2-methoxyethyl acrylate

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Nov 2015 - 24 Mar 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

2-methoxyethyl acrylate

EC Number:

221-499-3

EC Name:

2-methoxyethyl acrylate

Cas Number:

3121-61-7

Molecular formula:

C6H10O3

IUPAC Name:

2-methoxyethyl acrylate

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): 2-methoxyethyl acrylate
- Physical state: liquid, colourless
- Analytical purity: 99.9%
- Purity test date: 22 Nov 2012
- Expiration date of the lot/batch: 31 Mar 2016
- Storage condition of test material: Room temperature, in the dark, under nitrogen

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: TOAGOSEI CO., LTD
- Expiration date of the lot/batch: 31 Mar 2016
- Purity test date: 16 Sep 2015


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in the dark, under Nitrogen
- Stability under test conditions: confirmed by chemical analytics during conduct of the study
- Solubility and stability of the test substance in the solvent/vehicle: confirmed by chemical analytics during conduct of the study

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS Limited, UK
- Age at study initiation: 7 - 12 weeks
- Weight at study initiation: 150 - 200 g
- Assigned to test groups randomly: No
- Fasting period before study: No
- Housing: 5 animals of the same sex per cage in propylene cages with stainless stell mesh lids, bedding soft wood flakes
- Diet: Rodent 2014C Teklad Global Certified Diet (Envigo RMS Limited, UK), ad libitum
- Water: Drinking water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): at least 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

IN LIFE DATES: 24 Nov 2015 - 24 Mar 2016

ADDITIONAL INFORMATION: One animal (120 mg/kg) marginally exceeded the upper body weight range. Since the difference was very small, (less than 1.5% overweight) it was considered to have no impact on the study outcome.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Phosphate buffered saline (PBS)
- Concentration of test material in vehicle: 12, 24, 48 mg/mL
- Amount of vehicle: 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was freshly prepared before each administration as a solution at the appropriate concentration in phosphate buffered saline.

Duration of treatment / exposure:

2 single treatments within 24 hours

Frequency of treatment:

at 0 and 24 hours

Post exposure period:

Animals were sacrificed 4 hours after final treatment

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

120 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

240 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

480 mg/kg bw/day (nominal)

No. of animals per sex per dose:

7 (negative control and test item groups)
5 (positive control

Control animals:

yes, concurrent vehicle

Positive control(s):

N-methyl-N-nitrosourea
- Justification for choice of positive control(s): mentioned as positive control in OECD 489
- Route of administration: oral
- Doses / concentrations: 25 mg/kg bw

Examinations

Tissues and cell types examined:

Liver, non-glandular and glandular stomach

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Range finding study was performed to find maximum tolerated dose

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Animals were treated two times at 0 and 24 hours. 28 hours after first treatment, animals were sacrificed and tissues were prepared. Sub-samples of the liver, glandular and non-glandular stomach were taken from the vehicle control animals and the dose group animals and preserved in 10% buffered formalin for possible histopathological examination.

DETAILS OF THE SLIDE PREPARATION: Tissue samples were processed to provide single cell suspensions. Cell suspensions were mixed using low melting point agarose and placed onto pre-coated slides. Four slides/animal for each tissues were prepared. After cell lysis the slides were transferred to electrophoresis to allow the DNA to unwind for 20 minutes. Electrophoresis was conducted at approximately 0.7 V/cm, 300 mA for 20 minutes. Slides were fixed in 100% methanol after electrophoresis and stained with propidium iodide (20 µg/mL).

METHOD OF ANALYSIS: Two slides for each tissue per animal were analyzed using a fluorescence microscope combined with a CCD camera attached to a PC-based image analysis program (Comet IV version 4.3.1.) and a maximum of 200 cells per tissue and animal were scored primary for relative tail intensity. Tail length and tail moment were also considered. Each slide was assessed for the incidence of hedgehog to give an indication of cell integrity.

