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Description of key information

The susbtance is considered skin irritant category 2 and not eye irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was well performed and well documented. No OECD guideline was followed, however, the study is similar to the Draize test and the obtained scores can directly be interpreted for the classification according to the GLP regulation.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
Primary irritation to the skin is measured by a patch-test technique on the abraded and intact skin of albino rabbits.
Deviations:
yes
Remarks:
occlusive dressing; 24 h application; examination of erythema and edema according to Draize only 24 h after application;
GLP compliance:
not specified
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 2.5 - 3.0 kg
Type of coverage:
occlusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 mL
- Concentration (if solution): undiluted
Duration of treatment / exposure:
24 hours
Observation period:
24 and 72 hours after application
Number of animals:
6 for treatment on intact skin, 6 others for abraded skin
Details on study design:
TEST SITE
- Area of exposure: hairs were clipped, 2.5 x 2.5 cm
- Type of wrap: patches are fixed to the application site by means of adhesive tape and the entire trunk of the rabbits is wrapped with an impervious material to maintain the test patches in position and to retard evaporation of volatile substances.
- Abrasions: minor incisions through the stratum corneum, but not sufficiently deep to disturb the derma or to produce bleeding

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no, only removal of patches
- Time after start of exposure: 24 hours

SCORING SYSTEM:
Draize method for reading time point 24 h
A. Erythema and eschar formation
0: No erythema
1: Very slight erythema (barely perceptible)
2: Well-defined erythema
3: Moderate to severe erythema
4: Severe erythema (beet redness) to slight eschar formation (injuries in depth)

B. Edema formation
0: No edema
1: Very slight edema (barely perceptible)
2: Slight edema (edges of area well defined by definite)
3: Moderate edema (raised approximately 1 millimeter)
4: Severe edema (raised more than 1 millimeter and extending beyond the area of exposure)

CIVO-grading system, which comprises abnormalities of scaliness and/or necrosis for reading time point 72 h
0: No reaction at all
1: Very slight scaliness
2: Distinct scaliness or very slight incrustation
3: Distinct incrustation
4: Severe incrustation

Irritation parameter:
erythema score
Basis:
mean
Time point:
24 h
Score:
2.67
Max. score:
4
Remarks on result:
other: Test item on intact skin
Irritation parameter:
edema score
Basis:
mean
Time point:
24 h
Score:
0.67
Max. score:
4
Remarks on result:
other: Test item on intact skin
Irritation parameter:
erythema score
Basis:
mean
Time point:
24 h
Score:
2.17
Max. score:
4
Remarks on result:
other: Test item on abraded skin
Irritation parameter:
edema score
Basis:
mean
Time point:
24 h
Score:
1
Max. score:
4
Remarks on result:
other: Test item on abraded skin
Irritation parameter:
other: scaliness/incrustation (CIVO-grading)
Basis:
mean
Time point:
72 h
Score:
1.8
Max. score:
4
Remarks on result:
other: Test item on intact skin
Irritation parameter:
other: scaliness/incrustation (CIVO-grading)
Basis:
mean
Time point:
72 h
Score:
1.8
Max. score:
4
Remarks on result:
other: Test item on abraded skin
Irritant / corrosive response data:
The test item caused moderate skin irritation. Its dermal effects generally consisted of very slight to well-defined erythema, very slight ischemia, focal haemorrhages, slight to distinct scaliness, slight incrustation and very slight edema.

Individual and average skin irritation scores

 

A. Intact skin

Rabbit

24 hours - erythema

24 hours - edema

72 hours - scaliness/incrustation

8803

4

1

2

8804

2

1

2

8805

4

1

2

8806

1

0

2

8807

1

0

1

8808

4

1

2

Average

2.67

0.67

1.8

 

B. Abraded skin

Rabbit

24 hours - erythema

24 hours - edema

72 hours - scaliness/incrustation

8797

4

1

2

8798

2

1

2

8799

1

1

2

8800

4

2

2

8801

1

1

1

8802

1

0

2

Average

2.17

1

1.8
Interpretation of results:
other: Category 2 (irritant) based on CLP criteria
Conclusions:
On the basis of the present results it can be concluded that the test item is a moderate skin irritant.
Executive summary:

In the current study the test item was examined for skin irritation in New Zealand White albino rabbits. No OECD test guideline was followed, however the study is similar to the Draize test. The study was not GLP.

 

The test item was used undiluted and caused moderate skin irritation. Its dermal effects generally consisted of very slight to well-defined erythema, very slight ischemia, focal haemorrhages, slight to distinct scaliness, slight incrustation and very slight edema.

