Registration Dossier

Administrative data

Description of key information

Skin irritation: No irritating effects were observed in an acute dermal toxicity test. Also a human patch study testing the substance in a dilution of 20 % showed no irritating effects.  The OECD guideline 439 study showed after treatment with the test item irritating effects. The OECD 431 study showed after exposure of the tissues to the test item no corrosive effects. Both OECD guideline studies are reliable standardized test systems, testing the substance undiluted in a purity of 99.6 %. In conclusion it can be stated that the substance is irritant to the skin.
Eye irritation: One in vivo study equivalent to OECD guideline 405 showed no eye irritating effects. The substance was tested in a dilution of 20 %. The BCOP test according to OECD guideline 437 showed that the test item is not to be classified as serious eye damaging. In the OECD guideline 492 study irritating effects were observed following incubation with the test item. Under this experimental conditions tested the substance possesses an eye irritating potential.

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:
weight of evidence
Study period:
2015-05-29 to 2015-09-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline and GLP 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:
2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: Epi-200- SIT Kit Components, MTT-100 Assay Kit Components (human derived epidermal keratinocytes)
Strain:
other: not applicable
Details on test animals and environmental conditions:
Epi-200 SIT kits and MTT-100 assays diluent were purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts. EpiDerm™ tissues were shipped with cool packs on medium-supplemented agarose gels in a 24-well plate and reached Envigo CRS on June 30, 2015. On day of receipt the pre-incubation phase of the EpiDerm™ tissues started.
Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: No control animals because Human skin model. Negative and positive control were used in this test.
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 30 µL
Duration of treatment / exposure:
60 min
Observation period:
not applicable
Number of animals:
Human in vitro skin model, 3 tissues per dose
Details on study design:
Treatment
Time: 60 min
Incubator: Within this period the 6-well plates were put into the incubator for 35 min at 37 +/-1.5 °C, 5+/- 0.5 % CO2.
Rinsing: After the end of the treatment interval the inserts were removed immediately with DPBS (Dulbecco’s Phosphate Buffered Saline) at least 15 times in order to remove any residual test material.
Incubation time: 42 hours

MTT Assay
A volume of 300 µL of the MTT solution (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide) was added to each well and the plates were kept in an incubator (37 +/- 1 °C, 5 +/- 0.5% CO2) until further use.
Incubation period: 3 hours
Time: After the 42 hours incubation period was completed for all tissues and exposure groups, culture inserts were transferred from the holding plates to the MTT plates.
Rinsing: After 3 h incubation, wells were rinsed three times with DPBS (Dulbecco's Phosphate Buffered Saline) and were transferred onto new 24-well plates.
Extraction time: About 69 hours and 55 min at room temperature the formazan salt was extracted.
Extractant solution: Isopropanol

OD reading: OD was read in a microplate reader (Versamax Molecular Devices, Softmax Pro, version 4.7.1) with 570 +/- 1 nm filter. Mean values were calculated from the 3 wells per tissue.
Irritation / corrosion parameter:
other: other: Relative absorbance value compared to the relative absorbance value of the negative control
Value:
4.9
Remarks on result:
other:
Remarks:
Basis: other: test item, mean of 3 tissues. Time point: 60 min treatment. Max. score: 100.0. Reversibility: other: not examined. (migrated information)
Irritation / corrosion parameter:
other: other: Relative absorbance value compared to the relative absorbance value of the negative control
Value:
6.5
Remarks on result:
other:
Remarks:
Basis: other: positive control, mean of 3 tissues. Time point: 60 min treatment. Max. score: 100.0. Reversibility: other: not examined. (migrated information)
Irritant / corrosive response data:
The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 4.9% (threshold for irritancy: ≤ 50%), consequently the test item was irritant to skin.
Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item is irritating to skin according to EU CLP and UN GHS.
Executive summary:

