Registration Dossier

Administrative data

Description of key information

skin: does not show a skin irritation potential (EpiDerm™ skin corrosion/irritation test) (BASF, 2014)
eye: does not show an eye irritation potential (EpiOcular) (BASF, 2014)

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:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. certificate)
Species:
other: in vitro
Strain:
other: in vitro
Vehicle:
unchanged (no vehicle)
Details on study design:
METHODE
TEST SYSTEM
The EpiDerm TM 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 multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in viva. The EpiDerm TM tissues (surface 0.6 cm2)
are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm 0) and commercially available as kits (EpiDerm TM 200), containing 24 tissues on shipping agarose.
Skin model: Epi-200
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

TEST PROCEDURE

- Irritation test: Three tissues were treated with the test substance, the PC and NC, respectively. Thirty microliter (30 μL) of the undiluted liquid test substance (after heating at ca. 30 °C) was applied using a pipette.
Control tissues were concurrently applied with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, 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 incubated in 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 6 microtiter wells filled with isopropanol for each microtiter plate.

Individual and mean OD570 values, individual and mean viability values and standard deviations

Test substance

 

tissue 1

tissue 2

tissue 3

mean

SD

NC

mean OD570

2.078

2.215

2.189

2.160

 

viability

[% of NC]

96.2

102.5

101.3

100

3.37

14/0094-1

mean OD570

2.157

2.118

2.013

2.096

 

viability

[% of NC]

99.8

98.0

93.2

97

3.43

PC

mean OD570

0.076

0.072

0.074

0.074

 

viability

[% of NC]

3.5

3.3

3.4

3

0.09

The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 97%.

Irritation test

Mean tissue viability

(% of negative control)

Prediction

50

Irritant

>50

Non-irritant

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:
other: GLP guideline study
Guideline:
other: EpiOcular
GLP compliance:
yes (incl. certificate)
Species:
other: in vitro
Strain:
other: in vitro
Vehicle:
unchanged (no vehicle)
Details on study design:
METHOD
TEST SYSTEM

The EpiOcular™ model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinozytes used to model the human corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm2)are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm Ø ) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.

EXPERIMENT AL PROCEDURE
Two tissues are treated with each test substance, the PC and NC, respectively. There are two separate protocols for liquids and solids, differing in exposure time and post-incubation period. Due to the physical condition of the test substance the protocol for solids is applied.

The tissues will be transferred to sterile 6-well plates with 1 ml pre-warmed assay medium and preconditioned in the incubator at 37°C for 16 - 24 hours. After the pre-incubation the tissues are pre-treated with 20 μl of PSS in order to wet the tissue surface. The tissues are incubated at standard culture conditions for 30 minutes.
After the incubation I postincubation period, the assay medium is replaced by 0.3 ml MTT solution and the tissues are incubated in the incubator for 3 hours. After incubation, the tissues are washed with PBS to stop the MTT-incubation.
The formazan that is metabolically produced by the tissues will be extracted by incubation of the tissues in 2 ml isopropanol at room temperature overnight or for at least 2 hours on a plate shaker (ea. 120 rpm). After shaking the isopropanol extract and piercing the tissues, 2 aliquots of each extract per tissue will be transferred to a 96-well microtiter plate. The optical density (OD570) will be determined spectrophotometrically using a filter with a wavelength of 570 nm.

Individual and mean OD570 values, individual and mean viability values and inter-tissue variability

Test substance

 

tissue 1

tissue 2

mean

inter-tissue variability [%]

NC

mean OD570

1.809

1.818

1.813

 

viability

[% of NC]

99.7

100.3

100

0.5

14/0100-1

mean OD570

1.905

1.767

1.836

 

viability

[% of NC]

105.0

97.4

101

7.6

PC

mean OD570

0.455

0.562

0.508

 

viability

[% of NC]

25.1

31.0

28

5.9

The test substance is not able to reduce MTT directly.

The mean viability of the test-substance treated tissues was 101%.

Mean tissue viability

(%of negative control)

Prediction

 

50

Irritant

>5060

no prediction*

>60

non-irritant

*At presentnoprediction isperformedif the mean relative tissue viability with a test materialis> 50 ≤ 60%as the cut off valueiscurrently being evaluated toliein this range.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

skin irritation:

The potential of C 17 Methacrylate to cause dermal irritation was assessed by a single topical application of 30 μL of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). Three EpiDerm™ tissue samples 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 both values indicates the relative tissue viability. The EpiDerm skin irritation test showed the following results, the test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 97%. Based on the observed results and applying the evaluation criteria it was concluded, that C 17 Methacrylate does not show a skin irritation potential in the EpiDerm™ skin irritation test under the test conditions chosen.[BASF, 2014]

eye irritation:

The potential of C 17 Methacrylate 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™ tissue samples 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 EpiOcular™ eye irritation test showed the following results, the test substance is not able to reduce MTT directly. The mean viability of the test-substance treated tissues was 101%. Based on the observed results and applying the evaluation criteria it was concluded, that C 17 Methacrylate does not show an eye irritation potential in the [BASF, 2014]


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

Justification for selection of eye irritation endpoint:
GLP guideline study

Justification for classification or non-classification

Based on the available studies data on skin and eye irritating properties the test item would not have to be classified and labelled according to Regulation (EC) No 1272/2008 (CLP). Nevertheless, for the group of substances (monoalkyl or monoaryl or monoalkyaryl esters of methacrylic acid) an entry in Table 3.1 and 3.2 of Annex VI of Regulation (EC) No 1272/2008 exists which has to be adopted for heptadecyl methacrylate although obtainded data show the opposite. Thus, the substance is classified as skin irrit. cat. 2 (H315, causes skin irritation), eye irrit. cat. 2 (H319, causes serious eye irritation) and as STOT SE cat. 3 (May cause respiratory irritation) according to Regulation (EC) No 1272/2008 (CLP).