[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471 guideline study, Kr.2): negative with and without metabolic activation in S. typhimurium TA1535, TA1537, TA100 and TA98 strains and in E. coli WP2 uvrA strain.

In vitro chromosome aberrations study (GLP OECD 473 guideline study, Kr.2): positive without metabolic activation, negative with metabolic activation in Chinese Hamster Lung fibroblasts.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was performed similarly to the OECD 471 Guideline with acceptable restrictions.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

: no certificate of analysis, no historical control data, no true assessment of the cytotoxicity, only 2 plates/concentration, only the pre-incubation method is used, no indication on the precipitation.

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene for S. typhimurium strain, Trptophan for E. coli strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: additional mutations in rfa and uvrB genes. For the strains TA98, TA100 and WP2 uvrA presence of an additional plasmid pKM101 in order to enhance their sensitivity of detection of some mutagens. See Table 7.6.1/1

Metabolic activation:

with and without

Metabolic activation system:

Self-produced S9 fraction by induction of male SD rats' liver with administration of PB and 5,6-BF directly to stomach

Test concentrations with justification for top dose:

The amount used for the test was calculated as concentration of N-[2-(2-oxo-1-imidazolidinyl)ethyl]methacrylamide.
preliminary study: 0; 50; 100; 200; 500; 1000; 2000 and 5000 µg/plate
Main test: 313; 625; 1250; 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 2-(2-furyl)-3(5-nitro-2-furyl)acrylamide (AF-2); 2-methoxy-6-chloro-9-[3-(2-chloroehyl)-aminopropylamino]acridine.2HCl (ICR-191) and 2-aminoanthracene (2AA)

Remarks:

see details in table 7.6.1/2

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period:20 min
- Exposure duration: 48h
- Expression time (cells in growth medium): 48h
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: 2 plates(triplicate for the negative control), 2 independant experiments

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: no data

Evaluation criteria:

no data

Statistics:

no data

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See details in Table 7.6.1/3 to 7.6.1/6

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

As there was no decrease in the number of revertant, the test item can be considered as non cytotoxic.

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See details in Table 7.6.1/3 to 7.6.1/6

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

As there was no decrease in the number of revertant, the test item can be considered as non cytotoxic.

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See details in Table 7.6.1/3 to 7.6.1/6

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

As there was no decrease in the number of revertant, the test item can be considered as non cytotoxic.

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See details in Table 7.6.1/3 to 7.6.1/6

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

As there was no decrease in the number of revertant, the test item can be considered as non cytotoxic.

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See details in Table 7.6.1/3 to 7.6.1/6

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

As there was no decrease in the number of revertant, the test item can be considered as non cytotoxic.

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: > 50%
no other data

RANGE-FINDING/SCREENING STUDIES:the range-finding study was performed in the same conditions as the main study, the number of revertants was calculated by the mean of the 3 replicate plates perfomed for each concentration of test item. The range of concentration of the preliminary study is broader than in the main study. Thus, this preliminary can be considered as a mutagenic experiment as such.

COMPARISON WITH HISTORICAL CONTROL DATA: no historical control data

ADDITIONAL INFORMATION ON CYTOTOXICITY: no data

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 7.6.1/3:Number of revertants per plate in the absence of metabolic activation in the first test

Test item Concentration (µg/plate)	TA 1535	TA1537	TA 98	TA 100	WP2 uvrA
	Mean$	Mean$	Mean$	Mean$	Mean$
0	14	12	25	139	37
50	15	15	19	142	47
100	15	9	28	155	40
200	10	7	21	127	38
500	15	9	21	151	52
1000	8	9	24	151	47
2000	12	11	25	144	38
5000	11	8	20	138	53
Positive control**	259	1685	513	916	458

$: Mean of triplicate

**Mutagens positive controls:

- NaN₃(0.5 µg/plate) in TA1535 strain

- ICR-191 (1 µg/plate) in TA1537 strain

- AF-2 (0.01 µg/plate) in TA 100 and WP2 uvrA strains

- AF-2 (0.1 µg/plate) in TA 98 strain

 

 

Table 7.6.1/4: Number of revertants per plate in the presence of metabolic activation (S9 mix) in the first test 

Test item Concentration (µg/plate)	TA 1535	TA1537	TA 98	TA 100	WP2 uvrA
	Mean$	Mean$	Mean$	Mean$	Mean$
0	13	17	36	144	50
50	9	19	37	144	52
100	14	18	34	161	54
200	9	20	36	129	49
500	13	15	40	145	54
1000	11	15	43	143	61
2000	15	21	30	147	61
5000	14	26	47	203	58
Positive control**	202	172	200	948	547

$: Mean of triplicate

**Mutagens positive controls:

- 2AA (2 µg/plate) in TA1537 and TA1535 strains

- 2AA (1 µg/plate) in TA100 strain

- 2AA (0.5 µg/plate) in TA98 strain

- 2AA (10 µg/plate) in WP2 uvrA strain

 

Table 7.6.1/5: Number of revertants per plate in the absence of metabolic activation in the second test

Test item Concentration (µg/plate)	TA 1535	TA1537	TA 98	TA 100	WP2 uvrA
	Mean$	Mean$	Mean$	Mean$	Mean$
0	13	7	23	110	36
313	9	8	28	124	43
625	12	8	31	131	34
1250	10	11	20	123	39
2500	16	13	16	122	42
5000	12	11	27	130	42
Positive control**	257	1414	488	511	510

$: Mean of triplicate

**Mutagens positive controls:

- NaN₃(0.5 µg/plate) in TA1535 strain

- ICR-191 (1 µg/plate) in TA1537 strain

- AF-2 (0.01 µg/plate) in TA 100 and WP2 uvrA strains

- AF-2 (0.1 µg/plate) in TA 98 strain

 

Table 7.6.1/6: Number of revertants per plate in the presence of metabolic activation (S9 mix) in the second test

 

Test item Concentration (µg/plate)	TA 1535	TA1537	TA 98	TA 100	WP2 uvrA
	Mean$	Mean$	Mean$	Mean$	Mean$
0	11	20	38	122	43
313	12	17	26	131	47
625	14	18	29	142	53
1250	12	12	29	133	40
2500	12	16	44	143	39
5000	16	19	46	147	51
Positive control**	121	89	232	587	352

$: Mean of triplicate

**Mutagens positive controls:

- 2AA (2 µg/plate) in TA1537 and TA1535 strains

- 2AA (1 µg/plate) in TA100 strain

- 2AA (0.5 µg/plate) in TA98 strain

- 2AA (10 µg/plate) in WP2 uvrA strain

Conclusions:

Interpretation of results (migrated information):
negative

Under the test conditions, Reaction mass of methacrylic acid and N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide did not show any mutagenic activity in the bacterial reverse mutation test using Salmonella typhimurium and E. coli.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD No.471 guideline, Reaction mass of methacrylic acid and N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide diluted in water was tested in S. typhimurium TA1535, TA1537, TA100, TA98 and E. coli WP2 uvrA in the presence and the absence of mammalian metabolic activation (S9) using the preincubation method. The amount used for the test was calculated as concentration of N-[2-(2-oxo-1-imidazolidinyl)ethyl]methacrylamide (48.61%). Six known mutagens (Sodium azide; 2-(2-furyl)-3(5-nitro-2-furyl)acrylamide (AF-2); 2-methoxy-6-chloro-9-[3-(2-chloroehyl)-aminopropylamino]acridine.2HCl (ICR-191) and 2-aminoanthracene (2AA)) were used to check the sensitivity of the test system. They gave appropriate response, therefore the test was considered as valid.

In the first assay the test item was used at the following concentration: 0; 50; 100; 200; 500; 1000; 2000 and 5000 µg/plate (duplicate). There was a slight increase in the number of revertants in the TA98, TA100 and TA1537 strains with S9 mix at the highest dose, but this increase did not exceed twice the negative control (<1.5). Therefore it was not considered as relevant regarding the mutagenicity. For the other conditions, there was no increase in the number of revertants compared to the negative control.

