N,N'-methylenebis[methacrylamide]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-03-14 to 2017-03-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

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)

Version / remarks:

adopted July 26, 2013

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

December 09, 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-methylenebis[methacrylamide]

EC Number:

219-102-3

EC Name:

N,N'-methylenebis[methacrylamide]

Cas Number:

2359-15-1

Molecular formula:

C9H14N2O2

IUPAC Name:

N,N'-methylenebis(2-methylacrylamide)

Test material form:

solid

Specific details on test material used for the study:

The test item was suspended in a 0.9 % sodium chloride solution with a final concentration of 20n% (w/v).

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

not applicable

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL
- Concentration: 20 % (w/v)
VEHICLE
- Amount applied: 750 µL
- Concentration: 0.9 %
- Lot/batch no.: Batch no. 163548002; B. Braun Melsungen AG, 34212 Melsungen, Germany

Duration of treatment / exposure:

240 min

Number of animals or in vitro replicates:

3 corneas each

Details on study design:

SELECTION AND PREPARATION OF CORNEAS, QUALITY CHECK OF THE ISOLATED CORNEAS: For a detailed description please see section “any other information on materials and methods incl. tables”.
NUMBER OF REPLICATES: 3
NEGATIVE CONTROL USED: yes, 0.9 % sodium chloride solution (identical to solvent control).
POSITIVE CONTROL USED: yes, 20 % Imidazole in 0.9 % sodium chloride solution
APPLICATION DOSE AND EXPOSURE TIME: 750 µL of a 20 % (w/v) solution; 240 min
TREATMENT METHOD: open chamber
REMOVAL OF TEST SUBSTANCE: for a detailed description please see section “any other information on materials and methods incl. tables”.
- Number of washing steps after exposure period: at least three times.
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (OD490)
- For a detailed description please see section “any other information on materials and methods incl. tables”.
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
DECISION CRITERIA: for a detailed description please see section “any other information on materials and methods incl. tables”.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Test item

Value:

ca. -1.078

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not reported
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for solvent control: yes. The IVIS of the solvent control was 0.076.
- Acceptance criteria met for positive control: yes. The IVIS of the positive control was 79.071.

Any other information on results incl. tables

RESULTS

Corneal opacity was measured quantitatively as the amount of light transmission through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea, as detected in the medium in the posterior chamber. The measurements were used to calculate an in vitro irritancy score (IVIS), which was used to assign an in vitro irritancy hazard classification category for prediction of the in vivo ocular irritation potential of the test item.

Three corneas were used for each treatment group (test item, negative control and positive control). The corneas treated with the negative control item 0.9 % sodium chloride solution revealed a mean opacity value of -0.079 ± 0.890 and a mean permeability value of 0.010 ± 0.001. The calculated IVIS value of 0.076 ± 0.884 was well below the cut-off value of 3 (UN GHS no category).

The corneas treated with the positive control item 20 % Imidazole in 0.9 % NaCl solution revealed a mean opacity value of 59.176 ± 12.740 and a mean permeability value of 1.326 ± 0.107 compared to the solvent control. The calculated IVIS value of 79.071± 14.007 was within two standard deviations of the current historical mean and well above the cut-off value of 55. Hence, the acceptance criteria for the test were fulfilled.

Following treatment with the test item, a mean opacity of -1.183 ± 0.709 and a mean permeability value of 0.007 ± 0.004 compared to the negative control were determined. The calculated IVIS of -1.078 ± 0.773 is below the cut-off value of 3 (UN GHS no category). Hence, the test item did not show severely irritant or corrosive properties and consequently it is not classified as a severe irritant and is not corrosive according to UN GHS classification.

The opacity and permeability values are given in Tables 1 and 2. The IVIS values are given in Table 3.

Table 1: Opacity values

	Cornea No.	Opacity [opacity units]	Corrected opacity
				Mean of group	Standard deviation
0.9 % NaCl	1	0.917	-0.079	-0.079	0.890
	2	-0.797
	3	-0.358
20 % Imidazole	4	73.505	73.584	59.176	12.740
	5	49.323	49.402
	6	54.463	54.542
Test item (20 % w/v)	7	-0.637	-0.558	-1.183	0.709
	8	-1.116	-1.037
	9	-2.032	-1.953

 

Table 2: Permeability OD Values (490 nm)

	Cornea No.	Permeability [OD], mean of triplicates	Corrected permeability per cornea	Standard deviation per cornea	Corrected permeability per group (mean)	Standard deviation per group
0.9 % NaCl	1	0.010	0.010	0.002	0.010	0.001
	2	0.011	0.011	0.001
	3	0.010	0.010	0.002
20 % Imidazole	4	1.413	1.403	0.032	1.326	0.107
	5	1.214	1.204	0.020
	6	1.382	1.372	0.006
Test item (20 % w/v)	7	0.020	0.010	0.001	0.007	0.004
	8	0.019	0.009	0.003
	9	0.012	0.002	0.002

 

Table 3: In vitro irritancy score (IVIS)

	Cornea No.	Opacity	Permeability	IVIS
				Per cornea	Per group
					Mean	SD
0.9 % NaCl	1	0.917	0.010	1.067	0.076	0.884
	2	-0.797	0.011	-0.632
	3	-0.358	0.010	-0.208
20 % Imidazole	4	73.584	1.403	94.629	79.071	14.007
	5	49.402	1.204	67.462
	6	54.542	1.372	75.122
Test item (20 % w/v)	7	-0.558	0.010	-0.408	-1.078	0.773
	8	-1.037	0.009	-0.902
	9	-1.953	0.002	-1.923

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the present test conditions, the test substance tested in the in vitro BCOP test method, had an IVIS value of -1.078 ± 0.773, which is below the cut-off value of 3 (EU GHS no category). The test item is neither classified as a severe irritant nor as corrosive according to EU GHS classification. Therefore, GHS criteria are not met.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of N,N´-methylenebis[methacrylamide] by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted July 2013 and EU method B.47, December 2010.

The corneae were incubated with the test substance and controls for 240 min. The test was performed in triplicates. Opacity and permeability were determined. The in vitro irritancy score (IVIS) was calculated as mean opacity value + (15 x mean OD₄₉₀ value. A substance that induces an IVIS ≥ 55 is defined as a corrosive or severe irritant while a substance that induces an IVIS ≤ 3 refers to EU GHS “no category”.

The corneas treated with the negative control item 0.9 % sodium chloride solution revealed a calculated IVIS value of 0.076 ± 0.884 which was well below the cut-off value of 3 (EU GHS no category).

N,N´-methylenebis[methacrylamide] caused no increase of the corneal opacity and permeability. Under the present test conditions, N,N´-methylenebis[methacrylamide] had an IVIS value of -1.078 ± 0.773, which is below the cut-off value of 3 (EU GHS no category).

The corneas treated with the positive control item 20 % Imidazole revealed a calculated IVIS value of 79.071 ± 14.007. This was within two standard deviations of the current historical mean and well above the cut-off value of 55. Hence, the acceptance criteria for the test were fulfilled.

Under the present test conditions, N,N´-methylenebis[methacrylamide] it is not classified as a severe irritant and is not corrosive according to EU GHS classification.