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Administrative data

Description of key information

The test substance does not show a skin irritation potential.

The test substance shows an eye irritation 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:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Test system:
human skin model
Vehicle:
unchanged (no vehicle)
Details on test system:
Three dimensional human epidermis model
The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm ø) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
Tissue model: EPI-200
Tissue Lot Number: 23343 (Certificate of Analysis see appendix)
Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Irritation test:
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours. Three tissues were treated with the test substance, the PC and NC, respectively. Thirty microliter (30 μL) of the undiluted liquid test substance was applied using a pipette. Control tissues were concurrently treated with 30 μL of sterile PBS (NC) or with 30 μL of 5% SDS (PC). A nylon mesh was placed carefully onto the tissue surface afterwards. The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator. The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were placed into the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period. After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
irritation test
Value:
75.9

Irritation Test

Test substance

 

tissue 1

tissue 2

tissue 3

mean

SD

CV[%]

NC

mean OD570

2.268

2.194

2.033

2.165

 

 

viability

[% of NC]

104.7

101.3

93.9

100.0

5.5

5.5

16-0065-1

mean OD570

1.441

1.954

1.535

1.643

 

 

viability

[% of NC]

66.6

90.3

70.9

75.9

12.6

16.6

PC

mean OD570

0.052

0.053

0.051

0.052

 

 

viability

[% of NC]

2.4

2.4

2.4

2.4

0.0

1.5

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the observed results and applying the evaluation criteria it was concluded, that Isopropylidenglycerolmethacylate (IPGMA) does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.
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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Details on test animals or tissues and environmental conditions:
EpiOcular
The EpiOcularTM model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinocytes used to model the human corneal epithelium (compare Figure 1). The EpiOcularTM tissues (surface 0.6 cm²) are cultured on cell culture inserts (MILLICELLs®, 10 mm ø) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.
Tissue model: OCL-200
Tissue Lot Number: 23718 (Certificates of Analysis see appendix)
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
Vehicle:
unchanged (no vehicle)
Controls:
yes
Details on study design:
EpiOcular:
To assess the ability of the test material to directly reduce MTT a pretest (experimental conduct in accordance with GLP but without a GLP status) was performed. The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 3 hours. A negative control (de-ionized water) was tested concurrently. If the MTT solution color or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT. The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results. In case where direct MTT reduction occurred, two freeze-killed control tissues each were treated with the test article and the negative control, in the same way as described in the following section.
Several test substances were tested in parallel within the present test (test no. 77) using the same control tissues (NC and PC). Two tissues were treated with each, the test substance, the PC and the NC. There are two separate protocols for liquids and solids, differing in exposure time and postincubation period. Due to the physical state of the test substance the protocol for liquids was applied. On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours. After the pre-incubation, the tissues were pre-treated with 20 μL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes. Using a pipette, fifty microliter (50 μL) of the undiluted liquid test substance was applied covering the whole tissue surface. Control tissues were concurrently applied with 50 μL of sterile de-ionized water (NC) or with 50 μL of methyl acetate (PC). After application, the tissues were placed into the incubator until the total exposure time of 30 minutes was completed. To remove the test substance, the tissues were washed with sterile PBS. For this purpose the tissues were immersed and swiveled three times in each of three beakers filled with PBS. Washed tissues were immediately immersed into 12-well plates, pre-filled with 5 mL/well prewarmed medium (post-soak immersion) in order to remove residual test substance. After 12 minutes of post-soak immersion, each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium. Subsequently, the tissues were incubated at standard culture conditions for 2 hours (postincubation period). After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol at room temperature overnight or for at least 2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

Negative control (NC): De-ionized water, sterile
Positive control (PC): Neat methyl acetate (CAS No.: 79-20-9)

Remarks on result:
other: EpiOcular: Mean viability of the test-substance treated tissues was 49.9%.

The following results were obtained in the EpiOcular™ eye irritation assay:

The test substance is not able to reduce MTT directly.

The mean viability of the test-substance treated tissues was 49.9%.

Test substance

 

tissue 1

tissue 2

mean

inter-tissue variability [%]

NC

mean OD570

1.838

1.909

1.873

 

viability

[% of NC]

98.1

101.9

100.0

3.8

16/0065-1

mean OD570

0.926

0.944

0.935

 

viability

[% of NC]

49.4

50.4

49.9

1.0

PC

mean OD570

0.541

0.582

0.562

 

viability

[% of NC]

28.9

31.1

30.0

2.2

Summary of the individual test results of the in vitro eye irritation turnkey testing strategy:

Test Method

Test Result

Test Evaluation

Evaluation Test Strategy

BCOP Test

The mean IVIS of the test-substance treated corneas was 0.0

Not identified as corrosive or severe irritant

 

 

Ocular irritant

EpiOcular

Mean viability of the test-substance treated tissues was 49.9%

 

Irritant

 

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
Based on the results for EpiOcular Test and applying the evaluation criteria, Isopropylidenglycerolmethacrylate (IPGMA) shows an eye irritation potential in the in vitro eye irritation test under the test conditions chosen.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation (in vitro):

The objective was to assess the skin irritation and corrosion potential of Isopropylidenglycerolmethacrylate (IPGMA). Using the currently available methods a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential.

Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT). The potential of Isopropylidenglycerolmethacrylate (IPGMA) to cause dermal corrosion/irritation was assessed by a single topical application of 50 μL (corrosion test) or 30 μL (irritation test) of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). For the corrosion test two EpiDerm™ tissues were incubated with the test substance for 3 minutes and 1 hour, each. The irritation test was performed with three EpiDerm™ tissues, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test-substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The following results were obtained in the EpiDerm™ skin corrosion/irritation test: The test substance is not able to reduce MTT directly.

Corrosion test: The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 108.9%, and it was 99.7% after an exposure period of 1 hour.

Irritation test: The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 75.9%.

Based on the observed results and applying the evaluation criteria it was concluded, that Isopropylidenglycerolmethacrylate (IPGMA) does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

Eye irritation (in vitro):

The objective was to assess the eye irritating potential of Isopropylidenglycerolmethacrylate (IPGMA). Using the currently available methods a single in vitro assay is not sufficient to cover the full range of eye irritating potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test.

The potential of Isopropylidenglycerolmethacrylate (IPGMA) to cause ocular irritation or serious damage to the eyes (BCOP) was assessed by a single topical application of 750 μL of the undiluted test substance to the epithelial surface of isolated bovine corneas. Three corneas were treated with the test substance for 10 minutes followed by a 2-hours postincubation period. In addition to the test substance a negative control (NC; de-ionized water) and positive controls (PC1, 100% ethanol and PC2, 100% dimethylformamide) were applied to three corneas each. The mean IVIS of the test-substance treated corneas was 0.0.

The potential of Isopropylidenglycerolmethacrylate (IPGMA) to cause ocular irritation was assessed by a single topical application of 50 μL of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcular™). Two EpiOcular™ tissues were incubated with the test substance for 30 minutes followed by a 2-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test-substance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability. The test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues was 49.9%.

Based on the results for BCOP and EpiOcular Test and applying the evaluation criteria Isopropylidenglycerolmethacrylate (IPGMA) shows an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

Justification for classification or non-classification

Based on the results of the skin and eye irritation testing, the test item is classified as eye irritating cat. 2 (H319) according to Regulation (EC) No 1272/2008 (CLP).