Docosyl methacrylate

Endpoint summary

Description of key information

skin irritation /corrosion: not irritating

eye irritation: not irritating

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Vehicle:

unchanged (no vehicle)

Details on 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 ø) 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

On the day of arrival in the laboratory, the tissues will be transferred to sterile 6-well plates with 0.9 ml assay medium and preconditioned in the incubator at 37°C. After
1 hour, the pre-incubation medium is replaced with fresh medium and preconditioning
continues for 18 ± 3 hours.

Three tissues are treated with each test substance, the PC and the NC, respectively. Three additional tissues (KC) will be used for the test substance and the NC, respectively, if a positive reaction has been observed in the MTT reduction test. 25 μL sterile PBS are applied first. Due to the physical state of the test substance, application with a pipette or sharp spoon is not possible. Thus, a metal pin is covered with 50 μL undiluted liquid (at ca. 50°C heated) test substance. The pin is applied with direct
contact to the tissue, covering the whole tissue surface. Before application, the test
substance is cooled down to room temperature.
Control tissues are treated concurrently with 30 μL sterile PBS (NC, NC KC (if applicable)) or 5% SOS (PC) ortest substance (KC, if applicable). After application, a
nylon mesh is placed carefully onto each tissue surface of the NC and the PC and if
applicable, onto each tissue surface of the NC KC and the test substance KC.
The tissues are kept under the laminar flow hood at room temperature for 25 minutes and in the incubator for 35 minutes. The tissues are washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues are blotted on sterile absorbent paper and transferred into new 6-well plates pre-filled with 0.9 ml fresh medium. When all tissues are rinsed, the surface of each tissue is dried carefully with a sterile cotton swab. Subsequently, the tissues are incubated in the incubator at 37°C for 24 ± 2 hours.
After 24 ± 2 hours, the tissues are transferred into new 6-well plates pre-filled with 0.9 ml
fresh medium and placed into the incubator for an additional 18 ± 2-hour post-incubation
period. After the post-incubation period, the assay medium is replaced with 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 produced metabolically by the tissues will be extracted by incubation of the tissues in 2 ml isopropanol at room temperature on a plate shaker (ca. 120 rpm) for at least 2 hours. After shaking the isopropanol extract, 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.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

98.8

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the observed results and applying the evaluation criteria it was concluded, that Docosyl methacrylate does not show a skin irritation potential in the EpiDerm in vitro skin irritation test under the 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

Qualifier:

according to guideline

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)

GLP compliance:

yes (incl. QA statement)

Details on test animals or tissues and environmental conditions:

The EpiOcular™ 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. The EpiOcular™ tissues (surface 0.6 cm2) are cultured on cell culture inserts (MILLICELLs®, 10 mm 0) and are commercially available as kits (EpiOcular™ 200) containing 24 tissues on shipping agarose.
Tissue model: OCL-200
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Vehicle:

unchanged (no vehicle)

Details on study design:

In order to determine whether the test substance is able to reduce MTT directly, the test substance will be incubated with the substrate in a pretest (experimental conduct in accordance with GLP, but without a GLP status). 50 μL (liquid test substances) or ca. 50 μL bulk volume (solid test substances) are added to 0.9 ml MTT solution. The mixture is incubated in the dark at about 37°C for 3 hours. A negative control (50 μL deionized water) is tested concurrently. When the color of the mixture turns blue/purple, it is assumed that the test substance can reduce MTT directly. This might lead to a false optical signal if after incubation, sufficient amounts of the test substance remain in or on the skin. In case of water-insoluble substances, the coloration will be assessed at the border to the aqueous phase. In case that direct MTT reduction occurs, two freeze-killed control tissues (KC) are treated with each the test article and the negative control in the same way.
Several test substances will be tested in parallel within the present test (test no. 91) using the same control tissues (NC and PC). Two tissues are treated with each test substance, the PC and the NC, respectively. Two additional tissues (KCs) will be used for the test substance and the NC, respectively, if a positive reaction has been observed in the MTT reduction test. There are two separate protocols for liquids and solids differing in exposure time and postincubation 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 1 hour. After 1 hour, the pre-incubation medium is replaced with fresh medium, and preconditioning continues for
16 - 24 hours.
After pre-incubation, the tissues are pretreated with 20 μL PBS to wet the tissue surface. The tissues are incubated at standard culture conditions for 30 minutes.
Due to the physical state of the test substance, application with a pipette or sharp spoon is not possible. Thus, a metal pin is covered with 50 μL undiluted liquid (at ca. 50°C heated) test substance. The pin is applied with direct contact to the tissue, covering the whole tissue surface. Before application, the test substance is cooled down to room temperature. Control tissues are applied concurrently with 50 μL sterile deionized water (NC, NC KC (if applicable)) or with 50 μL methyl acetate (PC) ortest substance (KC, if applicable). After application, the tissues will be placed into the incubator until the total exposure time of 6 hours is completed.
In order to remove the test substance, the tissues are washed with sterile PBS. For this purpose, the tissues are immersed and swiveled thrice in each of three beakers filled with PBS. Washed tissues are immersed immediately into 12-well plates pre-filled with 5 ml/well pre-warmed medium (post-soak immersion) to remove residual test substance. After 25 minutes of post-soak immersion, each tissue is dried on absorbent paper and transferred to fresh 6-well plates filled with 1 ml/well pre-warmed medium.
After the incubation / post-incubation period, the assay medium is replaced with 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 produced metabolically 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 (ca. 120 rpm). After shaking the isopropanol extract, 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.

