2,3-dihydroxypropyl methacrylate

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:

19 June 2018 - 23 August 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

The method was designed to be compatible with the procedures indicated in the OECD 489 Guideline (2016)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

2,3-dihydroxypropyl methacrylate

EC Number:

227-642-6

EC Name:

2,3-dihydroxypropyl methacrylate

Cas Number:

5919-74-4

Molecular formula:

C7H12O4

IUPAC Name:

2,3-dihydroxypropyl 2-methylprop-2-enoate

Test animals

Species:

rat

Strain:

other: Wistar Han™ (HsdRCCHan™WIST)

Details on species / strain selection:

A range-finding test was performed to find suitable dose levels of the test item and the most appropriate sex. The Comet assay main test was conducted in male animals.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo (UK)
- Age at study initiation: approximately eight to ten weeks old
- Weight at study initiation: 187.3 to 215.5 g
- Assigned to test groups randomly: yes
- Fasting period before study: no information
- Housing: in groups of up to five by sex in solid-floor polypropylene cages with woodflake bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): set to achieve limits of 19 to 25 ºC . Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study
- Humidity (%): set to achieve limits of 30 to 70%. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study.
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


The groups of rats from each dose level were killed by humane euthanasia (carbon dioxide asphyxiation) approximately 4 hours following the second administration, 28 hours after the start of the test.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: distilled water
- Lot/batch no. (if required): 17K13BA1A
- Storage Conditions: Room temperature

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was freshly prepared as required as a solution at the appropriate concentration in distilled water. The test item was formulated within 2 hours of it being applied to the test system; it is assumed that the formulation was stable for this duration. This exception is considered not to affect the purpose or integrity of the study.

The positive control item was freshly prepared as required as a solution at the appropriate concentration in distilled water.

The Vehicle control (distilled water) was used as supplied.

Duration of treatment / exposure:

28 hours

Frequency of treatment:

Animals were dosed twice with a 24-hour interval

Post exposure period:

4 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Range -Finding Toxicity Test /2 Males and 2 Females

Doses / concentrations 2

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Range-finding Toxicity Test/ 2 Males/0 Females

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Comet Test (Animal Numbers 9-15)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Comet Test (Animal Numbers 16-20)

Doses / concentrations 5

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Comet Test (Animal Numbers 21-25)

No. of animals per sex per dose:

Comet Test:
2000 mg/kg bw/day (seven rats),
1000 mg/kg bw/day (five rats) and
500 mg/kg bw/day (five rats)

Control animals:

yes, concurrent vehicle

Positive control(s):

N-Nitroso-N-methylurea (MNU)
- Route of administration: oral
- Doses / concentrations: 25 mg/kg bw/day

Examinations

Tissues and cell types examined:

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

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION AND METHOD OF ANALYSIS:
Adequate numbers of slides were pre-coated with 0.5% normal melting point agarose and stored at room temperature. The slides were labelled for animal number, study number and tissue type prior to use for the comet assay.
Approximately 30 μL of the cell suspension was added to 270 μL of 0.5% low melting point (LMP) agarose, mixed thoroughly and 50 μL of this agarose/cell suspension mix was placed onto a pre-coated slide. Two gels were placed on each slide, and 4 gels were prepared for each tissue. Two of the gels were scored for Comets (A and B replicates) and two (C and D replicates) were kept in reserve in case further scoring was required or the gels were damaged during processing. The agarose/cell suspension mix was immediately covered with a glass cover slip, transferred to a cold room at approximately 4 °C in the dark for approximately 20 minutes to allow it to solidify.
Once the LMP agarose had set, the cover slips were removed and the slides gently lowered into freshly prepared lysing solution (pH 10) and refrigerated in the dark overnight. All slides went through the subsequent processing.
Following lysis, the slides were removed from the solution, briefly rinsed with neutralization buffer and placed onto the platform of an electrophoresis bath, which was filled with chilled electrophoresis buffer (pH>13), until the slide surface was just covered. The slides were then left for 20 minutes to allow the DNA to unwind, after which they were subjected to electrophoresis at approximately 0.7 V/cm (calculated between the electrodes), 300 mA for 20 minutes. The buffer in the bath was chilled during the electrophoresis period and the temperature of the electrophoresis buffer was monitored at the start of unwinding, the start of electrophoresis and the end of electrophoresis to ensure the electrophoresis solution was maintained at low temperature (2-10 °C).
At the end of the electrophoresis period, the bath was switched off, the slides gently removed and placed on to a draining surface and drop wise coated with a neutralization buffer and left for at least 5 minutes. The slides were then drained and a repeat of the addition of the neutralization buffer was performed twice. The slides were further drained and fixed in cold 100% methanol for 5 minutes and allowed to air dry.
Once dry the slides were stored prior to scoring. Two of the four processed slides were scored and the remaining slides were stored as backup slides.

Evaluation criteria:

Each slide was also assessed for the incidence of ‘hedgehog’ cells to give an indication of cell integrity. Hedgehogs are cells that exhibit a microscopic image consisting of a small or non-existent head, and large diffuse tails and are considered to be heavily damaged cells, although the etiology of the hedgehogs is uncertain.

Statistics:

Statistical analysis was considered un-necessary since there were no marked increases over the vehicle control values for the test item dose groups for the liver or glandular stomach. The positive control group demonstrated marked increases over the vehicle control group comparable with the current historical range for the liver and glandular stomach.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Mortality data from the range-finding test is presented below:

Dose Level (mg/kg bw/day)	Sex	Number of Animals Treated	Route	Deaths on Day		Total Deaths
				0	1
2000	Male	2	oral	0	0	0/2
2000	Female	2	oral	0	0	0/2
2000	Male	2	oral	0	0	0/2

There was no noticeable difference in clinical signs between the male and female animals and therefore only male animals were used for the main test.

Applicant's summary and conclusion

Conclusions:

The test item did not induce any significant increases in the percentage tail intensity or median percentage tail intensity compared to the vehicle control in the liver or glandular stomach. Therefore the test item was considered to be unable to induce DNA strand breakage to these tissues in vivo under the conditions of the test.