According to the CLP Regulation (EC) No 1272/2008 the main criteria for the skin irritant category is that at least 2 of 3 tested animals have a mean score of >= 2.3 - =< 4.0. In the present study 6 animals were tested, thus, by extrapolation 4 of 6 tested animals should have a mean score of >= 2.3 - =< 4.0. This is not the case. Nevertheless, the mean score for erythema after contact with the test item on an intact skin was 2.67. Therefore, in the current study the test item should be classified as moderately irriting.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
13.06.2016 - 06.10.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
July 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
human
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
- Source: MatTek Corporation (82105 Bratislava, Slovakia).
- EpiOcularTM tissue: normal, human-derived epidermal keratinocytes cultured to form a stratified squamous epithelium
- Surface: 0.6 cm²
- Shipment: at 2 - 8 °C on medium-supplemented agarose gels
- Equilibration step: 15 minutes at room temperature
- Inspection: each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used.
- Pre-incubation: standard culture conditions for 1 hour and after refreshing the Assay Medium at standard culture conditions overnight (21 h in the 1st experiment, 18 h in the 2nd experiment).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
- Volume: 50 μL
- Concentration: 83.3 μL/cm2
Duration of treatment / exposure:
30 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
2
Details on study design:
Details of the test procedure used
- RhCE tissue construct used, including batch number: EpiOcular Kit Lot No.: 23714 (1st experiment) and 23737 (2nd experiment)
- Conditions of exposure: 37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH
- Washing: extensively rinsing the tissues with Ca++Mg++-free DPBS
- Number of tissue replicates used per test chemical and controls: 2
- Wavelength and band pass used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm (OD570), Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1. No reference wavelength measurement was used.
- MTT assay: incubation with 0.3 mL of MTT solution for 180 minutes at standard culture conditions, after incubation with MTT the inserts were incubated with isopropanol at 2-8°C overnight after which MTT was extracted for 2-3 hours at room temperature
- Data evaluation: the following was calculated: mean of the blank control wells (ODBlk), ODBlk from each OD value of the same experiment (blank corrected values), mean of the two aliquots for each tissue (= corrected test item OD), mean of the two relating tissues for each control and test item (= corrected mean OD) (for further calculations only the corrected mean negative control OD value was needed), corrected OD value of the negative control corresponding to 100% viability (corrected negative control OD = Negative Control OD - ODBlk = 100% Viability)
- Description of evaluation criteria: If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is non-irritant and if the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labelled irritant.
A single test composed of at least 2 tissue replicates should be sufficient for a test chemical when the result is unequivocal. In cases of borderline results, such as non- concordant replicate measurements and/or mean percent tissue viability equal to 60±5%, a second test should be considered, as well as a third one in case of discordant results between the first two tests (according to OECD 492).
- Historical data positive control: Mean Viability: 32.3%; Rel. Standard Deviation: 11.1%; Range of Viabilities: 6.90% - 43.4%; Mean Absorption: 0.566; Rel. Standard Deviation: 0.283; Range of Absorbance: 0.107- 0.943
- Historical data negative control: Mean Absorption: 1.65; Rel. Standard Deviation: 0.295; Range of Absorbance: 1.27 – 2.16
- Acceptability of the Assay: The results are acceptable if (1) The negative control OD is > 0.8 and < 2.5, (2) The mean relative viability of the positive control is below 50% of the negative control viability. (3) The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the killed controls (items and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.
Irritation parameter:
in vitro irritation score
Remarks:
% tissue viability
Run / experiment:
First experiment
Value:
61.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: borderline result
Irritation parameter:
in vitro irritation score
Remarks:
% tissue viability
Run / experiment:
Second experiment
Value:
79.5
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:
The acceptance criteria were met.

Results after treatment for 30 minutes

Dose Group

Absorbance Well 1 (Tissue 1/2)

Absorbance Well 2 (Tissue 1/2)

Mean Absorbance* (Tissue 1/2)

Mean Absorbance * Tissue 1 and 2

Mean Absorbance of 2 Tissues*

Rel. Absorbance [%] Tissue 1 and 2**

Absolute Value of the Difference of the Rel. Absorbances [%] Tissue 1 and 2

Rel. Absorbance [% of Negative Control]**

First experiment

Blank

0.039

0.040

0.039

0.000

 

 

 

 

Negative Control

2.160

2.155

2.158

2.118

2.066

102.5

5.1

100.0

2.049

2.056

2.053

2.013

97.5

Positive Control

0.579

0.574

0.577

0.538

0.721

26.0

17.7

34.9

0.955

0.931

0.943

0.904

43.7

Test Item

1.377

1.321

1.349

1.310

1.272

63.4

3.7

61.5

1.275

1.271

1.273

1.233

59.7

Second experiment

Blank

0.039

0.039

0.039

0.000

 

 

 

 

Negative Control

2.110

2.057

2.084

2.045

2.023

101.1

2.2

100.0

2.027

2.052

2.040

2.001

98.9

Positive Control

0.943

0.921

0.932

0.893

0.878

44.2

1.6

43.4

0.908

0.892

0.900

0.862

42.6

Test Item

1.624

1.571

1.597

1.558

1.607

77.0

4.9

79.5

1.732

1.659

1.695

1.657

81.9

* Mean of two replicate wells after blank correction


** Relative absorbance [rounded values]: 100 x (absorbance test item / positive control) / absorbance negative control

Interpretation of results:
other: CLP criteria not met
Conclusions:
The test item is not eye irritant.
Executive summary:

In the current study the eye irritation potential of the test item was assessed by means of the Human Cornea Model Test. The test was according to OECD 492 and GLP.