In the second assay the test item was used at the following concentration: 313; 625; 1250; 2500 and 5000 µg/plate (duplicate). There was a slight increase in the number of revertants in the TA1537 without S9 mix at 2500 µg/plate, but the mean revertants did not exceed twice the mean obtained for the negative control and there was no dose-response relationship. Therefore it was not considered as relevant regarding the mutagenicity. For the other conditions, there was no increase in the number of revertants compared to the negative control.

As there was no decrease in the number of revertants compared to the negative control, the test item can be considered as non cytotoxic up to the recommended limit concentration of 5000 µg/plate.

Under the test conditions, Reaction mass of methacrylic acid and N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide did not show any mutagenic activity in the bacterial reverse mutation test using Salmonella typhimurium and E. coli. This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 471.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 20 October to 24 December 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was performed in compliance with the GLP and similarly to the OECD 473 Guideline with acceptable restrictions.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

: no certificate of analysis, no historical control data, no data on the precipitation, no assessment of the cytotoxicity in the main experiment, only one experiment was performed for the condition with the metabolic activation.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

mammalian cell line, other: Chinese Hamster Lung Fibroblast cells (CHL, Clone number 11)

Details on mammalian cell type (if applicable):

- Type and identity of media: eagle MEM medium (Nissui Pharmaceutical Corporation, Lot N° 318305). Newborn calf serum was added in the medium at 10%.
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction produced by induction of the rat Liver by an intraperitoneal injection of Phenobarbital and 5.6 Benzo-flavone.

Test concentrations with justification for top dose:

The amount used for the test was calculated as concentration of N-[2-(2-oxo-1-imidazolidinyl)ethyl]methacrylamid.
- S9 mix: 24h and 48 h treatments: 300; 450 and 600 µg/mL
+ S9 mix: 500; 1000 and 2000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water (distilled water purified)
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

pure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

MMC : Batch N° 876ABJ / CPA: Batch N° WDL7611

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: -S9 mix: 24 or 48h; +S9 mix: 6h
- Expression time (cells in growth medium): -S9 mix: 24 or 48h; +S9 mix: 24h
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): -S9 mix: 24h or 48h; +S9 mix: 24h

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): Colcemid added two hours prior to the completion of the culture
STAIN (for cytogenetic assays): Methanol and acetate liquid (3:1)

NUMBER OF REPLICATIONS: 2 slides per one Petri dish, 2 Petri dishes per concentration

NUMBER OF CELLS EVALUATED: 100 cells per Petri dish (i.e. 200 cells per concentration)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no

Evaluation criteria:

When the cells showing structural and numerical chromosome aberrations including gaps that have a frequency rate of less than 5 % (average value of two Petri dishes) the result is negative (-), over 5% yet less than 10% it is doubtful positive [ambiguous] (±), over 10% yet under 20% it is positive (+), over 20% yet under 50% it is positive (++), over 50% it is positive (+++). For dose groups showing results positive (+) or more, the frequency of the aberrant cells is considered to be a positive for the induction of chromosome aberrations when the result is dose-dependent and reproducible. However, in the event that the test results are doubtful positive [ambiguous], the test is repeated when it is positive only at a single concentration.

Statistics:

not used

Species / strain:

mammalian cell line, other: Chinese Hamster Lung Fibroblast cells (CHL, Clone number 11)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

mammalian cell line, other: Chinese Hamster Lung Fibroblast cells (CHL, Clone number 11)

Metabolic activation:

with

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
no data

RANGE-FINDING/SCREENING STUDIES: The concentration of the test substance causing 50 % cytostatis is approximately 570 µg/ml at 24 hour treatment with the direct method, it is approximately 350 µg/ml at 48 hours of treatment with the direct method, and for the metabolic activation method with concentrations below 2000 µg/ml (corresponding to 10 mM) the cell toxicity was too weak to be observed.The maximum concentration of the test substance that was determined to provide a sufficient number of cells when conducting the chromosome aberration test was 600 µg/ml at 24 and 48 hours with direct treatment and was 2000µg/ml under the metabolic activation method. See details in Table 7.6.1/1.

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: the cytotoxicity was not assessed during the main test.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The test has been determined to have been conducted normally due to the fact that each value taken from the two Petri dishes were not dispersed, the frequency in which the cell having aberrations in the negative contrast group was less than 5%, the frequency in which the aberration cell appears other than in the positive control group gap was over 10%, and that aberrations were not found in the control culture medium.

Table 7.6.1/1: Cytostasis Examination and Cell Proliferation Control Test Results: Range-finding study

 

Direct Method	24 Hour treatment	Test substance concentration (µg/ml)	0	100	300	400	500	600	800	1000	1500	2000
		Mitotic Index (%)*	100	83.3	64.5	54.4	53.5	49.1	46.5	35.5	18.4	3.5
		Mitotic cells Appearance	+++	+++	+++	++	++	+	±	±	-	-
	48 Hour treatment	Test substance concentration (µg/ml)	0	100	300	400	500	600	800	1000	1500	2000
		Mitotic Index (%)*	100	84.3	55.4	45.6	34.8	29.4	22.1	14.2	1.5	1.0
		Mitotic cells Appearance	+++	+++	++	++	+	+	±	-	-	-
Metabolic  activation method		Test substance concentration (µg/ml)	0	5	10	30	50	100	300	500	1000	2000
		Mitotic Index (%)*	100	92.6	81.0	99.0	91.1	87.7	96.1	108.6	119.0	107.4
		Mitotic cells Appearance	+++	+++	+++	+++	+++	+++	+++	+++	+++	+++

 *   Average from 2 Petri dishes

Table 7.6.1/2: Results of the chromosome aberration test without metabolic activation

	Treatment Duration (h)	Treatment Concentration (µg/ml)	Number of cells observed	Appearance number and frequency of cells with numerical anomalies (%)		Appearance number and frequency of cells with chromosome structure anomalies (%)*
						Gap	Chromatid Type		Chromosome Type		Others	Total		Decision
					Decision	Gap	ctb	cte	csb	ces		g-	g+
Solvent (pure Water)	24	0	100	1		1	0	0	0	0	0	0	1
			100	0		2	0	0	1	0	0	1	3
			200	1 (0.05)		3 (1.5)	0 (0.0)	0 (0.0)	1 (0.5)	0 (0.0)	0 (0.0)	1 (0.5)	4 (2.0)
	48	0	100	0		0	0	0	0	0	0	0	0
			100	0		1	0	0	0	0	0	0	1
			200	0 (0.0)		1 (0.5)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.5)
Test Substance	24	300	100	0	-	2	2	0	1	0	0	3	5	-
			100	0		1	1	2	0	0	0	3	4
			200	0 (0.0)		3 (1.5)	3 (1.5)	2 (1.0)	1 (0.5)	0 (0.0)	0 (0.0)	6 (3.0)	9 (4.5)
		450	100	0	-	5	7	3	0	0	0	10	15	+
			100	0		0	4	4	0	1	0	9	9
			200	0 (0.0)		5 (2.5)	11 (5.5)	7 (3.5)	0 (0.0)	1 (0.5)	0 (0.0)	19 (9.5)	24 (12.0)
		600	100	0	-	5	15	7	1	0	0	21	24	++
			100	0		7	15	6	0	1	0	21	24
			200	0 (0.0)		12 (6.0)	30 (15.0)	13 (6.5)	1 (0.5)	1 (0.5)	0 (0.0)	42 (21.0)	48 (24.0)
	48	300	100	1	-	0	0	0	0	0	0	0	0	-
			100	2		0	0	0	0	1	0	1	1
			200	3 (1.5)		0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.5)	0 (0.0)	1 (0.5)	1 (0.5)
		450	100	0	-	4	5	6	0	0	0	10	12	+
			100	0		1	2	4	0	0	0	5	5
			200	0 (0.0)		5 (2.5)	7 (3.5)	10 (5.0)	0 (0.0)	0 (0.0)	0 (0.0)	15 (7.5)	17 (8.5)
		600	100	1	-	7	22	38	0	1	0	47	50	++
			100	1		3	25	30	0	0	0	44	44
			200	2 (1.0)		10 (5.0)	47 (23.5)	68 (34.0)	0 (0.0)	1 (0.5)	0 (0.0)	91 (45.5)	94 (47.0)
Positive Control (MMC)	24	0.05	100	0	-	1	5	36	3	0	0	41	42	++
			100	1		1	14	48	1	0	0	57	57
			200	1 (0.5)		2 (1.0)	19 (9.5)	84 (42.0)	4 (2.0)	0 (0.0)	0 (0.0)	98 (49.0)	99 (49.5)
	48	0.05	100	1	-	1	14	64	0	0	0	69	69	+++
			100	0		1	30	71	2	3	0	82	83
			200	1 (0.5)		2 (1.0)	44 (22.0)	135 (67.5)	2 (1.0)	3 (1.5)	0 (0.0)	151 (75.5)	152 (76.0)

