Registration Dossier

Administrative data

Description of key information

Skin:
The test substance was found to be irritating to skin.
Eye:
The test substance was determined to be not eye irritating.

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:
2015-06-03 to 2015-07-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD 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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
human
Strain:
other: not applicable
Details on test animals and environmental conditions:
Test system:
Epi-200- SIT Kit (Lot No.: 21680)
MTT-100 Assay Kit
Source: both Kits MatTek Corporation (Bratislava, Slovakia)
Controls:
yes, concurrent vehicle
Amount / concentration applied:
TEST MATERIAL
- Amount applied: The applied volume of the test item was adjusted to purity, therefore 30.8 μL instead of 30 μL (47 μL/cm2 according to guideline) of the undiluted test item was dispensed directly atop the EpiDerm™ tissue and spread to match the surface of the tissue.
Duration of treatment / exposure:
60 minutes
Observation period:
The colour of the test item/water mixture was observed during the whole incubation period (60 min). The measurement of the OD of the test item in water at 570 nm was not required and consequently not performed.
Number of animals:
Three individual tissues were used for the test item and the positive control, respectively.
Details on study design:
SCORING SYSTEM:
The mean OD of the three negative control tissues was calculated after blank correction. This value corresponds to 100 % tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
Relative viability [%] = (mean OD (test item/positive control) /mean OD (negative control)) * 100
For the test item and the positive control the mean relative viability ± rel. standard deviation of the three individual tissues was calculated and used for classification according to the following prediction model:
For the current test, an irritation potential of a test item according to EU classification R38 (according to directive 67/548/EEC), H315 (according to regulation (EC) 1272/2008), and GHS category 2 according to UN GHS (published 2003, last (3rd) revision 2009) is recommended if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control.
Irritation / corrosion parameter:
other: other: relative absorbance ( % of negative control)
Value:
4.7
Remarks on result:
other:
Remarks:
Basis: other: mean of 3 tissues. Time point: 60 min. Max. score: 100.0. Reversibility: other: not examined. (migrated information)
Irritant / corrosive response data:
The mean relative absorbance after treatment with the test item was 4.7 (% of negative control). For the positive control the mean relative absorbance was found to be 6.8 (% of negative control).

Results

Dose Group

Exposure

Interval

Absorbance

570 nm

Tissue 1*

Absorbance

570 nm

Tissue 2*

Absorbance

570 nm

Tissue 3*

Mean

Absorbance

of 3 Tissues

Rel. Absor-bance [%] Tissue 1, 2 + 3**

 

Relative standard deviation

[%]

Rel.

Absorbance

[% of

Negative

Control]**

Negative control

60 min

1.751

1.579

1.844

1.724

101.5

91.5

106.9

7.8

100.0

Positive control

60 min

0.129

0.115

0.109

0.118

7.5

6.7

6.3

8.6

6.8

Test item

60 min

0.111

0.073

0.062

0.082

6.5

4.2

3.6

31.8

4.7

* Mean of three replicate wells after blank correction

** relative absorbance per tissue [rounded values]: (100x(absorbance tissue))/ mean absorbance negative control

*** relative absorbance per treatment group [rounded values]: (100x(mean absorbance test item/positive control))/ mean absorbance negative control

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was found to be irritating to skin according to UN GHS and EU CLP regulation.
Executive summary:

An in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test. The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

Each three tissues of the human skin model EpiDermTM were treated with the test item, the negative or the positive control for 60 minutes.

30.8 μL of the test item were applied to each tissue, and spread to match the surface of the tissue. 30 μL of either the negative control (DPBS) or the positive control (5 % SLS) were applied to each tissue. After treatment with the negative control the absorbance values were well in the required range of the acceptability criterion of mean OD≥0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system.

After treatment with the test item the mean relative absorbance value decreased to 4.7 % compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of ≤ 50 %. Therefore, the test item is considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the substance is irritating to skin.

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:
2015-05-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guideline and GLP.
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: bovine
Details on test animals or tissues and environmental conditions:
- Freshly isolated bovine cornea (at least 9 month old donor cattle)
- Source: Schlachthof Bensheim, 64625 Bensheim, Germany
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.75 mL
- Concentration: undiluted
Duration of treatment / exposure:
10 min
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
3 cornea per substance
Irritation parameter:
other: IVIS
Basis:
mean
Time point:
other: 10 min
Score:
0.23
Reversibility:
other: not applicable
Remarks on result:
other: BCOP test result

Results after 10 min incubation time

Test group

Opacity value = Difference (t130-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

Proposed in vitro Irritancy Score

 

 

Mean

 

Mean

 

 

 

Negative control

-1

-0.33

0.082

0.068

0.23

0.69

Not categorized

0

0.065

0.98

0

0.058

0.87

Positive control

55.33

1.348

70.55

79.37

Category 1

67.33

1.395

88.25

53.33

1.731

79.25

Test item

0.33

0

0.33

0.23

Not categorized

0.33

-0.006

0.24

0.33

-0.014

0.12

Opacity and permeability values of positive control and the test substance are shown as corrected values.

