Registration Dossier

Toxicological information

Genetic toxicity: in vivo

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Administrative data

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
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Reference
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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
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

Test material

Constituent 1
Reference substance name:
Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid
EC Number:
934-058-1
IUPAC Name:
Reaction mass of N-[2-(2-oxoimidazolidin-1-yl)ethyl]methacrylamide and methacrylic acid
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Reaction mass of N-[2-(2-Oxoimidazolidin-1-yl)ethyl] methacrylamide and methacrylic acid, other name: Sipomer wam II
- Storage condition of test material: at room temperature
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)

Test animals

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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Test results
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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.