The gap (g) includes both the chromatid type and the chromosome type.The total (-g) indicates the number and percentage excluding cells which possess a gap only. Even in the case where there are several anomalies inside one cell, that cell is counted as one abnormal cell. For example, if there are 2 breaks and 2 exchanges inside one cell, the calculation is made with one abnormal cell, one cell possessing a break and one cell possessing an exchange.ctb: chromatid type break; cts: chromatid type exchange; csb: chromosome type break cse: chromosome type

exchange         others:    fragmentation etc (excluding pulverization)

Conclusions:

Interpretation of results (migrated information):
positive without metabolic activation

Under the test conditions, Reaction mass of methacrylic acid and N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide induced chromosomal aberrations in Chinester Hamster fibroblasts without metabolic activation whereas the results were negative in the presence of S9 mix.

Executive summary:

In an in vitro chromosome aberrations study, performed according to the OECD No.473 guideline and in compliance with the GLP, Reaction mass of methacrylic acid and N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide diluted in water was tested in Chinese Hamster Lung fibroblasts in the presence and the absence of mammalian metabolic activation (S9 mix). The amount used for the test was calculated as concentration of N-[2-(2-oxo-1-imidazolidinyl)ethyl]methacrylamide (48.61%).

The cytotoxicity was tested in a preliminary test with concentrations of test item ranging from 0 to 2000 µg/mL. The concentration of the test substance causing 50 % cytostatis was approximately obtained for the concentration of 570 µg/ml and 350 µg/ml for the 24 hour and 48 hours treatment without S9 mix respectively. For the treatment with S9 mix (6h exposure) the cell toxicity was too weak to be observed below 2000 µg/ml (corresponding to10 mM, the maximal concentration recommended in the OECD guideline).The maximum concentration of the test substance that was determined to provide a sufficient number of cells when conducting the chromosome aberration test was 600 µg/ml at 24 and 48 hours without metabolic activation and 2000µg/ml under the metabolic activation method.

Positive controls such as mitomycin C and Cyclophosphamid were used to check the sensitivity of the test system. They gave appropriate response, so that the test was considered as valid.

During the main test, only three concentrations of test item were tested: 300; 450 and 600 without S9 mix (24 and 48 h treatment period) and 500; 1000 and 2000 µg/mL with S9 mix. Without metabolic activation, at both time of exposure (24 and 48h) the number of chromosome aberrations increased in a dose-response relationship. The number of aberrant cells was higher than the number obtained for the vehicle control for both treatment time exposure (without S9 mix): according to the evaluation criteria as the cells showing structural chromosome aberrations excluding gaps have a frequency rate over 20% at the highest concentration, the results were considered as positive.

With S9 mix, the number of chromosome aberration slightly increased but did not exceed 5%. Therefore the results with metabolic activation were considered as negative.

Under the test conditions, Reaction mass of methacrylic acid and N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide induced chromosomal aberrations in Chinese Hamster Lung fibroblasts without metabolic activation whereas the results were negative with S9 mix.

This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 473.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo mammalian comet assay (OECD 489 guideline study, Kr.1): negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18-Apr-2016 to 01-May-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Deviations:

yes

Remarks:

Slides were incubated for 10-32 minutes in the dark in the refrigerator. The quality of the slides was good and therefore this deviation had no effect on the results.

GLP compliance:

yes

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

Purity/composition correction factor: Yes, correction factor is 1.39 based on purity
Test item handling: No specific handling conditions required
Stability at higher temperatures: Not available
Chemical name (IUPAC), synonym:or trade name: Reaction mass of N-[2-(2-oxoimidazolidin-1- yl)ethyl]methacrylamide and methacrylic acid / SIPOMER WAM II
EC Number: 934-058-1
Molecular structure: Not indicated
Molecular formula: Not indicated
Molecular weight: 119 (The molecular weight is given as a mean value of the molecular weight of the components and additives of the reaction mass, based on the typical concentrations measured on a sample of the multiconstituant
substance)
Irritant or corrosive: Yes
Toxic to reproduction: Not indicated
Highly flammable: Not indicated
Highly reactive to water: Not indicated
Highly reactive to oxygen: Not indicated
Volatile: Not indicated
pH: 2-5 at concentration of 10%
Specific gravity/density: 1.1086 (relative density at 20°C)

Species:

rat

Strain:

other: Wistar Han

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
The Wistar Han rat strain (SPF) was used as this strain is recommended in the international guidelines for genotoxicity testing (i.e. micronucleus assay; OECD guideline 474). The animals were provided by Charles River, Sulzfeld, Germany.
- Age at study initiation:
Young adult animals were selected (6-7 weeks old at the start of treatment). The total number of animals used was 3 in the dose range finding study and 25 in the main study. In the Comet main study 5 male rats were treated in each group.
- Weight at study initiation:
The body weights of the rats at the start of the treatment with Reaction mass of N- [2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid were within 20% of the sex mean. The mean body weights were 151 ± 7.4 g (range 140 – 163 g).
- Assigned to test groups randomly:
The rats were identified by a unique number on the tail written with a marker pen. The animals were allocated at random to the treatment groups. On arrival and at the start of the treatment, all animals were clinically examined to ensure selected animals were in a good state of health.
- Fasting period before study: no
- Housing: The animals were housed in room number A 0.07 (dose range finding study) and A 0.08 (dose range finding study and main study). Group housing of maximum 5 animals per sex and per dose in labeled Macrolon cages (type
MIV height 180 mm, length 600 mm and width 330 mm) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son
(Wonham Mill Ltd), Surrey, United Kingdom).
- Diet (ad libitum): The animals had free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants of each batch were examined and archived.
- Water: The animals had free access to tap-water. Certificates of analysis (performed quarterly) were examined and archived.
- Acclimation period: The acclimatisation period was at least 5 days before the start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0C (actual range: 18.9 – 21.4°C)
- Humidity (%): 40 - 70% (actual range: 38 - 73%) . Due to e.g. cleaning procedures, temporary deviations from the maximum level for humidity (with max. 3%) occurred. Based on laboratory historical data these deviations are considered not to affect the study integrity.
- Air changes (per hr): approximately 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle

IN-LIFE DATES: From: 09 May 2016 To: 20 June 2016

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Elix water (Millipore Corp., Bedford, MA, USA)
- Justification for choice of solvent/vehicle: the test substance is soluble in water (999 g/L).
- Concentration of test material in vehicle: 50-200 mg/ml
- Amount of vehicle (gavage): 10 ml/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Negative control:
The negative control was Elix water (Millipore Corp., Bedford, MA, USA), the vehicle of the test item. The route and frequency of administration and the volume administered of the negative control were the same as those of the test item
Test item preparation:
Test item concentrations were corrected for the purity (factor 1.39). Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid was dissolved in Elix water. The specific gravity of Elix water is 1.0 g/ml. Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid concentrations was dosed to the animals within 2 hours after preparation.
Positive control:
The positive control for the Alkaline Comet test was Ethyl Methanesulfonate (EMS; CAS no. 62-50-0; Sigma Aldrich, Steinheim, Germany) at 200 mg/kg body weight dissolved in physiological saline. EMS was used within 2 hours after preparation and was orally administered. The dosing volume was 10 mL/kg body weight.