Interpretation of results:
study cannot be used for classification
Remarks:
Migrated information
Conclusions:
From the results of the in vitro BCOP study it was concluded that the test substance is not to be categorized.
Executive summary:

An in vitro study with fresh bovine cornea was conducted according to OECD 437 to investigate the eye irritation properties of the test substance. After a first opacity measurement of the fresh bovine corneae (t0), the neat test item, the positive and the negative controls were applied to corneae and incubated for 10 minutes at 32 ± 1 °C. After the incubation phase the substances were rinsed from the corneae. The corneae were incubated for another 120 minutes at 32 ± 1 °C in incubation medium, and opacity was measured a second time (t130). After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C. With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 0.69). The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (mean IVIS = 79.37) corresponding to a classification as serious eye damaging (CLP Cat 1). The test item was tested undiluted. Relative to the negative control, the test item did not cause an increase of the corneal opacity or permeability. The calculated mean IVIS was 0.23 (threshold for serious eye damage: IVIS55). According to OECD 437 the test item is not categorized.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

Four studies were considered to characterize the skin irritating properties of the test substance: Two in vitro studies using human skin models, one in vivo test with rabbits and one human patch test.

 

An in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test. Each three tissues of the human skin model EpiDermTM were treated with the test item (30.8 µL) , the negative (30 µL DPBS) or the positive control (30 µL 5% SLS) for 60 minutes. After treatment with the test item the mean relative absorbance value decreased to 4.7 % compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of ≤ 50 %. Therefore, the test item is considered to possess an irritant potential. In conclusion, it can be stated that in this study and under the experimental conditions reported, the substance is irritating to skin.

 

Another in vitro Human Skin Model Test with EpiDerm™ (according to OECD 431) was performed to assess the corrosive potential of the test substance. Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 min and 1 hour. After exposure of the tissues to the test item the relative absorbance value decreased to 93.6% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was 101.4%. Both values did not touch the threshold for corrosivity, which is defined to be < 50% after the 3 minutes exposure and < 15% after the 1 hour exposure. Therefore, the test item was not considered to be corrosive. In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item was non corrosive to skin according to EU CLP and UN GHS.

 

To assess the test substance in an in vivo skin irritation study 6 New Zealand White-rabbits were used. 0.5 mL of a 10 % solution in dipropylenglycol were applied. The exposure time of the test sample on the shorn skin (scarified and intact) was 4 hours. Reactions (erythema and edema), were noticed at 30' - 60', 24, 48 and 72 h after bandage removal. As a result, no erythema or edema formation was observed. Therefore, the test substance was considered not to be irritating to skin.

 

The test material was further tested for its potential to cause skin irritation in the human patch test. The concentration of the substance was 10 % (suspension in white petrolatum). The test area on the body was the shoulder blade. As testing patch Leukosilk was used. The time of exposure was 24 hours and skin reactions were evaluated immediately after removal of the testing patch and 24 hours later. The interpretation of the test reaction was based on the criteria of the International Contact Dermatitis Research Group. The preparation was tested on 43 persons (18 male, 25 female). 23 persons had eczema of various kinds. As a result, no skin reactions were observed and therefore the material was considered to not cause skin irritation. For details please refer to IUCLID5, section 7.10.5.

 

The in vitro tests showed that the pure (100 %) test substance has potential skin irritating properties but did not induce skin corrosion. In in vivo tests with rabbits and humans the test substance did not induce skin irritation. As both in vivo tests were conducted with a low concentration of the test substance (10 %) and the worst case scenario was considered, the test substance was established to be skin irritating based on the in vitro test results. Therefore the test substance is classified for skin irritation Category 2 (H315: Causes skin irritation).

 

 

Eye irritation:

An in vitro study with fresh bovine cornea was conducted according to OECD 437 to investigate the eye irritation properties of the test substance. With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 0.69). The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (mean IVIS = 79.37) corresponding to a classification as serious eye damaging (CLP Cat 1). The test item was tested undiluted. Relative to the negative control, the test item did not cause an increase of the corneal opacity or permeability. The calculated mean IVIS was 0.23 (threshold for serious eye damage: IVIS≥55). According to OECD 437 the test item is not categorized.

 

To assess the irritation potential of the test substance at a concentration of 10 %, 0.1 mL of the solution were instilled each in the right eye of a rabbit in an in vivo study. Six New Zealand White rabbits were used in the test. The right eye served as control. Readings of ocular reactions were made 1 h, 24 h, 48 h, 72 h after treatment. In addition the eyes were examined for potential eye lesions 24 h and 72 h p.a. by means of 1 % fluorescein-dilution (1 drop/eye). As a result, the 24, 48 and 72 h mean scores over all animals for cornea, iris, conjunctivae redness and chemosis were 0, 0, 1.2, 0.9, respectively. Therefore, the test material at the concentration of 10 % was considered to be not irritating to eyes.

 

The in vitro study with fresh bovine cornea (OECD 437) showed that the test item is not categorized as serious eye damaging (CLP Cat 1) and the irritation test with 10 % test substance showed that it is not classified as eye irritating (CLP Cat 2).

Therefore it was concluded that the test substance is not classified as damaging or irritating to the eye.

 


Justification for selection of skin irritation / corrosion endpoint:
A weight of evidence approach was done. The resulting classification is based on this study.

Justification for selection of eye irritation endpoint:
The test substance showed the same effect in in vivo and in vitro studies.

Effects on skin irritation/corrosion: irritating

Justification for classification or non-classification

Skin irritation

In a weight of evidence approach the test substance was established to be skin irritating based on the in vitro test results. Therefore the test substance is classified for skin irritation Category 2. (H315: Causes skin irritation) under Regulation (EC) No 1272/2008 as amended for the seventh time in Regulation (EC) No 2015/1221.

 

Eye irritation

The available data did not indicate an irritating property of the test substance to the eyes. Therefore the substance is not to be classified for eye irritation under Regulation (EC) No 1272/2008 (CLP) as amended for the seventh time in Regulation (EC) No 2015/1221.