo study is not needed. The results obtained from this in vitro study allow a conclusive decision on the classification of the substance.

Justification for classification or non-classification

Based on the results of an in vitro eye irritation/corrosion study and an in vitro skin irritation study, the substance does not need to be classified for skin/eye irritation/corrosion according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) and Regulation (EC) No 1272/2008 (CLP Regulation).