2,3-dihydroxypropyl methacrylate

Endpoint summary

Description of key information

The test substance shows no skin irritation in animal studies.

The test substance shows an eye irritation potential.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

no guideline followed

GLP compliance:

not specified

Species:

rabbit

Strain:

not specified

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 62 months
- Weight at study initiation: approx. 2 kg

Type of coverage:

not specified

Preparation of test site:

shaved

Vehicle:

water

Controls:

not specified

Amount / concentration applied:

TtEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): 25 and 35 wt% in water

Duration of treatment / exposure:

twice daily for 7 days

Observation period:

7 days

Details on study design:

30 µL of the dentin primers was applied twice a day for 7 days to the shaved skin of white rabbits. Twenty min after the final application, 3 of the rabbits were sacrificed to examine possible pathological changes and the others were sacrificed after an additional 7 days without primer application in order to observe any recovery in cutaneous changes.

Remarks on result:

other: no obvious changes were recognized

Other effects:

No morphological changes were observed in the shaved skin of rabbits.

Interpretation of results:

other: CLP/EU GHS criteria are not met, no classification required according to Regulations (EC) No 1272/2008.

Conclusions:

No morphological changes were observed in the shaved skin of rabbits after repeated application of GM solution.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

Pretest for Murine Local Lymphnode Assay (LLNA)

GLP compliance:

yes (incl. QA statement)

Species:

mouse

Strain:

CBA

Vehicle:

other: methyl ethyl keton2

Details on study design:

Pretest for LLNA:
A pretest with test-substance concentrations of 5, 50 and 100% (undiluted test substance) was performed. Two animals per concentration were treated once daily for three consecutive days with the respective test substance concentrations by a topical application on the dorsum of both ears.
In the pretest, clinical signs were recorded after each application as well as on day 5. Signs of local irritation were recorded on day 1,2 and 5. Prior to the first application of the test item, (day 0), on day 2 and before sacrifice (day 5), the ear thickness was determined by using a micrometer. The ears were punched after sacrifice at the apical area by using a biopsy punch (ø 0.8 cm) and were immediately pooled per animal and weighed by using an analytical balance. The weight of the pooled lymph nodes from both sides were determined for each animal.

Remarks on result:

other: Regarding irritation/inflammation of the application site, the test substance did not elicit excessive local skin irritation/inflammation up to the tested concentration of 100% (undiluted test item).

At the tested concentrations the animals did not show relevant signs of local irritation as confirmed by the ear weights (compared to current vehicle values) and ear thickness measurements. However, an accumulation of the applied undiluted test item with concomitant erythema of the affected skin in the shoulder and neck region was observed on study day 2 and 5.  

Regarding irritation/inflammation of the application site, the test substance did not elicit excessive local skin irritation/inflammation up to the tested concentration of 100% (undiluted test item).

Interpretation of results:

other: CLP/EU GHS criteria are not met, no classification required according to Regulations (EC) No 1272/2008.

Conclusions:

Regarding irritation/inflammation of the application site, the test substance did not elicit excessive local skin irritation/inflammation up to the tested concentration of 100% (undiluted test item).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

BCOP
Isolated bovine cornea: The test system is the isolated bovine cornea. Bovine eyes are obtained as a by-product of freshly slaughtered cattle (age of the animals: minimum 12 months, maximum 60 months).
Schlachthof Mannheim, Schlachthofstr. 21, 68165 Mannheim, Germany

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Details on study design:

BCOP:
Several test substances were tested in parallel within the present test (test no. 209) using the same control corneas (NC and PC).
Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagle’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour.
After the equilibration period the medium in both chambers was replaced with fresh prewarmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that showed macroscopic tissue damage or an opacity value < 548 opacity units1 were discarded. The remaining corneas were then distributed into negative control, positive control and treatment groups. Each corneal holder was uniquely identified with a number on the chambers.
Each treatment group (test substance, NC and PC) consisted of 3 corneas. Before application, the medium in the anterior chamber was removed using a syringe. 750 μL of the undiluted liquid test substance was applied into the anterior chamber using a pipette. For the control tissues 750 μL of de-ionized water (negative control, NC) or 750 μL of 100% ethanol / 100% dimethylformamide (positive controls, PC 1 and PC 2), were applied into the anterior chamber using a pipette.
The corneas were incubated in a horizontal position at about 32 °C for approximately 10 minutes (liquids and surfactants). The test substance, NC and PC were then removed from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red).
The corneas were incubated for further 2 hours at about 32 °C. After the incubation period the medium was removed and both chambers were then refilled with fresh Eagle’s MEM.
Before measurement, each cornea was observed visually and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.
For determination of permeability the medium in the anterior chamber was replaced by 1 mL sodium fluorescein solution (4 mg/mL for liquid test substances and surfactants) and incubated for 90 ± 5 min in a horizontal position at about 32 °C.
The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was measured spectrophotometrically. Three aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined.