OTHER: Due to statistical increase on percentage tail intensity in glandular and non-glandular stomach tissue, additional histopathological examinations of the non-glandular and glandular stomach were conducted. Therefore tissue samples were processed to paraffin wax, sectioned and stained with hematoxylin and eosin.

Evaluation criteria:

EVALUATION CRITERIA:
Acceptability Criteria:
- The concurrent negative control is comparable with the laboratory historical negative control range.
- The positive controls induce responses that are comparable with those in the laboratory positive control range.
- Adequate numbers of cells and doses have been analysed.
- The highest dose level selected meets the requirements of the guideline and the study plan.

Evaluation of results
Clearly negative if:
- None of the test concentrations exhibits a significant increase compared with the concurrent negative control.
- There is no evidence of a dose- related response.
- The results are within the laboratory historical vehicle control range.
- There is evidence, direct or indirect, to demonstrate exposure or toxicity to the target tissue has been achieved.

Clearly positive if:
- At least one of the test doses exhibits a statistical significant increase compared to the concurrent negative control.
- The response is considered to be dose- related.
- The results are substantially outside the laboratory historical vehicle control range.

Statistics:

Students t-test on transformed data using a transformation √(x+1)

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

positive

Remarks:

Statistically significant increase in the % tail intensity in the mid and high dose groups

Toxicity:

yes

Remarks:

1/7 animals died in the high dose group. Hunched posture was observed in the remaining 6/7 animals of the high dose group 1 hour after dosing

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
A range-finding test was performed to find suitable dose levels of the test item following a double oral administration at zero and 24 hours and to select the most appropriate sex for use in the main test. The upper dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg bw.
- Dose range: 400, 480, 500, 600 mg/kg bw
- Clinical signs of toxicity in test animals: mortality was observed in males (2/3 animals, one animal was dosed only once) at 600 mg/kg and females at 500 (1/1 animal) and 600 mg/kg bw (1/1 animal), hunched posture was observed at 480-600 mg/kg bw
- Rationale for exposure: starting dose based on oral LD50 in rats (404 mg/kg bw)

RESULTS OF DEFINITIVE STUDY
- Percentage tail intensity: No significant increase in percentage tail intensity was observed in liver tissue of treatment groups compared with the negative control (Table 3).

A significant increase in the mean of median percentage tail intensity in the glandular stomach tissue was noted in all dose groups, and in the mean percentage tail intensity in the mid- and high dose group, compared with the control group respectively (1.4 - 3.6 fold compared with mean of controls) (Table 1). However, the increase fell within the historical negative control values (2.67 - 12.74% mean % tail intensity).
A significant increase in the percentage tail intensity was also observed in the non-glandular stomach tissue of the mid- and high-dose group, compared with the control group (1.7 - 2.3 fold compared to mean of controls, for mean percentage tail intensity and mean of median percentage tail intensity, Table 2). The individual variation in each dose group and in the control groups was quite high in stomach tissues.
Histopathological findings (Table 4):
- Glandular stomach: Erosion of the glandular epithelium observed in males treated with 240 mg/kg bw (4/7 animals) or 480 mg/kg bw (6/6 animals), inflammation of submucosa observed in males treated with 240 mg/kg bw (3/7 animals) and 480 mg/kg bw (6/6 animals), ulceration in 1 male treated with 480 mg/kg bw, myofiber degeneration observed in males treated with 480 mg/kg bw (4/6 animals)
- Non-glandular stomach: Ulceration observed in males (3/6 animals) treated with 480 mg/kg bw, erosion observed in males (2/6 animals) treated with 480 mg/kg bw, vacuolation of the non-glandular epithelium in males treated with 480 mg/kg bw (3/6 animals) and in 1 male treated with 240 mg/kg bw; Ulceration of the limiting ridge in males treated with 240 mg/kg bw(2/7 animals) and 480 mg/kg bw (4/6 animals), mucosal necrosis in males treated with 480 mg/kg bw (2/6 animals), vacuolation of the epithelium at the limiting ridge in males treated with 120 mg/kg bw (3/7 animals) or 240 mg/kg bw (3/7 animals) , epithelial hyperplasia in 1 male treated with 240 mg/kg bw, inflammation observed treated with 240 mg/kg bw (1/7 animals) and 480 mg/kg bw (6/6 animals), myofiber degeneration in males treated with 480 mg/kg bw (6/6 animals)
- Statistical evaluation: Significant increase in percentage tail intensity (mean percentage tail intensity and mean of median percentage tail intensity) was observed in the glandular- and non-glandular stomach tissues at 240 and 480 mg/kg bw, and at 120 mg/kg bw in the glandular stomach tissue (mean of median percentage tail intensity).

