Dodecanoic acid, 3-[[2-(2-hydroxyethoxy)ethyl]imino]-2,2-dimethylpropyl ester

Endpoint summary

Description of key information

Sika Hardener LG was examined for its skin corrosion and ocular 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: in vitro / ex 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)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Section 4, No. 431, “In Vitro Skin Corrosion: Human Skin Model”

Version / remarks:

2004

Deviations:

no

Guideline:

other: Commission Regulation (EC) No 440/2008, Annex Part B, B.40Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EPISKIN model has been validated for corrosivity testing in an international trial, it is considered to be suitable for this study

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The EPISKIN Standard Model™ from Skinethic (France) is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum.
- Tissue batch number: 09-EKIN-013

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Each skin unit was rinsed to remove the test item by use of PBS solution (0.9 % NaCl) and returned to the plate in the culture medium.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA

- Barrier function: IC 50 = 2.1 mg/mL
- Morphology: differentiated epidermis consisting of a basal layser, several spinous and granular layers and a thick stratum corneum
- Contamination: no
- Reproducibility: proven

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE - not used

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
If the mean value is >35% and the variability is less that 50% = Non Corrosive
If the mean value is <35% and the variability is less that 50% = Corrosive

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL

NEGATIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 9 g/L saline

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration:

Duration of treatment / exposure:

4 hours at room temperature (18-28°C)

Duration of post-treatment incubation (if applicable):

none

Number of replicates:

3 disks

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1st experiment

Value:

148

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: The test to verify if the test item was capable of inducing the colour reaction produced a visible blue precipitation, which indicates that residual test item within the rinsed skin disk could have resulted in a higher OD value
- Colour interference with MTT: not tested

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro EPISKIN model test with Sika Hardener LG, the results indicate that the test item is not 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)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

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

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

GLP compliance:

yes

Species:

cattle

Strain:

not specified

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration: 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.

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
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.
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 ± 1 °C 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.

QUALITY CHECK OF THE ISOLATED CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 0.9% sodium chloride solution

POSITIVE CONTROL USED: ethanol

APPLICATION DOSE AND EXPOSURE TIME: 750 µL for 10 min

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Using a syringe to create a whirlpool effect each cornea was rinsed with fresh complete MEM
- POST-EXPOSURE INCUBATION: 120 min in MEM

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others: visual observations

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 ± 1 °C 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 492 nm (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.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
IVIS 0 - 25 = mild irritant
IVIS 25.1 -55 = moderate irritant
IVIS 55.1 and above = severe irritant

Irritation parameter:

in vitro irritation score

Run / experiment:

1st experiment

Value:

1.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

0.9

Positive controls validity:

valid

Remarks:

77.1

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

The in vitro Irritancy scores are summarised as follows:

Treatment	In Vitro Irritancy 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:

GHS criteria not met

Conclusions:

Comparison of the in vitro irritancy scores of the test substance, negative control and positive control showed that the test substance is not an eye 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. Based on the obtained results, the test item is not an eye irritant and no further tests are required.

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. Based on the obtained results, the test item is not an eye irritant and no further tests are required.

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 Regulation (EC) No 1272/2008, as amended for the fifteenth time in Regulation (EU) No 2020/1182.