Duration of treatment / exposure:

three consecutive days

Frequency of treatment:

once a day

Post exposure period:

sampling times:
- somatic tissues: Approximately 3-4 hours after the third treatment with the test compound or vehicle and second treatment with EMS, liver and glandular stomach tissues were collected/isolated and examined for DNA damage with the alkaline Comet assay.
- Complementary organ sampling and storing: A liver lobe and a small piece of the glandular stomach (approx. 1/5 of the glandular stomach) were collected and fixed and stored in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).
In addition gonads were collected and stored in 10% buffered formalin for potential subsequent analyses.

Remarks:

0,500, 1000 and 2000 mg/kg b.w. Main study

Remarks:

0, 2000 mg/kg b.w. Dose range finding study

No. of animals per sex per dose:

Main study: 5 males
Dose range finding study: 3 males and 3 females

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethylmethanesulphonate (EMS), 5 males

- Justification for choice of positive control(s): known mutagen.
- Route of administration: oral gavage
- Doses / concentrations: 200 mg/kg

The rats were dosed for two consecutive days (once daily) with the positive control EMS by oral gavage (oral intubation with a plastic gavage needle). Approximately 3-4 hours after the second treatment with EMS, liver and glandular stomach tissues were collected/isolated and examined for DNA damage with the alkaline Comet assay.

Tissues and cell types examined:

TISSUES EXAMINED:
Liver and glandular stomach tissues were examined for DNA damage with the alkaline Comet assay.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Selection of an adequate dose range for the Comet main test was based on a dose range finding study. The test procedure and conditions were similar to those applied in the main test. Based on the dose range finding study, three doses were selected for the main study. Since there were no substantial differences in toxicity between sexes only males were used in the main study. Five male rats were used in each treatment group. The animals were dosed with vehicle or Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid for three consecutive days and twice with EMS according to the following testing scheme.

TREATMENT AND SAMPLING TIMES :
Approximately 3-4 hours after the third treatment with the test compound or vehicle and second treatment with EMS, liver and glandular stomach tissues were collected/isolated and examined for DNA damage with the alkaline Comet assay.
A liver lobe and a small piece of the glandular stomach (approx. 1/5 of the glandular stomach) were collected and fixed and stored in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands). In addition gonads were collected and stored in 10% buffered formalin for potential subsequent analyses.

DETAILS OF CELL SUSPENSIONS PREPARATION:
Isolation of liver cells
The isolation method was based on the publication of Hu et al. (2002). A portion of 0.4-0.7 gram from the liver was removed and minced thoroughly on aluminium foil in ice. The minced liver tissue was added to 10 mL of collagenase (20 Units/mL) dissolved in HBSS (Ca2+ - and Mg2+-free) and incubated in a shaking waterbath at 37 °C for 20 minutes. Thereafter, a low centrifugation force was applied two times to remove large undigested liver debris (40 g for 5 min). The supernatant was collected and centrifuged to precipitate the cells (359 g for 10 min). The supernatant was removed and the cell pellet was resuspended in ice cold HBSS (Ca2+ - and Mg2+-free) and kept on ice.

Isolation of (glandular) stomach cells
The stomach was cut open,washed free from food using cold Hank’s Balanced Salt Solution (HBSS; Ca++, Mg++ free)and stored on ice in HBSS. The stomach was transferred to a tube containing mincing buffer, consisting of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution (HBSS) (Ca++, Mg++ free, and phenol red free if available), pH 7.5 (DMSO was added immediately before use). The stomach was incubated in mincing buffer on ice for 15-30 minutes.

After incubation, the fore-stomach was removed and discarded. The surface epithelia of the glandular epithelia was gently scraped once. This layer was discarded since the lifetime of these cells is very short in the body with a maximum of 3 days. Therefore this layer contains a high amount of apoptotic cells which disturb the interpretation in the Comet assay. Moreover, since the lifetime of these cells is very short it is unlikely that these cells play a role in carcinogenesis.
The stomach was then minced thoroughly on aluminium foil in ice. The minced tissue was added to 10 mL of mincing buffer and incubated in a shaking waterbath at 37 °C for 15 minutes. Thereafter, a low centrifugation force was applied two times to remove undigested debris (40 g for 5 min). The supernatant was collected and filtered through a 100 µm Cell Strainer to purify the cell suspension. The purified cell suspension was centrifuged to precipitate the cells (359 g for 10 min). The supernatant was removed and the cell pellet was resuspended in ice cold HBSS (Ca2+ - and Mg2+-free) and kept on ice.

Determination of cell viability
The viability of the cells after isolation was determined by manually counting the number of viable cells using trypane blue staining. Only one animal per group was checked.

DETAILS OF SLIDE PREPARATION:
To 20 µL of the cell suspension, 280 µL melted low melting point agarose (LMAgarose; Trevigen, Gaithersburg, USA) was added. The cells were mixed with the LMAgarose and 50 µL was layered on a precoated Comet slide (Trevigen) in duplicate. Three slides per tissue were prepared. The slides were marked with the study identification number, animal number and group number. The slides were incubated for 11-32 minutes (study plan deviation 1) in the refrigerator in the dark until a clear ring appeared at the edge of the Comet slide area.
The cells on the slides were overnight (approximately 16-17 h) immersed in prechilled lysis solution (Trevigen) in the refrigerator. After this incubation period, the slides were immersed/rinsed in neutralization buffer (0.4M Tris-HCl pH 7.4) for approximately 5 minutes. The slides were then placed in freshly prepared alkaline solution for 30 minutes at room temperature in the dark.
The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 1 Volt/cm. The electrophoresis was performed for 30 minutes under constant cooling (actual temperature 4.0 – 4.5°C). After completion of electrophoresis, the slides were immersed/rinsed in the neutralization buffer. The slides were subsequently immersed for 5 minutes in Absolut ethanol (Merck, Darmstadt, Germany) and allowed to dry at room temperature. The slides were stained for approximately 5 minutes with the fluorescent dye SYBR® Gold (Life Technologies, Bleiswijk, The Netherlands) in the refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark.

METHOD OF ANALYSIS:
To prevent bias, slides were randomly coded before examination of the Comets. An adhesive label with study identification number and code was placed over the marked slide. The slides were examined with a fluorescence microscope connected to a Comet Assay IV image analysis system (Perceptive instruments Ltd, Suffolk, United Kingdom). One hundred fifty Comets per slide (50 comets of each replicate LMAgarose circle) were examined.
The following criteria for scoring of Comets were used:
- Only horizontal orientated Comets were scored, with the head on the left and the tail on the
right.
- Cells that showed overlap or were not sharp were not scored.

Evaluation criteria:

VALIDITY CRITERIA:
The in vivo comet is considered acceptable if it meets the following criteria:
a) The percentage Tail Intensity of the solvent control should reasonably be within the laboratory historical control data range.
b) The positive control EMS should produce at least a statistically significant increase in the percentage Tail Intensity compared to the negative control treated animals. The positive control data were analysed by the Students t test (one-sided, p < 0.05).

EVALUATION CRITERIA:
A test compound is considered positive in the Comet assay (in a tissue) if the following criteria are met:
It induces a biologically as well as a statistically significant (Dunnett’s test, one-sided, p < 0.05) dose-dependent increase in percentage Tail Intensity. In case of other non-dosedependent significant increases the data interpretation is made on a case by case base.