Any other information on results incl. tables

Table 1: Summary Table Comet Assay – Glandular Stomach

Dose Level	Group Mean % Hedgehogs	Group Mean % Tail Intensity	Group Mean of Mean of Median % Tail Intensity per Animal
Vehicle	3.84 ± 1.44	2.05 ± 0.62	0.69 ± 0.42
480 mg/kg bw	2.65 ± 1.03	3.98 ± 1.71^a	2.52 ± 1.61^b
240 mg/kg bw	4.18 ± 1.33	2.92 ± 0.79^b	1.22 ± 0.63^c
120 mg/kg bw	4.00 ± 1.40	2.72 ± 0.99	1.18 ± 0.64^c
Positive (MNU)	6.04 ± 1.01	21.09 ± 1.81^a	19.28 ± 1.88^a

^a= P < 0.001

^b= P < 0.01

^c= P < 0.05

 

Table 2: Summary Table Comet Assay – Non-Glandular Stomach

Dose Level	Group Mean % Hedgehogs	Group Mean % Tail Intensity	Group Mean of Mean of Median % Tail Intensity per Animal
Vehicle	6.12 ± 2.32	6.68 ± 1.88	4.35 ± 1.74
480 mg/kg bw	6.41 ± 1.07	11.42 ± 3.16^a	9.30 ± 3.87^a
240 mg/kg bw	3.93 ± 1.04	11.92 ± 3.58^a	10.29 ± 3.97^a
120 mg/kg bw	4.83 ± 1.28	7.92 ± 2.42	5.92 ± 2.42
Positive (MNU)	7.78 ± 1.71	41.68 ± 3.60^a	41.90 ± 4.21^a

^a= P < 0.001

Table 3: Summary Table Comet Assay – Liver

Dose Level	Group Mean % Hedgehogs	Group Mean % Tail Intensity	Group Mean of Mean of Median % Tail Intensity per Animal
Vehicle	1.16 ± 0.71	0.34 ± 0.06	0.01 ± 0.01
480 mg/kg bw	0.25 ± 0.27	0.34 ± 0.08	0.01 ± 0.01
240 mg/kg bw	0.21 ± 0.26	0.32 ± 0.08	0.00 ± 0.00
120 mg/kg bw	0.69 ± 0.26	0.41 ± 0.23	0.01 ± 0.00
Positive (MNU)	1.04 ± 1.01	16.08 ± 3.85^a	15.56 ± 4.07^a

^a= P < 0.001

 

Table 4: Histopathological results of glandular and non glandular stomach (No affected animals/total No. examined)