Controls BCOP
Negative control (NC): De-ionized water
Positive control (PC): 100% ethanol (PC 1) for liquid test substances and surfactants; 100% dimethylformamide (PC 2) was used additionally to increase the historic database of the PC.

Remarks on result:

other: The mean IVIS of the test-substance treated corneas was 27.3.

Result BCOP:

Test substance	Mean Opacity Value	Mean Permeability Value	Mean In Vitro Irritancy Score
16/0064-1	19.6	0.510	27.3
NC	5.6	0.002	5.6
PC 1	20.0	1.020	35.3
PC 2	90.0	0.670	100.1

Summary table for turnkey test strategy evaluation:

Test Method	Test Result	Test Evaluation	Evaluation Test Strategy
BCOP Test	The mean IVIS of the test-substance treated corneas was 27.3.	Not identified as corrosive or severe irritant	Ocular irritant
EpiOcular	Mean viability of the test-substance treated tissues was 5.5%.	Irritant

 

Interpretation of results:

other: not identified as corrosive or severe irritant

Conclusions:

Based on the results for BCOP and applying the evaluation criteria, GMMA was not identified as corrosive or severe irritant under the test conditions chosen.

The irritation potential of the test substance was assessed in the BCOP assay. Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 27.3. Thus, the test substance is considered to be non-corrosive, but no prediction on the irritation potential can be made and further evaluation and/or data generation is required.

Reference 2

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

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, concurrent positive control

yes, concurrent negative control

Details on study design:

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 waterphase, 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: Mean viability of the test-substance treated tissues was 5.5%.

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 5.5%.

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
16/0064-1	mean OD570	0.117	0.089	0.103
	viability [% of NC]	6.2	4.8	5.5	1.5
PC	mean OD570	0.541	0.582	0.562
	viability [% of NC]	28.9	31.1	30.0	2.2

Summary table for turn key test strategy evaluation:

Test Method	Test Result	Test Evaluation	Evaluation Test Strategy
BCOP Test	The mean IVIS of the test-substance treated corneas was 27.3.	Not identified as corrosive or severe irritant	Ocular irritant
EpiOcular	Mean viability of the test-substance treated tissues was 5.5%	Irritant

Interpretation of results:

other: irritating potential

Conclusions:

Based on the results of the EpiOcular Test and applying the evaluation criteria described, Glycerol Monomethacrylate (GMMA) shows an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

Under the conditions of the conducted test, the test substance is considered to possess an irritating potential towards human cornea in the EpiOcular™ model but the result is not conclusive with respect to classification of the test substance as eye irritant (Eye Irritant Cat. 2) or serious eye damage (Eye Damage Cat. 1) and therefore requires further evaluation and/or data generation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

The dentin primer was applied twice a day for 7 days to the shaved skin of white rabbits (30 µL). Twenty min after the final application, 3 of the rabbits were sacrificed to examine possible pathological changes and the others were sacrificed after an additional 7 days withour primer application in order to observe any recovery in cutaneous changes. No morphological changes were observed in the shaved skin of rabbits after repeated application of GM solution (Manabe et al., 1990).

In the pretest for Murine Local Lymphnode Assay (LLNA) two animals per concentration (5, 50 and 100 % of the undiluted test substance) were treated once daily for three consecutive days by a topical application on the dorsum of both ears (BASF SE, 2017c). At the tested concentrations the animals did not show relevant signs of local irritation as confirmed by the ear weights (compared to current vehicle values) and ear thickness measurements. However, an accumulation of the applied undiluted test item with concomitant erythema of the affected skin in the shoulder and neck region was observed on study day 2 and 5. Regarding irritation/inflammation of the application site, the test substance did not elicit excessive local skin irritation/inflammation up to the tested concentration of 100% (undiluted test item).

Eye irritation:

The objective was to assess the eye irritating potential of Glycerol Monomethacrylate (GMMA). 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 (BASF SE, 2017a; BASF SE, 2017b). The potential of Glycerol Monomethacrylate (GMMA) to cause ocular irritation or serious damage to the eyes 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 post-incubation period. In addition to the test substance a negative control (NC; de-ionized water) and a positive control (PC 1: 100% ethanol / PC 2: 100% dimethylformamide) were applied to three corneas each. Corneal opacity was measured quantitatively as the amount of light transmitted through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. the mean IVIS of the test-substance treated corneas was 27.3. The potential of Glycerol Monomethacrylate (GMMA) 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 testsubstance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability. 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 5.5%. Based on the results for BCOP and EpiOcular Test and applying the evaluation criteria, Glycerol Monomethacrylate (GMMA) shows an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

Justification for classification or non-classification

The available data on skin irritation/corrosion do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.

The available data on eye irritation/corrosion meet the criteria for classification as Eye Irrit. 2 (H319) according to Regulation (EC) No 1272/2008 (CLP).