A test compound is considered as negative in the Comet assay (in a tissue) if the following criteria are met:
None of the tested concentrations shows a statistically significant (Dunnett’s test, onesided, p < 0.05) dose-dependent increase in percentage Tail Intensity.

Data were normally distributed thus no transformation (y = 1/y) of the data was necessary. In addition no Cochran Armitage trend test (p < 0.05) was performed.

Statistics:

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data.

Sex:

male

Genotoxicity:

negative

Remarks:

liver and glandular stomach

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In the dose range finding test, three male and three female animals were dosed via oral gavage with 2000 mg Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid per kilogram body weight once daily for three consecutive days. The animals showed no treatment related clinical signs or mortality. Based on the results of the dose range finding study dose levels of 500, 1000 and 2000 mg/kg body weight were selected as appropriate doses for Comet main test. Five male animals were used in each treatment group.

RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route:
Dose levels: 0,500, 1000 and 2000 mg/kg b.w.
Route: oral gavage (oral intubation with a plastic gavage needle). The route of administration was selected taking into account the possible route of human exposure during manufacture, handling and use.

- Formulation analysis:
The accuracy of preparations, homogeneity and stability of the test substance in the formulations was determined.
The concentrations of Group B-D were in agreement with target concentrations (i.e. mean accuracies of 100% respectively). No test substance was detected in group A (vehicle) formulation. Overall it was concluded that the concentrations were acceptable. The formulations were considered homogenous, as demonstrated by Group B (i.e. coefficient of variation of 0.81%). Analysis of group B after storage at room temperature under normal laboratory light for 4 hours yielded a relative difference of -0.77%. Based on this it is concluded that formulations were stable when stored at room temperature under normal laboratory light conditions for at least 4 hours.

- Mortality and toxic signs:
The animals of the groups treated with 500, 1000 and 2000 mg Reaction mass of N-[2-(2- oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid/kg body weight and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality.

- Comet slide analysis
After treatment, single cell suspensions from liver and stomach were prepared. The viability of one single cell suspension per tissue per group was assessed by using trypan blue. The viability of the single cell suspensions was 100% for liver and in the range 97 – 99% for stomach.
Comet slides were prepared and analysed.

Liver
No statistically significant increase in the mean Tail Intensity (%) was observed in liver cells of Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid-treated male animals at any of the dose levels tested compared to the vehicle treated animals.
The mean Tail Intensity (%) in liver cells of vehicle treated male rats was 4.65%. The positive control EMS, showed a mean Tail Intensity of 91.23% (20-fold statistically significant induction; Students t test p<0.001; ). The negative and positive control Tail Intensities were within the historical control data range. Hence, all criteria for an acceptable assay were met.

Glandular stomach
No statistically significant increase in the mean Tail Intensity (%) was observed in stomach cells of Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid-treated male animals at any of the dose levels tested compared to the vehicle treated animals.
The mean Tail Intensity (%) in stomach cells of vehicle treated male rats was 54.53%. The positive control EMS, showed a mean Tail Intensity of 97.64% (1.8-fold statistically significant induction; Students t test p<0.001; ). The negative and positive control Tail
Intensities were within the historical control data range. Hence, all criteria for an acceptable assay were met.

Table1                              Mean body weight immediately prior to dosing with Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid and EMS

Group code	Dose (mg/kg/bw)	Day 1 Body weight (Mean ± S.D.)		Day 2 Body weight (Mean ± S.D.)	Day 3 Body weight (Mean ± S.D.)
A	0	150.6	± 8.3	149.4 ± 8.3	150.6 ± 8.6
B	2000	155.6	± 8.3	155.4 ± 8.9	156.2 ± 10.4
C	1000	148.4	± 7.6	147.2 ± 9.5	148.6 ± 9.0
D	500	149.2	± 5.4	148.2 ± 4.5	152.0 ± 3.7
E	200 (EMS)	^1		154.4 ± 7.2	148.6 ± 6.7

¹Not dosed on day 1. Dosing with EMS was started on Day 2

 

Table2               Viability single cell suspensions

animal number_group code	Dose (mg/kg/bw)	Organ	Viability (%)	Organ	Viability (%)
1A	0	Liver	100	Stomach	98
6B	2000	Liver	100	Stomach	99
11C	1000	Liver	100	Stomach	97
16D	500	Liver	100	Stomach	98
21E	200 (EMS)	Liver	100	Stomach	99

 

Table3               Overview Tail Intensity in liver cells of male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	4.65	0.25
Test Item 500 mg/kg	5.93	1.42
Test Item 1000 mg/kg	5.47	1.10
Test Item 2000 mg/kg	4.70	0.33
EMS 200 mg/kg	91.23	2.09

 

Table4               Overview Tail Intensity in stomach cells of male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	54.53	3.64
Test Item 500 mg/kg	41.21	8.38
Test Item 1000 mg/kg	41.87	14.67
Test Item 2000 mg/kg	51.72	10.36
EMS 200 mg/kg	97.64	0.53

Conclusions:

Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid shows a negative result (non-genotoxic) in the Comet assay in liver and glandular stomach post dosing of male rats up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions.

Executive summary:

Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid was tested in the alkaline in vivo Comet assay in male rats, to evaluate its potential genotoxic effect in liver and glandular stomach.

The study procedures described in this report were based on the most recent version of OECD guideline No. 489 “In Vivo Mammalian Alkaline Comet Assay”.

Batch MWAM152801 of Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid was an amber liquid with a purity of 71.9%. The test item was dissolved in water. Test item concentrations were corrected for the purity.

Formulation analysis was performed to determine the accuracy of preparation, homogeneity and stability of the test substance in formulations. The concentrations analysed in the formulations of the three treatment groups were in agreement with target concentrations (i.e. mean accuracy 100%). No test substance was detected in the vehicle control. The formulations were homogeneous (i.e. coefficient of variation 0.81%) and stable when stored at room temperature under normal laboratory light conditions for at least 4 hours.

In the dose range finding study, 3 males and females were dosed via oral gavage with 2000 mg Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid per kg body weight once daily for three consequtive days. The animals showed no treatment related clinical signs or mortality after dosing. Since there were no substantial differences in toxicity between sexes only males were used in the main study.

In the main study, 25 male animals were dosed by oral gavage with vehicle or 500, 1000 and 2000 mg Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid per kg body weight once daily for three consecutive days. A positive control group was dosed twice by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS) per kg body weight. In total 5 treatment groups were used, each consisting of 5 animals. No treatment-related clinical signs or mortality were noted after dosing in any animal treated with 500, 1000 or 2000 mg/kg Reaction mass of N-[2-(2-oxoimidazolidin-1- yl)ethyl]methacrylamide and methacrylic acid or control animals receiving vehicle or EMS.

Approximately 3-4 hours after the second dose of EMS and third dose of the vehicle or Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid, liver and glandular stomach tissues were collected. The animals were sacrificed by abdominal aorta bleeding under isoflurane anaesthesia. Single cell suspensions from liver and stomach were made followed by Comet slide preparation. The slides were analyzed by an image analysis system and the Tail Intensity (%) was assessed.

No statistically significant increase in the mean Tail Intensity (%) was observed in liver or glandular stomach cells of Reaction mass of N-[2-(2-oxoimidazolidin-1- yl)ethyl]methacrylamide and methacrylic acid-treated male animals at any of the dose levels tested compared to the vehicle treated animals.

The mean Tail Intensity in liver and stomach cells of vehicle-treated rats was 4.65% and 54.53%, respectively. The positive control EMS, showed a mean Tail Intensity of 91.23% (20-fold statistically significant induction; Students t test p<0.001) in liver and of 97.64% (1.8-fold statistically significant induction; Students t test p< 0.001) in stomach tissue. The negative and positive control Tail Intensities were within the historical control data range. Hence, all criteria for an acceptable Comet assay were met.