Remarks on result:

other: EpiOcular: Mean viability of the test-substance treated tissues was 81%

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results for EpiOcular Test and applying the evaluation criteria, Docosyl methacrylate does not show an eye irritation potential in the in vitro eye irritation test under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

The objective of the present in vitro method is to assess the skin irritation potential of the test substance Docosyl methacrylate using the reconstructed human epidermal model EpiDerm ™ Skin Irritation Test. The mean viability of the test substance treated tissues determined was 98.8%. Based on the observed results and applying the evaluation criteria it was concluded, that Docosyl methacrylate does not show a skin irritation potential in the EpiDerm in vitro skin irritation test under the conditions chosen (BASF SE, 2018).

In an acute dermal irritation / corrosion study according OECD 404 New Zealand White rabbits were dermally exposed to undiluted 0.5 g Propenoic acid, 2-methyl-, C11-14-isoalkyl esters, C13-rich for 4 hours under semi-occlusive conditions. Animals then were observed for 8 or 9 days. The mean erythema score (average value of the single scores (animals 1-3; erythema; intact skin, 24h, 48 h and 72 h)  was determined to be 0.33 out of 4 and, accordingly, the mean edema score 1.33 out of 4 (Evonik Röhm GmbH UNTER 89-010).

In a supporting primary dermal irritation study New Zealand White rabbits were dermally exposed (intact and scarified skin) to 0.5 mL undiluted 2-Propenoic acid, 2-methyl-, C12-18-alkyl esters for 24 hours under occlusive conditions. Animals then were observed for 72 hours. Irritation was scored by the method of Draize et al, 1959.

The mean erythema score (animals 1-6; erythema; intact skin, 24 h and 72 h) was determined to be 1.00 out of 4 and the mean edema score 0.334 out of 4. Slight formation of edema could be observed in one of the test animals within 24 and 72 hours (Evonik Oil Additives GmbH UNTER 77-003).

In a further supporting primary dermal irritation study albino rabbits were dermally exposed for 24 hours (intact and scarified skin) to 0.5 mL undiluted 2-Propenoic acid, 2-methyl-, C12-16-alkyl esters for 24 hours under occlusive conditions. Animals then were observed for 72 hours. Irritation was scored by the method of Draize et al, 1959.

The mean erythema score (animals 1-6; erythema; intact skin, 24h and 72 h) was determined to be 1.25 out of 4 and the mean edema score 0.08 out of 4. Slight formation of edema could be observed in one of the test animals within 72 hours (Evonik RohMax Additives GmbH UNTER 77-006).

Performance of the above mentioned supporting studies does not comply with requirements of the relevant recent EU and OECD guidelines, where semi-occlusive dressing, an exposure period of 4 hours, treatment of only intact skin and a recovery period of up to 14 days is stipulated. These studies are therefore of limited adequacy for C&L purposes due to intensity of the exposure regime and too short recovery periods.

Eye irritation

The objective of the present in vitro method is to assess the eye irritation potential of the test substance Docosyl methacrylate using the reconstructed human ocular model EpiOcular™ Eye Irritation Test. The mean viability of the test substance treated tissues determined was 81%. Based on the results for EpiOcular Test and applying the evaluation criteria, Docosyl methacrylate does not show an eye irritation potential in the in vitro eye irritation test under the test conditions chosen (BASF SE, 2018).

In an OECD 405 guideline study, 2-Propenoic acid, 2-methyl-, C11-14-isoalkyl esters, C13-rich (0.1 mL) were placed in the conjunctival sac of the right eye of three New Zealand White rabbits. The lids were then gently held together for one second. The test eyes were not washed out following the instillation. The left eye remained untreated for control. The eyes were examined at 1, 24, 48 and 72 hours as well as 8 days from beginning of test. Eye irritation was scored for signs of corneal damage (density, area), iris reaction and lesions of the conjunctivae (erythema, chemosis, discharge). Additionally, the cornea was examined with the aid of fluorescein after recording the observations at 24 hours. One hour after dosing, one animals exhibited slight conjunctival redness and slight discharge. A second animal exhibited slight discharge one hour after dosing. At 24 hours following dosing all signs of irritation had resolved. The remaining third animal showed no signs of irritation at any time during the test (Evonik Röhm GmbH UNTER 89-011).

In a further primary eye irritation study ( according to Appraisal of the Safety of Chemicals in foods, drugs and cosmetics, FAD Draize (1959)) 0.1 mL undiluted 2-Propenoic acid, 2-methyl-, C12-16-alkyl esters was instilled into the conjunctival sac of the left eye of 6 New Zealand White rabbits for 72 hours (not rinsed). Animals were observed for 7 days. Irritation was scored according to Draize scoring and re-evaluated according OECD-GHS criteria. An initial slight irritation of the conjunctiva within the first 8 hours (irritation scores 0-1) disappeared entirely within the first 24 h. No further irritation was observed (Evonik RohMax Additives GmbH UNTER 77-005).

Justification for classification or non-classification

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 not considered to be classified for skin irritation/corrosion or eye irritaion under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.