Reaction mass of nonyl methacrylate and decyl methacrylate and undecyl methacrylate

Endpoint summary

Description of key information

skin irritation: not irritating

eye irritation: not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: 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:

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: 25800
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 pre-incubation medium was replaced by fresh medium and reconditioning continued for 18 ± 3 hours. Three tissues were treated with the test substance, the PC and the NC, respectively. Thirty microliters (30 μL) undiluted liquid test substance were applied by using a pipette. Control tissues were concurrently treated with 30 μL sterile PBS (NC) or with 30 μL 5% SDS (PC). A nylon mesh was carefully placed 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 fresh medium and placed into the incubator for an additional 18 ± 2-hour 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 spectrophotometrically determined. 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:

110

Test substance		tissue 1	tissue 2	tissue 3	mean	SD	CV[%]
NC	mean OD570	1.756	1.639	1.739	1.711
	viability [% of NC]	102.6	95.8	101.6	100.0	3.7	3.7
16/0068-1	mean OD570	1.914	1.837	1.896	1.882
	viability [% of NC]	111.9	107.3	110.8	110.0	2.4	2.2
PC	mean OD570	0.016	0.038	0.044	0.043
	viability [% of NC]	2.7	2.2	2.5	2.5	0.2	9.6

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results observed and by applying the evaluation criteria, it was concluded that C9-11 Methacrylate 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

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:

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: 23769
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Vehicle:

unchanged (no vehicle)

Controls:

yes

Details on study design:

In order 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 MTT solution. The mixture was incubated in the dark at about 37°C for 3 hours. A negative control (deionized 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 of direct MTT reduction, two freeze-killed control tissues were treated with the test article and the negative control each in the same way as described in the following section.
Several test substances were tested in parallel within the present test (test no. 84) by 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 pre-incubation medium was replaced by fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours. After pre-incubation, the tissues were pretreated with 20 μL PBS to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes. By using a pipette, fifty microliters (50 μL) undiluted liquid test substance were applied covering the whole tissue surface. Control tissues were concurrently applied with 50 μL sterile deionized water (NC) or with 50 μL methyl acetate (PC). After application, the tissues were placed into the incubator until the total exposure time of 30 minutes was completed. By using a pipette, fifty microliters (50 μL) undiluted liquid test substance were applied covering the whole tissue surface. Control tissues were concurrently applied with 50 μL sterile deionized water (NC) or with 50 μL methyl acetate (PC). After application, the tissues were placed into the incubator until the total exposure time of 30 minutes was completed. In order 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 pre-warmed medium (post-soak immersion) 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 overnight incubation of the tissues in isopropanol at room temperature or by incubation for at least 2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was spectrophotometrically determined. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

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

Remarks on result:

other: Mean viability of the tissues treated with the test substance was 105.3%.

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

The test substance is not able to directly reduce MTT.

The final mean viability of the tissues treated with the test substance was 105.3%.

Test substance		tissue 1	tissue 2	mean	inter-tissue variability [%]
NC	mean OD570	1.651	1.614	1.632
	viability [% of NC]	101.1	98.9	100.0	2.3
16/0068-1	mean OD570	1.798	1.639	1.719
	viability [% of NC]	110.2	100.4	105.3	9.8
PC	mean OD570	0.325	0.438	0.382
	viability [% of NC]	19.9	26.8	23.4	6.9

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results observed in the EpiOcular Test alone and by applying the evaluation criteria, it was concluded that C9-11 Methacrylate does not show an eye irritation potential in the in vitro eye irritation test strategy 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 / corrosion:

The objective was to assess the skin irritation and corrosion potential of C9-11 Methacrylate. By 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). However, in the current case for C9-11 Methacrylate the results derived with SIT alone were sufficient for a final assessment. Therefore, further testing in SCT was waived. The potential of C9-11 Methacrylate to cause dermal corrosion/irritation was assessed by a single topical application of 30 μL (irritation test) of the undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™). The irritation test was performed with three EpiDerm™ tissues which were incubated with the test substance for 1 hour followed by a 42-hour post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by 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 test substance is not able to directly reduce MTT. The final mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour with an about 42-hour post-incubation was 110%. Based on the results observed and by applying the evaluation criteria, it was concluded that C9-11 Methacrylate does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen (BASF, 2017).

Eye irritation:

The objective was to assess the eye irritating potential of C9-11 Methacrylate. By using the methods currently available 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. However, in case of C9-11 Methacrylate, the results derived with EpiOcular alone were sufficient for a final assessment. Therefore, further testing in BCOP was waived.

The potential of C9-11 Methacrylate to cause ocular irritation was assessed by a single topical application of 50 μL 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-hour post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by 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 epidermal tissues treated with the test substance 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 directly reduce MTT. The final mean viability of the tissues treated with the test substance was 105.3%. Based on the results observed in the EpiOcular Test alone and by applying the evaluation criteria, it was concluded that C9-11 Methacrylate does not show an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen (BASF, 2017).

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 of Annex VI of Regulation (EC) No 1272/2008 exists. 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).