This 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 (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide) 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 EpiDerm™ were treated with the test item, the negative or the positive control for 60 minutes. 30 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, Dulbecco's Phosphate Buffered Saline) 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 4.9% 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 test item is irritant 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:
weight of evidence
Study period:
2015-07-09 to 2016-01-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline and GLP study
Qualifier:
according to
Guideline:
other: OECD guideline 492
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: EpiOcular Kit Components, MTT-100 Assay Kit Components (human derived epidermal keratinocytes)
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
EpiOcular™ kits and MTT-100 assays are purchased from MatTek Corporation (Ashland, MA 01721, USA). The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts. EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. An appropriate volume of EpiOcular™ Assay Medium was warmed to approximately 37 °C. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates.
Vehicle:
unchanged (no vehicle)
Controls:
other: No control animals because Human skin model. Negative and positive control were used in this test.
Amount / concentration applied:
50 μL of the test item
Duration of treatment / exposure:
30 min
Observation period (in vivo):
Not applicable
Number of animals or in vitro replicates:
Duplicate tissues
Details on study design:
Treatment
Time: 30 min
Incubator: Incubated at standard culture conditions for 30 min
Rinsing: At the end of the 30 minutes treatment time, the test item was removed by extensively rinsing the tissues with Ca++Mg++-free DPBS (Dulbecco’s Phosphate Buffered Saline) (brought to room temperature).

MTT Assay
A volume of 300 µL of the MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide) solution was added to each 24 well plate.
Incubation period: 180 min
Time: Each insert was removed from the 24-well plate after 180 minutes.
Extraction time: To extract the MTT, the plates were placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature.
Extractant solution: Isopropanol

OD reading: OD was read in a microplate reader (Versamax Molecular Devices, Softmax Pro, version 4.7.1) with 570 +/-1 nm filter. Mean values were calculated from the 2 wells per tissue.
Irritation parameter:
other: relative absorbance (% of negative control)
Basis:
other: positive control, mean of 2 tissues
Time point:
other: 30 min
Score:
38.3
Max. score:
100
Reversibility:
other: not examined
Irritation parameter:
other: relative absorbance (% of negative control)
Basis:
other: test item, mean of 2 tissues
Time point:
other: 30 min
Score:
4.8
Max. score:
100
Reversibility:
other: not examined
Irritant / corrosive response data:
The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 4.8% (threshold for irritancy: ≤ 60%), consequently the test item was not irritant to eye.
Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item possesses an eye irritating potential.
Executive summary:

This in vitro study was performed to assess the eye irritation potential of the test item by means of the Human Cornea Model Test according to OECD guideline 492. The test item did not prove to be an MTT (3 -(4,5 -dimethylthiazole-2 -yl)-2,5 -diphenyl-tetrazoliumbromide) reducer in the MTT pre-test. Also its intrinsic colour was not intensive and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed or viable tissues did not have to be performed. Each 50 μL of the test item, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissue. After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD > 1.0 and < 2.6 thus showing the quality of the tissues. Treatment with the positive control induced a decrease below 60% compared with the negative control value in the relative absorbance thus ensuring the validity of the test system. The difference of viability between the two relating tissues of a single test item was < 20% in the same run (for positive and negative control tissues and tissues of single test items). Irritating effects were observed following incubation with test item. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues decreased below 60% (4.8%). In conclusion, it can be stated that in this study and under the experimental conditions reported, test item possesses an eye irritating potential.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A weight of evidence approach was performed. One non standard in vivo skin irritation study is available testing the substance in a probably pure quality. Also a human patch test with the test substance is available. 2 additional in-vitro skin irritation studies according to OECD guidelines were performed to support the results.

Skin Irritation

The study was carried out on male and female rats to determine the acute dermal toxicity and the skin irritation potential of the test item. The test item was applied to the skin, which was clipped. The substance was given to a scarified group and to a non-scarified group. Each group consisted of 5 males and 5 females. The substance was left on for 24 hours. The post-treatment observation period lasted 14 days. No mortality occurred. The LD50 was determined to be >15000 mg/kg bw. Also no skin irritating potential was determined. An erythema score of 0 was determined. Also the edema score was 0.

Human Patch test

In a human patch test the skin irritation potential of the test item was tested in a 20 % suspension in vaseline on 50 persons. The test area on the body was the shoulder blade. The test was performed by the patch skin test. The preparation was tested on 50 persons (21 female, 29 male). 40 persons had eczema of various kinds. Reactions were read at patch removal and again 24 hours after patch removal.Therefore the test was conducted under especially “hard” conditions. No adverse skin irritation reactions were observed.

In vitro skin irritation (OECD 439)

This 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 EpiDerm™ were treated with the test item, the negative or the positive control for 60 minutes. 30 μL of the test item were applied to each tissue, and spread to match the surface of the tissue. 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 4.9% 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 test item is irritant to skin.