It is concluded that Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid shows a negative result (non-genotoxic) in the Comet assay in liver and glandular stomach post dosing of male rats up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29-Oct-2018 to 16-Jan-2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

Purity/composition correction factor: Yes, correction factor is 1.39 based on purity
Test item handling: No specific handling conditions required
Stability at higher temperatures: Not available
Chemical name (IUPAC), synonym:or trade name: Reaction mass of N-[2-(2-oxoimidazolidin-1- yl)ethyl]methacrylamide and methacrylic acid / SIPOMER WAM II
EC Number: 934-058-1
Molecular structure: Not indicated
Molecular formula: Not indicated
Molecular weight: 119 (The molecular weight is given as a mean value of the molecular weight of the components and additives of the reaction mass, based on the typical concentrations measured on a sample of the multiconstituant
substance)
Irritant or corrosive: Yes
Toxic to reproduction: Not indicated
Highly flammable: Not indicated
Highly reactive to water: Not indicated
Highly reactive to oxygen: Not indicated
Volatile: Not indicated
pH: 2-5 at concentration of 10%
Specific gravity/density: 1.1086 (relative density at 20°C)

Species:

rat

Strain:

other: Wistar Han

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
The Wistar Han rat strain (SPF) was used as this strain is recommended in the international guidelines for genotoxicity testing. The animals were provided by Charles River, Sulzfeld, Germany.
- Age at study initiation:
Young adult animals were selected (6 weeks old at the start of treatment). The total number of animals used in the main study was 25. In the Comet main study 5 male rats were treated in each group.
- Weight at study initiation:
The body weights of the rats at the start of the treatment with Reaction mass of N- [2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid were within 20% of the sex mean. The mean body weights were 149 ± 9.8 g (range 130 – 164 g).
- Assigned to test groups randomly:
The rats were identified by a unique number on the tail written with a marker pen. The animals were allocated at random to the treatment groups. On arrival and at the start of the treatment, all animals were clinically examined to ensure selected animals were in a good state of health.
- Fasting period before study: no
- Housing: Group housing of maximum 5 animals per sex and per dose in labeled Macrolon cages (type MIV height 180 mm, length 600 mm and width 330 mm) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- Diet (ad libitum): The animals had free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants of each batch were examined and archived.
- Water: The animals had free access to tap-water. Certificates of analysis (performed quarterly) were examined and archived.
- Acclimation period: The acclimatisation period was at least 5 days before the start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0C (actual range: 20 – 21°C)
- Humidity (%): 40 - 70% (actual range: 49 - 60%).
- Air changes (per hr): approximately 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle

IN-LIFE DATES: From: 30 October 2018 To: 01 November 2018

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Elix water (Millipore Corp., Bedford, MA, USA)
- Justification for choice of solvent/vehicle: the test substance is soluble in water (999 g/L).
- Concentration of test material in vehicle: 50-200 mg/ml
- Amount of vehicle (gavage): 10 ml/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Negative control:
The negative control was Elix water (Millipore Corp., Bedford, MA, USA), the vehicle of the test item. The route and frequency of administration and the volume administered of the negative control were the same as those of the test item
Test item preparation:
Test item concentrations were corrected for the purity (factor 1.39). Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid was dissolved in Elix water. The specific gravity of Elix water is 1.0 g/ml. Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid concentrations was dosed to the animals within 3 hours after preparation.
Positive control:
The positive control for the Alkaline Comet test was Ethyl Methanesulfonate (EMS; CAS no. 62-50-0; Sigma Aldrich, Steinheim, Germany) at 200 mg/kg body weight dissolved in physiological saline. EMS was used within 2 hours after preparation and was orally administered. The dosing volume was 10 mL/kg body weight.

Duration of treatment / exposure:

three consecutive days

Frequency of treatment:

once a day

Post exposure period:

sampling times:
- somatic tissues: Approximately 3-4 hours after the third treatment with the test compound or vehicle and second treatment with EMS, glandular stomach was collected/isolated and examined for DNA damage with the alkaline Comet assay.
- Complementary organ sampling and storing: A small part of stomach from the animals (with exception of the positive control) used (after isolation of a part for the comet assay) was collected, fixed and stored in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No histotechnology and histopathology was performed.

Remarks:

0,500, 1000 and 2000 mg/kg b.w.

No. of animals per sex per dose:

5 males

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethylmethanesulphonate (EMS), 5 males

- Justification for choice of positive control(s): known mutagen.
- Route of administration: oral gavage
- Doses / concentrations: 200 mg/kg

The rats were dosed for two consecutive days (once daily) with the positive control EMS by oral gavage (oral intubation with a plastic gavage needle). Approximately 3-4 hours after the second treatment with EMS, glandular stomach was collected/isolated and examined for DNA damage with the alkaline Comet assay.

Tissues and cell types examined:

TISSUES EXAMINED:
Glandular stomach was examined for DNA damage with the alkaline Comet assay.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The doses selected were those tested in the initial study (Study number 512210).

TREATMENT AND SAMPLING TIMES :
Approximately 3-4 hours after the third treatment with the test compound or vehicle and second treatment with EMS, glandular stomach was collected/isolated and examined for DNA damage with the alkaline Comet assay.
A small part of stomach from the animals (with exception of the positive control) used (after isolation of a part for the comet assay) was collected, fixed and stored in 10% buffered
formalin (neutral phosphate buffered 4% formaldehyde solution). No histotechnology and histopathology was performed.

DETAILS OF CELL SUSPENSIONS PREPARATION:
Isolation of (glandular) stomach cells
This isolation method for glandular stomach is based on the JACVAM Comet validation study (Uno et al., 2015). The stomach was cut open and washed free from food using cold Hank’s Balanced Salt Solution (HBSS; Ca++, Mg++ free, Life Technologies, Breda, the Netherlands). The forestomach was removed and discarded. The glandular stomach was stored on ice in mincing
buffer incomplete (HBSS containing 20 mM EDTA (Merck, Darmstadt, Germany).
The glandular stomach was then transferred to a petri-dish on ice containing 10 mL mincing buffer incomplete. The surface epithelia of the glandular epithelia was gently scraped 3-4 times with a cell scraper. This layer was discarded since the lifetime of these cells is very short in the body with a maximum of 3 days. Therefore this layer contains a high amount of apoptotic cells which disturb the interpretation in the Comet assay. Moreover, since the lifetime of these cells is very short it is unlikely that these cells play a role in carcinogenesis.
The glandular stomach was then rinsed with mincing buffer incomplete and transferred to a petri-dish containing 10 mL mincing buffer. The stomach was then scraped multiple times with a cell scraper and the cells were collected in the mincing buffer present in the petri-dish. The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution Ca++, Mg++ free, pH 7.5 (DMSO (Merck) was added immediately before use).
The cell suspension was filtered through a 100 μm Cell Strainer (Falcon, Corning life Sciences, Tewksbury, United States) to purify the cell suspension and collected in a tube and stored on ice.

DETAILS OF SLIDE PREPARATION:
To the cell suspension, melted low melting point agarose (LMAgarose; Trevigen, Gaithersburg, USA) was added (ratio 10:140). The cells were mixed with the LMAgarose and 50 μL was layered on a pre-coated Comet slide (Trevigen) in duplicate. Three slides per tissue per animal were prepared. The slides were marked with the study identification number, animal number and group number. The slides were incubated for 19 – 21 minutes in the refrigerator in the dark until a clear ring appears at the edge of the Comet slide area.
The cells on the slides were overnight (approximately 17 h) immersed in pre-chilled lysis solution (Trevigen) in the refrigerator. After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4). The slides were then placed in freshly prepared alkaline solution for 20 minutes at room temperature in the dark. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 0.7 Volt/cm. The electrophoresis was performed for 20 minutes under constant cooling (actual temperature 4.5 °C). After electrophoresis the slides were immersed/rinsed in neutralization buffer for 5 minutes. The slides were subsequently immersed for 5 minutes in Absolut ethanol (99.6%, Merck) and allowed to dry at room temperature. The slides were stained for approximately 5 minutes with the fluorescent dye SYBR® Gold (Life Technologies, Bleiswijk, The Netherlands) in the refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark and fixed with a coverslip.

