Registration Dossier

Administrative data

Description of key information

Sika Hardener LG was examined for its skin corrosion and occular irritancy potential in an in vitro skin corrosion: human skin model (EPISKIN) and in Bovine Corneal Opacity and Permeability (BCOP) test, respectively. Sika Hardener LG was shown to be not corrosive in the in vitro skin irritation test and not corrosive / irritating in the in vitro eye irritation test. 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation / corrosion
Remarks:
in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-03-31 to 2009-07-13
Reliability:
1 (reliable without restriction)
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals, Section 4, No. 431, “In Vitro Skin Corrosion: Human Skin Model” adopted 13 April 2004.
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
2008
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
other: Not applicable as EPISKIN is an in vitro method, nevertheless the skin samples were of human origin.
Strain:
other: See above
Details on test animals and environmental conditions:
The EPISKIN Standard Model™ is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum.
Each reconstructed epidermis unit consists of a plastic well and a human collagen (Types III and I) matrix, representing the dermis, covered with a film of Type IV human collagen, upon which stratified differentiated epidermis derived from human keratinocytes has been laid.
The EPISKIN model has been validated for corrosivity testing in an international trial, it is considered to be suitable for this study
Type of coverage:
other: Not applicable
Preparation of test site:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: Not applicable as the test in an in vitro test. Nevertheless, it was indicated the three negative control skin units and three positive control skin units were used.
Amount / concentration applied:
Three skin units were used for each test or control material.
- 50 µL of test item was added to each of the three test skin units
- 50 µL NaCI (9 g/L saline) was added to each of the three negative control skin units
- 50 µL glacial acetic acid was added to each of the three positive control skin units
Duration of treatment / exposure:
The lid was replaced and the plate was incubated for 4 hours at room temperature (18-28°C).
Observation period:
When whole assay procedures came to an end (see below in details on study design)
Number of animals:
Not applicable
Details on study design:
At 4 hours the lid was removed and each skin unit removed and rinsed to remove the test item by use of PBS solution (0.9 % NaCl) and returned to the plate in the culture medium.
After all units were rinsed, the media was removed and remaining PBS on the surface of the skin was carefully removed.
MTT solution (2.2 ml of 0.3 mg/ml MTT) was added to each cell below the skin units. The lid was replaced and the plate incubated at room temperature (20-28°C ) for 3 hours, protected from light.
At the end of incubation with MTT a formazan extraction was undertaken.
Irritant / corrosive response data:
However, taking into account of the actual results and the physical appearance of the epidermis after incubation, it was considered that the weight of evidence indicated that the test item was not corrosive in this in vitro model.
Other effects:
No other effects

No remarks

Interpretation of results:
other: not corrosive
Remarks:
Criteria used for interpretation of results: OECD GHS
Conclusions:
In this in vitro EPISKIN model test with Sika Hardener LG, the results indicate that the test item is not a skin corrosive.
Executive summary:

Disks of EPISKIN were treated with positive control, negative control or test item and incubated for 4 hours. After rinsing, viability of each disk was assessed by use of an MTT method. Viability below 35% of the negative control is considered to indicate a corrosive effect. The positive and negative control results were found to meet the acceptability criteria. The positive control result showed zero viability and the negative control result was at least greater then the OD of the extraction solution alone. The test item did not show a reduced cell viability in comparison to the negative control. In this in vitro EPISKIN model test with Sika Hardener LG results indicated that the test item is not a skin corrosive.

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:
2009-03 to 2009-06-05
Reliability:
1 (reliable without restriction)
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: Environmental Protection Agency. 1996. Label Review Manual: 2nd Edition. EPA737-B-96-001. Washington, DC: U.S. Environmental Protection Agency
Deviations:
no
Qualifier:
according to
Guideline:
other: European Union. 2001. General Classification and Labelling Requirements for Dangerous Substances and Preparations. In Directive 2001/59/EC (28th adaption); Annex 6 of Council Directive 67/548/EEC on the approximation of the laws, regulations and administr
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes
Test material information:
Composition 1
Species:
other: Eyes from cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee and placed in Hanks’ Balanced Salt Solution (HBSS) containing the antibiotics Penicillin (100 IU/mL) and Streptomycin (100 µg/mL) and transported to the laboratory on ice packs. The eyes were used within 5 hours of slaughter.

All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed (epithelial side uppermost) in a dish containing HBSS until they were mounted in BCOP holders.
The anterior and posterior chambers of each BCOP holder were filled with fresh complete MEM and plugged. The holders were incubated at 32 ±1ºC for at least 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.
Vehicle:
other: sodium chloride
Controls:
not required
Amount / concentration applied:
For the purpose of this study the test material was prepared as a 10% v/v concentration in 0.9% w/v sodium chloride solution.
0.75 mL of the test material or control materials were applied directly to the epithelial surface of the appropriate corneas.
Duration of treatment / exposure:
The test material was applied as a 10% v/v concentration for 10 minutes followed by a post incubation period of 120 minutes with MEM (without the tets substance).
Observation period (in vivo):
The condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement.
Number of animals or in vitro replicates:
Not applicable
Details on study design:
Application of Sodium Fluorescein:
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from both chambers (anterior chamber first) was removed. The posterior chamber was refilled with fresh complete MEM and 1 mL of 4 mg/mL sodium fluorescein was applied to the anterior chamber. The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1ºC for 90 minutes ± 5 minutes.