Organ	Effect	control	480 mg/kg bw	240 mg/kg bw	120 m/kg bw
Glandular stomach	Erosion - minimal - slight	0/7 0/7	2/6 4/6	4/7 0/7	0/7 0/7
	Ulceration - slight	0/7	1/6	0/7	0/7
	Inflammation submucosa - minimal - slight	0/7 0/7	6/6 0/6	3/7 0/7	0/7 0/7
	Myofiber degeneration - minimal - slight	0/7 0/7	3/6 1/6	0/7 0/7	0/7 0/7
Non-glandular stomach	Ulceration - slight - marked	0/7 0/7	1/6 2/6	0/7 0/7	0/7 0/7
	Erosion - slight - moderate	0/7 0/7	1/6 1/6	0/7 0/7	0/7 0/7
	Vascuolation - minimal - slight	0/7 0/7	3/6 0/6	0/7  1/7	0/7 0/7
	Vacuolation limiting ridge - minimal - slight	0/7 0/7	0/6 0/6	1/7 2/7	3/7 0/7
	Epithelial Hyperplasia - slight	0/7	0/6	1/7	0/7
	Ulceration, Limiting Ridge - minimal - slight	0/7 0/7	2/6 2/6	1/7 1/7	0/7 0/7
	Mucosal necrosis, Limiting ridge - slight	0/7	2/6	0/7	0/7
	Inflammation submucosa - minimal - slight	0/7 0/7	0/6 6/6	0/7 1/7	0/7 0/7
	Myofiber Degeneration - minimal - slight - moderate	0/7 0/7 0/7	2/6 2/6 2/6	0/7 0/7 0/7	0/7 0/7 0/7

Applicant's summary and conclusion

Conclusions:

The results in non-glandular stomach were considered to be an intrinsic genotoxic response.

Executive summary:

A Comet Assay was conducted according to the OECD TG 489 and in compliance with GLP. The primary target tissues of this assay were liver, glandular stomach and non-glandular stomach. A range-finding test was performed to find suitable dose levels of the test item and to investigate if there were any differences between the sexes. The initial dose selection for the range-finding test was based on the documented LD50 data from an oral rat study. The comet assay main test was performed using male rats only and groups, each of seven rats for the vehicle control group and the test item dose groups, and five rats for the positive control group were dosed twice at 0 and 24 hours with the test item via the oral route. The Comet assay main test was conducted with the test item at the maximum tolerated dose (MTD) 480 mg/kg with 240 mg/kg and 120 mg/kg as the lower dose levels. Animals were killed 4 hours after the second dose administration and samples of the liver, glandular stomach and non- glandular stomach tissues were processed to produce single cell suspensions. These were processed to produce the comet slides which were subsequently scored for the presence of Comets.

Further groups of rats were given a double oral dose of Phosphate Buffered Saline (seven rats) or N-Nitroso-N-methylurea (five rats), to serve as vehicle and positive controls, respectively.

The primary target tissues of this assay were liver, glandular stomach and non-glandular stomach.

Formulation analysis was performed on the test item formulations of the main test.

 

In the liver, no significant change in the % tail intensity was observed between the treatment groups and control group.

In the glandular stomach tissue, a dose-related significant increase in the mean of median % tail intensity was noted in all dose groups, and in the mean % tail intensity in the mid and high dose groups, compared with the control group. However, the increase fell within the range of the historical control data, which is composed by a limited dataset (only 11 animals) thus its adequacy is questionable.

In the non-glandular stomach tissue, a significant increase in the % tail intensity was also observed of the mid and high dose groups. At the site of contact: inflammation and degeneration of the glandular and non-glandular stomach tissues in the mid and high dose animals are considered to be the result of the corrosive properties of the substance and were more severe in non-glandular stomach than in glandular stomach. A statistically significant increase in the mean % tail intensity in the non-glandular stomach was observed already at the lowest dose, showing only minimal concomitant histopathological findings in the non-glandular stomach. Moreover, in the non-glandular stomach, the increase in cytotoxicity was clearly dose-related, but did not correlate with an increased genotoxic response. The genotoxicity effects were higher at the mid dose. If the genotoxicity effects were only the result of a cytotoxic response, the highest % tail intensity in the Comet assay would have been expected to be in the highest dose group, but this is not the case. Marked cytotoxicity including ulceration and necrosis were only observed at 480 mg/kg bw (highest dose) and not at 240 mg/kg bw (mid dose).Tthese results suggest that the genotoxic response cannot only be explained by cytotoxicity. Therefore, the results in non-glandular stomach were considered to be an intrinsic genotoxic response.