METHOD OF ANALYSIS:
To prevent bias, slides were randomly coded before examination of the Comets. An adhesive label with study identification number and code was placed over the marked slide. The slides were examined with a fluorescence microscope connected to a Comet Assay IV image analysis system (Perceptive instruments Ltd, Suffolk, United Kingdom). One hundred fifty Comets per slide (50 comets of each replicate LMAgarose circle) were examined.
The following criteria for scoring of Comets were used:
- Only horizontal orientated Comets were scored, with the head on the left and the tail on the right.
- Cells that showed overlap or were not sharp were not scored.

In addition the frequency of hedgehogs was determined and documented based on the visual scoring of at least 150 cells per tissue per animal in the repeat experiment. The occurrence of hedgehogs was scored in all treatment groups and the control. Since there was no effect of the test item Hedgehogs data was not reported and maintained in the raw data.

Evaluation criteria:

VALIDITY CRITERIA:
The in vivo comet is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The positive control EMS should produce at least a statistically significant increase in the percentage Tail Intensity compared to the vehicle treated animals. The response should be compatible with the data in the historical control database. The positive control data will be analyzed by the Students t test (one-sided, p < 0.05) in case of homogeneous variances or by the Welch t test in case of inhomogeneous variances (one-sided, p < 0.05).
c) Adequate numbers of cells and doses have been analysed
d) The highest test dose is the MTD or 2000 mg/kg/day

EVALUATION CRITERIA:
A test item is considered positive in the Comet assay if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p <0.05) increase in percentage Tail Intensity is detected compared with the concurrent
negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative in the Comet assay if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in percentage Tail Intensity is detected compared with the concurrent negative
control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.

Data were normally distributed thus no transformation (y = 1/y) of the data was necessary.

Statistics:

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data.

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

glandular stomach

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS:
- Appropriateness of dose levels and route:
Dose levels: 0,500, 1000 and 2000 mg/kg b.w.
Route: oral gavage (oral intubation with a plastic gavage needle). The route of administration was selected taking into account the possible route of human exposure during manufacture, handling and use.
- Formulation analysis:
Formulation analysis was performed to determine the accuracy and homogeneity of preparation of the test item in formulations. The concentrations analyzed in the formulations of the high dose, mid dose and low dose in male animals were in agreement with target concentrations (all 97% of target). No test substance was detected in the vehicle control. The high and low dose formulation were homogeneous (relative standard deviation of concentrations of <3.2%). Overall it was concluded that the accuracy and homogeneity of the preparations were acceptable

- Mortality and toxic signs:
The animals of all the groups treated with the test item and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality.

- Comet slide analysis
No statistically significant increase in the mean Tail Intensity (%) was observed in glandular stomach cells of the test item treated male animals at any of the dose levels tested compared to the vehicle treated animals.
The mean Tail Intensity in glandular stomach cells of vehicle-treated rats was 2.84 ± 0.23% (mean ± SD) which is within the 95% control limits of the distribution of the historical control data for the vehicle control. The positive control EMS was significantly increased and showed a mean Tail Intensity of 63.17% ± 6.40% (mean ± SD, 22-fold induction; p<0.001 Students t test). The mean positive control Tail
Intensity was within the 95% control limits of the distribution of the historical positive control database.
Adequate numbers of cells (150 cells per animal) and doses were analyzed and the highest test dose was the maximum dose required by the guidelines. Hence, all criteria for an acceptable assay were met.

Table 1: Mean body weight immediately prior to dosing with Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid and EMS

Group code	Dose (mg/kg/bw)	Day 1 Body weight (Mean ± S.D.)		Day 2 Body weight (Mean ± S.D.)	Day 3 Body weight (Mean ± S.D.)
A	0	149.6	± 14.1	14.0 ± 14.1	150.8 ± 13.9
B	2000	146.2	± 12.3	142.8 ± 14.1	142.4 ± 16.0
C	1000	144.2	± 3.1	140.8 ± 1.8	140.8 ± 6.7
D	500	154.2	± 7.2	149.2 ± 6.8	154.0 ± 6.8
E	200 (EMS)	#		137.8 ± 5.9	138.8 ± 8.8

#Not dosed on day 1. Dosing with EMS was started on Day 2

 

Table 2: Overview Tail Intensity in glandular stomach cells of male Rats

	Tail Intensity (%)	S.D.
Vehicle Control	2.84	0.23
Test Item 500 mg/kg	3.78	0.58
Test Item 1000 mg/kg	2.32	0.97
Test Item 2000 mg/kg	2.34	0.87
EMS 200 mg/kg	63.17***	6.40

*** p<0.001 Student’s t test; EMS 

Conclusions:

Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid is not genotoxic in the Comet assay in glandular stomach cells when sampled approximately 3-4 hours post dosing, of male rats that were dosed via oral gavage for three consecutive days up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Executive summary:

The objective of this study was to obtain information on the potential genotoxicity of Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid when administered to rats at the maximum recommended dose in accordance with current regulatory guidelines, by measuring the increase in DNA strand breaks in glandular stomach.

This study was specifically requested by the European Chemicals Agency (ECHA Decision Number TPE-D-2114439314-53-01/F). A previous Alkaline in vivo comet study conducted in this laboratory with the same test material investigated liver and glandular stomach and gave negative results (THE ALKALINE IN VIVO COMET ASSAY WITH REACTION MASS OF N-[2-(2-OXOIMIDAZOLIDIN-1-YL)ETHYL]METHACRYLAMIDE AND METHACRYLIC ACID IN LIVER AND GLANDULAR STOMACH OF WISTAR HAN RATS; Study number 512210; Report dated 01 May 2017). In 2017, study 512210 was evaluated by ECHA as a follow-up of the registration dossier update. While the results for the Comet assay in liver were found acceptable, ECHA considers that results for the Comet assay in glandular stomach do not fulfil the quality criteria based on the relatively high Tail Intensity in stomach.

In order to comply with the information requested by ECHA for glandular stomach this new study was initiated with a modified method (electrophoresis parameters were optimized as well as the isolation method) and adopted acceptability criteria as requested by ECHA. The same dose levels as in the previous study were tested.

The Wistar Han rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic items. Moreover, historical control background data has been generated with this strain.

The study procedures described in this report were based on the most recent OECD guidelines.

Batch MWAM180121 of the test item was an amber thick liquid with a purity of 71.7%. The test item was dissolved in Elix water.

Male rats were dosed once daily by oral gavage with vehicle or with 500, 1000 and 2000 mg Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid per kg body weight for three consecutive days. A positive control group was dosed twice by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS) per kg body weight. In total 5 treatment groups were used, each consisting of 5 animals. No treatment related clinical signs or mortality were noted in any animal treated with the test item or control animals receiving vehicle or EMS.

Approximately 3-4 hours after the second dose of EMS and third dose of the vehicle or the test item, the animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia and glandular stomach was isolated. Single cell suspensions from glandular stomach were made followed by comet slide preparation.

No statistically significant increase in the mean Tail Intensity (%) was observed in glandular stomach cells of the test item treated male animals at any of the dose levels tested compared to the vehicle treated animals.

The mean Tail Intensity in glandular stomach cells of vehicle-treated rats was 2.84 ± 0.23% (mean ± SD) which is within the 95% control limits of the distribution of the historical control data for the vehicle control. The positive control EMS was significantly increased and showed a mean Tail Intensity of 63.17% ± 6.40% (mean ± SD, 22-fold induction; p<0.001 Students t test). The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database. Adequate numbers of cells and doses were analyzed and the highest test dose was the maximum dose required by the guidelines. Hence, all criteria for an acceptable assay were met.