In vitro skin corrosion (OECD 431)

This in vitro study was performed to assess the corrosive potential of the test item by means of the Human Skin Model Test with EpiDerm™ tissues models. 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 minutes and 1 hour, respectively. 50 μL of the test item were dispensed directly onto duplicate EpiDermTM tissue surface, and spread to match the surface of the tissue. After exposure of the tissues to the test item the relative absorbance value decreased to 78.9% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 48.9%. Both values did not exceed 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.

Conclusion

No irritating effects were observed in a non-standard acute dermal toxicity test. Also a human patch study testing the substance at 20 % in a formulation showed no irritating effects. The OECD guideline 439 study showed irritating effects. The OECD guideline 431 study showed after exposure of the tissues to the test item no corrosive effects. A purity of 99.6 % was tested in the in-vitro studies. In contrast, in human patch tests a 20 % formulation was tested. Classification of the pure substance can thus only be made based on the new in vitro studies. The in-vivo skin irritation study with rats was performed in the context of an acute dermal toxicity test and is a non-standard test. Therefore the result of this study can be used as rough indication of skin irritation. The in-vitro studies are reliable, actual and standardized test systems, testing the substance undiluted in a purity of 99.6 %. In conclusion it can be stated that the pure test substance is irritating to skin. Based on the results of the in vivo study it can be concluded, that a 20 % mixture of the substance is not irritating to the skin.

Eye Irritation

A weight of evidence approach was done for this endpoint. One in vivo eye irritation study equivalent to OECD guideline 405 was performed testing the substance in a dilution of 20 % in peanut oil. Further studies according to OECD guideline 437 and OECD guideline 492 were performed to support the result.

OECD guideline 405

In a study equivalent to OECD guideline 405 the eye irritation with 6 New Zealand rabbits was tested. The test item was applied in an amount of 0.1 mL. Only a 20 % mixture of the test substance was tested. The room temperature was kept constantly at 20 °C with the relative humidity verying between 45 and 55 %. The duration of lighting was 12 hours/day. The animals were observed over a period of 1, 2, 8 hours and 1, 2, 3, 4, 5,6 and 7 days after application. The right eye served as control. Eye irritation was evaluated according to the criteria of Draize et al.

The mean scores at 24, 48 and 72 h for all animals were as following:

Cornea: 0

Iris: 0

Conjunctivae: 0

Chemosis: 0

No eye irritation effects were observed.

OECD guideline 437

This in vitro study was performed to assess the corneal damage potential of the test item by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437. With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed. The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)). Relative to the negative control, the test item caused an increase of the corneal opacity and distinctive increase permeability. The calculated mean in vitro irritancy score was 31.63. According to OECD 437 the test item is not classified as serious eye damaging (CLP/EPA/GHS (Cat 1) but the test item’s hazard for eye damaging cannot be predicted.

OECD guideline 492

This in vitro study was performed to assess the eye irritation potential of the test item by means of the Human Cornea Model Test. Treatment with the positive control induced a decrease below 60% compared with the negative control value in the relative absorbance thus ensuring the validity of the test system. The difference of viability between the two relating tissues of a single test item was < 20% in the same run (for positive and negative control tissues and tissues of single test items). Irritating effects were observed following incubation with test item. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues decreased below 60% (4.8%). In conclusion, it can be stated that in this study and under the experimental conditions reported, test item possesses an eye irritating potential.

Conclusion

One in vivo study equivalent to OECD guideline showed no eye irritating effects testing the substance at 20 % in a formulation. In the OECD guideline 437 in-vitro study the test substance was not classified as serious eye damaging (CLP/EPA/GHS (Cat 1). In the OECD guideline 492 in-vitro study irritating effects were observed. A purity of 99.6 % was tested in the OECD guideline in-vitro studies. In contrast in the in-vivo study only a 20 % formulation was tested. Classification of the pure substance can thus only be made based on the new in vitro studies. Both OECD guideline in-vitro studies are reliable, actual and standardised test systems, testing the substance undiluted in a purity of 99.6 %. In conclusion, it can be stated that the pure test item possesses an eye irritating potential and has to be classified in cat. 2.


Justification for selection of skin irritation / corrosion endpoint:
Guideline and GLP study

Justification for selection of eye irritation endpoint:
Guideline and GLP study

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: irritating

Justification for classification or non-classification

Skin irritation

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the pure substance is considered to be classified for skin irritation cat. 2 H315 under Regulation (EC) No 1272/2008. A 20 % mixture of the test substance has not to be classified for skin irritation according to Regulation (EC) No 1272/2008.

Eye irritation

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is considered to be classified for eye irritation cat. 2 H319 under Regulation (EC) No 1272/2008.