Important to mention is that the first experiment did not give a clear result, because of this a second confirmation experiment was performed.

In the pre-tests, the test item did not reduce MTT and the intrinsic colour of the substance was not intensive and did not color water or isopropanol. Therefore, additional tests with freeze-killed or viable tissues did not have to be performed.

For the main test, 50 μL of the test item, the negative control (deionised water) or the positive control (methyl acetate) was applied to tissue for 30 minutes and the test was performed in duplicate.

The absorbance values after treatment with the negative control were well within the required acceptability criterion showing the quality of the tissues, while treatment with the positive control induced a decrease below 50% compared to the negative control ensuring the validity of the test system.

The test was also valid as the difference in viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues).

Treatment with the test item did not indicate irritating effects. In the first experiment the relative mean absorption value corresponding to the viability of the tissues was 61.5%. This result lies within the borderline range of 60±5%, thus, a a second test was performed. In the second experiment the mean adsorption was 79.5%, giving a clear result.

In conclusion, in this study and under the experimental conditions reported, the test item is not eye irritant.

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

Additional information

Skin Irritation

For the skin irritation endpoint there are two studies available.

In the key study rabbits were used to assess the skin irritant potential of the test item. No OECD test guideline was followed, however, the study is similar to the Draize test. The test item was applied undiluted for 24 h under occlusive bandage and caused moderate skin irritation. The dermal effects were very slight to well-defined erythema, very slight ischemia, focal haemorrhages, slight to distinct scaliness, slight incrustation and very slight edema.

According to the CLP Regulation (EC) No 1272/2008 the main criteria for the skin irritant category is that at least 2 of 3 tested animals have a mean score of >= 2.3 - =< 4.0. The mean score for erythema was 2.67 at 24 h. Therefore, the test item should be classified as moderately irriting.

In the supporting study the irritant and/or corrosive effect(s) were tested according to the OECD 404 in an acute dermal irritation/corrosion test with 3 rabbits. The skin was exposed to the test substance at 10% in white vaseline for 4 h and animals were examined for signs of erythema and edema at 30-60 min, 24, 48 and 72 h. 

No test article-dependent findings were observed in this study.

The test item is considered to be skin irritant and will be classified accordingly.

Eye Irritation

For this endpoint there are two studies available.

In the key study the eye irritation potential of the test item was assessed by the Human Cornea Model Test according to OECD 492 and GLP. The test was performed twice as the result of the first experiment was borderline.

The test item did not reduce MTT and did not show interference with the staining materials.

50 μL of the test item, the negative control (deionised water) or the positive control (methyl acetate) was applied for 30 minutes. The negative control was well within the required acceptability criterion showing the quality of the tissues, and the positive control induced a clear decrease ensuring the validity of the test system. The test item did not indicate irritating effects. In the first experiment the relative mean absorption value was 61.5%, what lies within the borderline range of 60±5%, thus, a second test was performed. In the second experiment the mean adsorption was 79.5%, giving a clear result, and indicating that the test item is not eye irritant.

In the supporting study the eye irritant potential was assessed according to the OECD guideline 405 in 3 rabbits. 0.1 mL of the test substance at 10% in white vaseline was applied in the conjunctival sac of 1 eye and the untreated eye serving as a control. Examination was done at 1, 24, 48 and 72 h. The mean scores at 24/48/72 h were: Cornea: 0 0 0; Iris: 0 0 0; Conjunctival redness: 0 0.33 0.33; and Chemosis: 0 0 0. The substance should not be classified as eye irritant.

In conclusion the test item is not eye irritant.

Justification for classification or non-classification

Skin Irritation

In the key study similar to the Draize test, the mean score for erythema was 2.67 and based on the CLP Regulation (EC) No 1272/2008 criteria the substance is classified as moderately irriting because according to the CLP Regulation a substance is considered skin irritant category 2 when at least 2 of 3 tested animals have a mean score of >= 2.3 - =< 4.0.

Eye Irritation

In an in vitro study the mean adsorption was 79.5%, which is above the cut off value of 60 %, indicating that the test item is not eye irritant.

Additionally, in an in vivo study with 10% test substance in white vaseline the mean scores were: Cornea: 0 0 0; Iris: 0 0 0; Conjunctival redness: 0 0.33 0.33; and Chemosis 0 0 0.

According to the CLP Regulation (EC) No 1272/2008 the substance should not be classified as eye irritant because a substance is considered to be irritant to the eyes (category 2) if a substance produces at least in 2 of 3 animals, a positive response of:

- corneal opacity >= 1 and/or

- iritis >= 1 and/or- conjunctival redness >= 2 and/or

- conjunctival oedema (chemosis) >= 2

calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material, and which fully reverses within an observation period of 21 days.