Formulation analysis was performed to determine the accuracy and homogeneity of preparation of the test item in formulations. The concentrations analyzed in the formulations of the high dose, mid dose and low dose in male animals were in agreement with target concentrations (all 97% of target). No test substance was detected in the vehicle control. The high and low dose formulations were homogeneous (relative standard deviation of concentrations of <3.2%). Overall it was concluded that the accuracy and homogeneity of the preparations were acceptable.

The present study was performed with a modified method compared to the previous Alkaline in vivo comet study conducted in this laboratory with the same test material and therefore resulted in low negative control tail intensities (<20%) as requested by ECHA. The present study gave negative results in glandular stomach, consequently, confirmed the result of the earlier study (Study number 512210; Report dated 01 May 2017).

In conclusion, the test is valid and Reaction mass of N-[2-(2-oxoimidazolidin-1 -yl)ethyl]methacrylamide and methacrylic acid is not genotoxic in the Comet assay in

glandular stomach cells when sampled approximately 3-4 hours post dosing, of male rats that were dosed via oral gavage for three consecutive days up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Reaction mass of methacrylic acid and N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide was assessed for its genotoxic potential in in vitro studies (Ames test and chromosome aberration test):

Ames Test:

On study of reliability 2 according to Klimisch cotation critera is available (Yukko, 1993) and was selected as a key study. In a reverse gene mutation assay in bacteria, performed according to the OECD No.471 guideline, Reaction mass of methacrylic acid and N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide diluted in water was tested in S. typhimurium TA1535, TA1537, TA100, TA98 strains and E. coli WP2 uvrA strain in the presence and the absence of mammalian metabolic activation (S9 mix) using the preincubation method. Six known mutagens were used to check the sensitivity of the test system and gave appropriate response. The test was therefore considered as valid.

In the first assay the test item was used at the following concentration: 0; 50; 100; 200; 500; 1000; 2000 and 5000 µg/plate (duplicate). There was a slight increase in the number of revertants in the TA98, TA100 and TA1537 strains with S9 mix at the highest dose, but this increase did not exceed twice the negative control (<1.5). Therefore it was not considered as relevant regarding the mutagenicity. For the other conditions, there was no increase in the number of revertants compared to the negative control.

In the second assay the test item was used at the following concentration: 313; 625; 1250; 2500 and 5000 µg/plate (duplicate). There was a slight increase in the number of revertants in the TA1537 without S9 mix at 2500 µg/plate, but the mean revertants did not exceed twice the mean obtained for the negative control and there was no dose-response relationship. Therefore it was not considered as relevant regarding the mutagenicity. For the other conditions, there was no increase in the number of revertants compared to the negative control.

As there was no decrease in the number of revertants compared to the negative control, the test item can be considered as non cytotoxic up to the recommended limit concentration of 5000 µg/plate. Based on these data it can be concluded that the registered substance is not mutagenic in bacteria.

In vitro chromosome aberration test:

On study of reliability 2 according to Klimisch cotation critera is available (Shouzou, 1993) and was selected as a key study. In a GLP-compliant in vitro chromosome aberration test performed according to the OECD 473 Guideline, Reaction mass of methacrylic acid and N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide diluted in water was tested in Chinese Hamster Lung fibroblasts in the presence and the absence of mammalian metabolic activation (S9 mix). The cytotoxicity was tested in a preliminary test with concentrations of test item ranging from 0 to 2000 µg/mL. The concentration of the test substance causing 50 % cytostatis was approximately obtained for the concentration of 570 µg/ml and 350 µg/ml for the 24 hour and 48 hours treatment without S9 mix respectively. For the treatment with S9 mix (6h exposure) the cell toxicity was too weak to be observed below 2000 µg/ml.The maximum concentration of the test substance that was determined to provide a sufficient number of cells when conducting the chromosome aberration test was 600 µg/ml at 24 and 48 hours without metabolic activation and 2000µg/ml under the metabolic activation method.

Positive controls such as mitomycin C and Cyclophosphamid were used to check the sensitivity of the test system. They gave appropriate response, so that the test was considered as valid.

During the main test, three concentrations of test item were tested: 300; 450 and 600 without S9 mix (24 and 48 h treatment period) and 500; 1000 and 2000 µg/mL with S9 mix (6h treatment period). Without metabolic activation, at both time of exposure (24 and 48h) the number of chromosome aberrations increased in a dose-response relationship. The number of aberrant cells was higher than the number obtained for the vehicle control for both treatment time exposure (without S9 mix): according to the evaluation criteria as the cells showing structural chromosome aberrations excluding gaps have a frequency rate over 20% at the highest concentration, the results were considered as positive.

With S9 mix, the number of chromosome aberration slightly increased but did not exceed 5%. Therefore the results with metabolic activation were considered as negative.

Since positive results were obtained in vitro in the chromosome aberration test in the absence of metabolic activation, an in vivo mammalian alkaline comet assay is conducted according to the OECD No. 489 test guideline.

In vivo mammalian alkaline comet test:

Two studies of reliability 1 according to Klimisch cotation critera are available (W.M.A Westerink, 2017, 2019) and were selected as a key studies. In the alkaline in vivo Comet assay, performed according to the OECD No. 489 guideline, Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid was tested in male rats to evaluate its potential genotoxic effect in liver and glandular stomach. Of note, the second study (2019) was specifically requested by the European Chemicals Agency (ECHA Decision Number TPE-D-2114439314-53-01/F) because ECHA considered that the results for the first Comet assay (2017) in glandular stomach did not fulfil the quality criteria based on the relatively high Tail Intensity for the vehicle treated animals. In order to comply with the information requested by ECHA for glandular stomach a new study was initiated with a modified method (electrophoresis parameters were optimized as well as the isolation method) and adopted acceptability criteria as requested by ECHA.

In both studies, 25 male animals were dosed by oral gavage with vehicle or 500, 1000 and 2000 mg Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid per kg body weight once daily for three consecutive days. A positive control group was dosed twice by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS) per kg body weight. In total 5 treatment groups were used, each consisting of 5 animals. No treatment-related clinical signs or mortality were noted after dosing in any animal treated with 500, 1000 or 2000 mg/kg Reaction mass of N-[2-(2-oxoimidazolidin-1- yl)ethyl]methacrylamide and methacrylic acid or control animals receiving vehicle or EMS.

Approximately 3-4 hours after the second dose of EMS and third dose of the vehicle or Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid, liver and glandular stomach tissues were collected (only glandular stomach in the second study). The animals were sacrificed by abdominal aorta bleeding under isoflurane anaesthesia. Single cell suspensions from liver and/or stomach were made followed by Comet slide preparation. The slides were analyzed by an image analysis system and the Tail Intensity (%) was assessed.

No statistically significant increase in the mean Tail Intensity (%) was observed in liver or glandular stomach cells of Reaction mass of N-[2-(2-oxoimidazolidin-1- yl)ethyl]methacrylamide and methacrylic acid-treated male animals at any of the dose levels tested compared to the vehicle treated animals.

The mean Tail Intensity in liver and stomach cells of vehicle-treated rats was 4.65% and 54.53%, respectively in the first study and was 2.84% in the stomach cells in the second study. The positive control EMS, showed a mean Tail Intensity of 91.23% (20-fold statistically significant induction; Students t test p<0.001) in liver and of 97.64% (1.8-fold statistically significant induction; Students t test p<0.001) and 63.17% (mean ± SD, 22-fold induction; p<0.001 Students t test)

in stomach tissue in the first and second study, respectively. The negative and positive control Tail Intensities were therefore within the historical control data range. Hence, all criteria for an acceptable Comet assay were met.

It is concluded that Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid shows a negative result (non-genotoxic) in the Comet assay in liver and glandular stomach post dosing of male rats up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Justification for classification or non-classification

Harmonized classification:

No harmonized classification is available according to the Regulation (EC) No 1272/2008.

Self classification:

Based on the available data (in vitro and in vivo tests), Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid is not classified for the genotoxicity according to the Regulation (EC) 1272/2008 (CLP).