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Description of key information

Skin irritation (OECD439): not irritating

Eye irritation (OECD437): not irritating

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:
09 October 2017 - 16 October 2017
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:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
20 July 2012
Deviations:
no
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
other: epidermal keratinocytes
Cell source:
other: SkinEthic Laboratories, Lyon, France.
Source strain:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM, 0.38 cm^2
- Tissue batch number: 17-EKIN-041
- Twenty five μL of the undiluted test substance was added into 12-well plates on top of the skin tissues.
- The test item was applied topically to the corresponding tissues ensuring uniform covering.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 36.5 - 37.2°C

REMOVAL OF TEST SUBSTANCE
- Washing: phosphate buffered saline
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
- 42 hours

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.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Test material
- Applied volume: 25 μL
Duration of treatment / exposure:
15-Minute exposure period
Duration of post-treatment incubation (if applicable):
42 hours post-exposure incubation period.
Number of replicates:
A total of 9 tissues were used: Triplicate tissues were treated with: test substance, positive control or negative control.
Irritation / corrosion parameter:
% tissue viability
Value:
110
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The relative mean tissue viability compared to the negative control tissues (100%).
Other effects / acceptance of results:
OTHER EFFECTS:
- Colour interference with MTT and Direct-MTT reduction: The test item was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no color changes were observed it was concluded that the test item did not interact with the MTT endpoint.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.
- Acceptance criteria met for positive control: The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 10%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was < 7%, indicating that the test system functioned properly.
Interpretation of results:
GHS criteria not met
Remarks:
According to Regulation (EC) No. 1272/2008.
Conclusions:
The 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 test 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 Standard Model) performed according to OECD439 guideline and GLP principles, the influence of the test substance on the viability of human skin was tested. The test substance was applied directly to 0.38 cm^2 cultured skin (25 μL). After 15 minutes, the substance was removed and cells were cultured for 42 hours. 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 10% whereas the test substance showed cell viability of 110%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it can be concluded that the test substance is not irritating in the in vitro skin irritation test and the substance does not have to be classified for skin irritation according to Regulation (EC) No. 1272/2008.

Endpoint:
skin corrosion: in vitro / ex vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
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:
17 October 2017
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:
26 July 2013
Deviations:
no
GLP compliance:
yes
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL per cornea


Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
Three corneas were selected at random for each treatment group.
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

QUALITY CHECK OF THE ISOLATED CORNEAS
The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED
Physiological saline.

POSITIVE CONTROL USED
Ethanol.

APPLICATION DOSE AND EXPOSURE TIME
750 μL for 10 minutes.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: two
- POST-EXPOSURE INCUBATION: 120 ± 10 minutes

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
- Corneal permeability: Following the final opacity measurement, permeability of the cornea to Nafluorescein was evaluated. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader).
- Others: Each cornea was inspected visually for dissimilar opacity patterns.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) = mean opacity value + (15 x mean OD490 value).

DECISION CRITERIA:
A test substance that induces an IVIS ≤ 3 is not classified for eye irritancy (UN GHS: no category);
A test substance that induces an IVIS > 55 is defined as a corrosive or severe irritan (UN GHS: catgeory 1);
For a test substance that induces an IVIS >3 and ≤ 55, no prediction on irritant potency can be made.
Irritation parameter:
in vitro irritation score
Run / experiment:
Single run
Value:
0
Vehicle controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: The corneas were clear after the 10 minutes of treatment with Ammonium di hydrogen citrate.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.
- Acceptance criteria met for positive control: The mean in vitro irritancy score of the positive control (Ethanol) was 48 and within two standard deviations of the current historical positive control mean.
It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
Interpretation of results:
GHS criteria not met
Remarks:
According to Regulation (EC) No 1272/2008.
Conclusions:
Based on the outcome of a Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD 437 guideline and GLP principles, it is concluded that the substance is not irritant or corrosive for the eye.

Executive summary:

A Bovine Corneal Opacity and Permeability test (BCOP) was performed with the substance according to OECD guideline 437 and GLP principles. The substance was applied undiluted (750 μL/ cornea, n=3).

The mean in vitro irritancy score of the positive control (ethanol) was 48, and the mean in vitro irritancy score of the negative control (physiological saline) was 1.1. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The substance did not induce ocular irritation through opacity or permeability, resulting in a mean in vitro irritancy score of 0.0 after 10 minutes of treatment. Since the substance induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage according to Regulation (EC) No 1272/2008.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

In an in vitro skin irritation test using a human skin model (EPISKIN Standard Model) performed according to OECD439 guideline and GLP principles, the influence of the test substance on the viability of human skin was tested. The test substance was applied directly to 0.38 cm^2 cultured skin (25 μL). After 15 minutes, the substance was removed and cells were cultured for 42 hours. 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 10% whereas the test substance showed cell viability of 110%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it can be concluded that the test substance is not irritating in the in vitro skin irritation test.

Eye irritation:

An Bovine Corneal Opacity and Permeability test (BCOP) was performed with the substance according to OECD guideline 437 and GLP principles. The substance was applied undiluted (750 μL/ cornea, n=3).

The mean in vitro irritancy score of the positive control (ethanol) was 48, and the mean in vitro irritancy score of the negative control (physiological saline) was 1.1. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The substance did not induce ocular irritation through opacity or permeability, resulting in a mean in vitro irritancy score of 0.0 after 10 minutes of treatment. Since the substance induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Skin corrosion: the substance is not corrosive in the absence of skin and eye irritation.

Justification for classification or non-classification

Based on the results, the substance does not have to be classified for skin irritation/corrosion or eye irritation according to Regulation (EC) No. 1272/2008 and its amendments.