Permeability Determinations:
After incubation all the medium in the posterior chamber of each holder was decanted using a syringe with a needle attached and thoroughly mixed in tubes, pre-labelled according to holder number, so that a representative sample was obtained for the OD492 determination.
360 µL of medium representing each cornea was applied to a designated well on a 96 well plate. Two wells were designated as blanks remained empty.These were used for blank subtraction purposes. The optical density at 492nm (OD492) was measured using the Anthos 2001 micro plate reader.
A sodium fluorescein calibration curve was performed to determine the linear range of the testing facilities microplate reader.
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: The condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement.
Score:
1.6
Reversibility:
other: not applicable
Remarks on result:
other: Regarding the maximal score, a substance that induces an IVIS (in vitro score) >= 55.1 is defined as a corrosive or severe irritant.
Irritant / corrosive response data:
Following assessment of the data for all endpoints the test material was considered to be a mild ocular irritant (see table below) and same as for the negative control.
Other effects:
No other effects

The in vitro Irritancy scores are summarised as follows:

Treatment

In VitroIrritancy Score

Classification

Test Material

1.6

Mild Irritant

Negative Control

0.9

Mild Irritant

Positive Control

77.1

Severe Irritant

As it appears in the table, the test material and the negative control scores are relatively similar and therefore were classified identically in this table. Thus, the test item is practically not irritating.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
Comparison of the in vitro irritancy scores of the test substance, negative control and positive control showed that the test substance is not a skin irritant.
Executive summary:

The study was performed to assess the ocular irritancy potential of the test substance to isolated bovine cornea. The study followed the procedures of the US Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) recommended protocol for the Bovine Corneal Opacity and Permeability (BCOP) test method. The test material was prepared as a 10% v/v concentration in 0.9% w/v sodium chloride solution. 0.75 mL of the test material or control materials were applied directly to the epithelial surface of the appropriate corneas (ones that were found at the beginning of the study to be free of damage) for 10 minutes, followed by a post incubation period of 120 minutes with MEM (without the test substance). The condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement. Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. Once the opacity and mean permeability (OD490) values have been corrected for background opacity and the negative control permeability OD490 values, the mean opacity and permeability OD490 values for each treatment group was combined in an empirically-derived formula to calculate an in vitro score (IVIS) for each treatment group. The in vitro irritancy scores for the test material (1.6) and the negative control (0.9% sodium chloride solution) (0.9) were relatively similar while the result of the positive control (ethanol) was as expected high (77.1). A substance that induces an IVIS (in vitro score) ≥ 55.1 is defined as a corrosive or severe irritant, therefore the test material was not considered to be a severe skin irritant. Results of the BCOP test can indicate of positive results - a severe skin irritant or corrosive and when the test results are negative (not skin irritating not corrosive) an in vivo study is required. As under the REACH requirements for the test substance tonnage (REACH section 8.2 column 2) only an in vitro study for eye irritation is required, the study conclusion defined the test substance as a mild irritant although it is practically not irritant (see classification and scores of of test substance and negative control in the table of remarks on results in IUCLID 5 7.3.2)) thereby avoiding the possibility of testing Sika Hardener LG in an in vivo eye irritation study which is anyway not required under REACH.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation / corrosion

Sika Hardener LG was examined for its skin corrosion effects in an in vitro skin corrosion: human skin model according to the EU method B.40 and OECD guideline no. 431. Disks of EPISKIN were treated with positive control (glacial acid), negative control (saline) or test item (50 µL per skin unit) and incubated for 4 hours. After rinsing, viability of each disk was assessed by use of an MTT method. Viability below 35% of the negative control was considered to indicate a corrosive effect.

The positive and negative control results were found to meet the acceptability criteria. The positive control result showed zero viability and the negative control result was at least greater then the OD of the extraction solution alone. The test item did not show a reduced cell viability in comparison to the negative control. Thus, results of this in vitro EPISKIN model test with Sika Hardener LG indicated that the test item was not a skin corrosive.

Eye irritation / corrosion

The study was performed to assess the ocular irritancy potential of the test substance to isolated bovine cornea. The study followed the procedures of the US Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) recommended protocol for the Bovine Corneal Opacity and Permeability (BCOP) test method. The test material was prepared as a 10% v/v concentration in 0.9% w/v sodium chloride solution. 0.75 mL of the test material or control materials were applied directly to the epithelial surface of the appropriate corneas (ones that were found at the beginning of the study to be free of damage) for 10 minutes, followed by a post incubation period of 120 minutes with MEM (without the test substance). The condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement. Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. Once the opacity and mean permeability (OD490) values have been corrected for background opacity and the negative control permeability OD490 values, the mean opacity and permeability OD490 values for each treatment group was combined in an empirically-derived formula to calculate an in vitro score (IVIS) for each treatment group. The in vitro irritancy scores for the test material (1.6) and the negative control (0.9% sodium chloride solution) (0.9) were relatively similar while the result of the positive control (ethanol) was as expected high (77.1). A substance that induces an IVIS (in vitro score) ≥ 55.1 is defined as a corrosive or severe irritant, therefore the test material was not considered to be a severe skin irritant. Results of the BCOP test can indicate of positive results - a severe skin irritant or corrosive and when the test results are negative (not skin irritating not corrosive) an in vivo study is required. As under the REACH requirements for the test substance tonnage (REACH section 8.2 column 2) only an in vitro study for eye irrittaion is required, the study conclusion defined the test substance as a mild irritant although it is practically not irritant (see classification and scores of of test substance and negative control in the table of reamrks on results in IUCLID 5 7.3.2)) thereby avoiding the possibility of testing Sika Hardener LG in an in vivo eye irritation study which is anyway not required under REACH.


Justification for selection of skin irritation / corrosion endpoint:
Only one study available.

Justification for selection of eye irritation endpoint:
Only one study available.

Justification for classification or non-classification

Based on the results of corrosion / irritation studies, Sika Hardener LG was not classified and labelled for skin or eye irritation /corrosion, according to Directive 67/548/EEC (DSD) and Regulation 1272/2008EC (CLP).